Clinical trial NCT02711826
Conditions: Kidney Transplant Adult Living Donor Kidney Transplant Recipients Renal Transplant Living Kidney Donor
Keywords: graft inflammation Treg polyclonally expanded Tregs (polyTregs) calcineurin inhibitors (CNIs) mTOR inhibitors
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2016-09-20; Completion date: 2023-08-04
Enrollment: 32 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is:

* To see if polyTregs can reduce inflammation in a transplanted kidney.
* To find out what effects, good or bad, polyTregs will have in the kidney recipient.
* To find out what effects, good or bad, taking everolimus after polyTregs will have in the kidney recipient.

Detailed description:
Inflammation occurs when the body's defense system recognizes a foreign object (such as a transplanted kidney), and responds by sending white blood cells to attack the foreign object. These cells and the substances they produce can damage the transplanted kidney. There is currently no standard treatment for inflammation in the kidney; some transplant centers do not treat inflammation at all. Rejection is a more severe form of inflammation and injury. Both inflammation and rejection are diagnosed by looking at a piece of kidney (a kidney biopsy) under a microscope. Kidneys that have inflammation and/or rejection do not work as well or last as long as kidneys without injury.

People who have a transplant take immunosuppressive drugs (IS) to prevent inflammation and rejection. Although kidney transplant recipients usually do well in the first five years after transplant, transplant researchers are interested in finding ways to prevent inflammation and rejection without IS, or with lower doses of IS in order to avoid side effects.

While some white blood cells cause inflammation, other types of white blood cells, called T regulatory cells (Tregs), can control inflammation. Tregs may have an important role in controlling or preventing inflammation and rejection. A person's Tregs can be grown in the laboratory to increase their number (polyTreg). These Tregs can be given back through a needle placed in a vein (IV). PolyTregs, when given to the recipient, might reduce inflammation in the transplanted kidney. However, this effect has not yet been shown.

One of the IS drugs used in kidney transplant is Everolimus. Everolimus has been shown to help Tregs survive better than other types of IS drugs.

This is a randomized open-label trial to determine the safety and efficacy of a single dose of autologous polyTregs in renal transplant recipients with subclinical inflammation (SCI) in the 3 to 7 months post-transplant allograft protocol biopsy compared to control patients treated with CNI-based immunosuppression. The efficacy of the Treg therapy will be assessed by the reduction of graft inflammation on biopsies performed at 7 months after study group allocation compared to the eligibility biopsy.

Arms: arm 1: Standard of care

(N=7 participants in this group) arm 2: Subjects to receive polyTregs (550 ± 450 x 10\^6). After receiving at least 300X10\^6 polyTregs infusion, eligible subjects will start mammalian Target of Rapamycin (mTOR) inhibitor.

Target tacrolimus trough levels prior to conversion to everolimus are 4-11 μg/dl, which falls within standard of care. For eligible participants in polyTregs and darTregs groups, the tacrolimus dose will be reduced by 50% when everolimus is initiated. Tacrolimus will be discontinued 4 weeks after initiation of everolimus therapy.

Everolimus, a mTOR inhibitor immunosuppressant, will be initiated at a dose of 1.5 mg orally twice daily and titrated, as needed. Participants will begin everolimus with target trough levels of 3-8μg/L for 4 weeks while still taking tacrolimus. Everolimus target trough levels will be 6-10 μg/L when tacrolimus is discontinued.

(N=7 participants in this group)
Interventions: intervention 1: Polyclonal Regulatory T Cells intervention 2: Everolimus intervention 3: Tacrolimus intervention 4: Mycophenolate mofetil intervention 5: Mycophenolic acid intervention 6: Acetaminophen intervention 7: Diphenhydramine intervention 8: Biopsy, Kidney intervention 9: Blood Draw intervention 10: Leukapheresis intervention 11: IS regimen conversion
Intervention descriptions: intervention 1: Participants randomized to polyTregs group will receive a single infusion of 550 ± 450 x 10\^6 polyTregs. intervention 2: None intervention 3: None intervention 4: All enrolled participants will be on MMF (or MPA below) at the time of study entry at a minimum dose of 1000mg per day. intervention 5: All enrolled participants will be on MPA (or MMF above) at the time of study entry at a minimum dose of 720mg per day. intervention 6: 650 mg acetaminophen, administered 30-60 minutes prior to infusion as pre-medication. intervention 7: 25-50 mg diphenhydramine intravenously or by mouth, administered 30-60 minutes prior to infusion as pre-medication. intervention 8: None intervention 9: None intervention 10: None intervention 11: Conversion from Tacrolimus, a calcineurin inhibitors (CNI), to Everolimus, an mTOR inhibitor.

Protocol documents:
ICF_000.pdf: 
National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 1 of 9 
 
 
 
1. TITLE OF CLINICAL RESEARCH STUDY 
Full Title:  “Treg Adoptive Therapy in Subclinical Inflammation in Kidney Transplantation” (TASK) 
Protocol Number:   CTOT-21 
Short Title:  Tregs in Subclinical Inflammation 
 
2. YOUR PARTICIPATION IS VOLUNTARY 
We will explain this research study to you.  You may ask questions.   
Research is different from regular medical care.  When you receive standard medical care, your physician makes 
decisions based only on what is in your best interest. When you are in a research protocol, your physician is still 
bound to act in your best interest, but some medical decisions will be directed by the research study. 
• 
Taking part in this study is your decision. 
• 
You may change your mind about being in this study at any time. 
• 
You will be given a copy of this consent form for your records 
 
3. PRINCIPAL INVESTIGATOR 
(Insert site Principal Investigator as well as other investigators if desired.) 
 
4. KEY INFORMATION FOR THE STUDY 
This document contains information that will help you decide whether to take part in a research study.  We 
encourage you to read the entire document.  All the information is important, but here are some key points to 
help you understand the study.  Additional information is available in the document, in the sections noted in 
italics, below.  
 
• 
This research study is for people who received a kidney transplant between 3 and 7 months after 
transplant, and who recently had a kidney biopsy showing inflammation.  See Section 5, Introduction and 
Background  
• 
The study will test whether a new product called donor-reactive T-regulatory cells (DRTregs), given one 
time through an intravenous line, is safe and will reduce inflammation in the kidney.  See Section 5, 
Introduction and Background; and Section 6, Purpose of the Study  
• 
The study asks kidney donors to make 1 to 2 office visits.  However, kidney recipients can still be in the 
study if donors are not able to take part.  See Section 8B, Study Overview/Visit Schedule 
• 
Kidney donors who choose to be in this study will have: 
o Blood draw  
o Physical examination and interview. See 7C, Study Procedures 
• 
The risks of receiving DRTregs are not known because they have only been given to a very small number 
of people (fewer than 10 people).  The reason for giving them is that they might act on the immune 
system to prevent or decrease damage to the transplanted kidney. However, they might affect the 
immune system in other ways.  For example, the immune system might be less able to fight off infection 
or cancer. There is also a risk for an immediate bad reaction such as rash, swelling, or breathing problems 
when the DRTregs product is given.  
• 
There might be no direct medical benefit to you or your kidney recipient for being in this study.  The 
information learned from this study might someday benefit future kidney transplant recipients.  See 
Section 8, Potential Benefits 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 2 of 9 
 
 
 
 
5. INTRODUCTION/BACKGROUND 
You are being asked to take part in this study because you donated a kidney to a recipient who may qualify to be 
in this study.  This research study is for people who received a kidney transplant about 3-7 months ago, and who 
recently had a kidney biopsy showing inflammation.  One blood draw is needed from you, the kidney donor in 
order for the recipient to participate.   
 
 
Inflammation occurs when the body’s defense system recognizes a foreign object (such as a transplanted kidney), 
and responds by sending white blood cells to attack the foreign object.  These cells and the substances they 
produce can damage the transplanted kidney.  Rejection is a more severe form of inflammation and injury.  Both 
inflammation and rejection are diagnosed by looking at a piece of kidney (a kidney biopsy) under a microscope.  
Kidneys that have inflammation and/or rejection do not work as well or last as long as kidneys without injury. 
 
People who have a transplant take immunosuppressive drugs (IS) to prevent inflammation and rejection.  
Transplant researchers are interested in finding ways to prevent inflammation and rejection without IS, or with 
lower doses of IS in order to avoid side effects.   
 
While some white blood cells cause inflammation, other types of white blood cells, called T regulatory cells 
(Tregs), can control inflammation.  Tregs may have an important role in controlling or preventing inflammation 
and rejection.  A person’s Tregs can be grown in the laboratory to increase their number.  These Tregs can be 
given back through a needle placed in a vein (IV).  Researchers can expose a transplant recipient’s Tregs to the 
donor’s cells, which results in Tregs that recognize the donor.  These are called donor reactive Tregs (DRTreg).  
Both Tregs and DRTreg, when given to the recipient, might reduce inflammation in the transplanted kidney.  The 
DRTregs are thought to be more effective because they might be able to help the body specifically recognize and 
accept the donated kidney.  
 
One of the IS drugs used in kidney transplant is everolimus.  Everolimus has been shown to help Tregs survive 
better than other types of IS drugs. 
 
6. PURPOSE OF THE STUDY 
The purpose of this study is:   
• 
To see if Tregs and/or “donor reactive” DRTregs can lower inflammation in the transplanted kidney.   
• 
To find out what effects, good or bad, Tregs or DR Tregs will have in the kidney recipient. 
• 
To find out what effects, good or bad, taking everolimus after Tregs or DRTregs will have in the kidney 
recipient. 
Kidney transplant recipients who agree to participate in this study will be assigned to one of three groups.   
Group 1 will receive standard of care IS.  This means that Group 1 will receive the same IS drugs that are given to 
kidney transplant recipients who are not in the study.  Group 1 subjects will not receive Treg or DRTregs.  Group 
2 will receive Tregs, and Group 3 will receive DRTregs.  Participants who are assigned to group 2 or 3 will have a 
kidney biopsy 2 weeks after receiving Tregs or DRTregs.  If that biopsy shows inflammation is reduced, their IS 
drugs will be changed to include everolimus. 
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 3 of 9 
 
 
 
You, your recipient, and the study doctor cannot choose which group the recipient will be in.  The assignment is 
made using a computer program and will be based on whether the donor is available and how many people are 
in the study already.   
 
 
7. STUDY COMPONENTS 
A. Number of Study Sites and Participants 
This study is sponsored by the National Institute of Allergy and Infectious Diseases (NIAID).  The lead study doctor 
is Dr. Flavio Vincenti at the University of California, San Francisco.  Dr. <insert site principal investigator name> is 
in charge of the study at <insert institution>.  Up to <insert expected number for your site> subjects will be 
enrolled at <insert institution>.  We plan to treat 45 kidney transplant recipients in this study at 6 transplant 
centers in the United States.  For the recipients in Group 3 to participate, their kidney donors must be willing to 
give one blood sample so that we can grow up the recipient’s Tregs that react to the kidney donor.   
B. Study Procedures 
Blood Draw 
If the recipient is fully eligible for the study and is assigned to Group 3, you will be asked to have 100ml (just 
under 7 tablespoons) of blood drawn at the transplant center.  Some of the cells in the blood will be removed 
from the blood specimen to make DRTregs for infusion in your recipient.  Some of the cells will be stored for tests 
to determine if your recipient has a detectible reaction against you, as his / her kidney donor, and if this changes 
after receiving Treg cells.  
 
At the same study visit, you will have additional blood (1.5 tablespoons, 23 ml) to test for infections that could be 
transmitted in the cells you donate.  This testing is required by law since the cells from your blood will be used to 
make the Treg infusion to be given to the recipient.  The tests will include human immunodeficiency virus, 
hepatitis B or C virus, syphilis, cytomegalovirus, Epstein Barr Virus, and human T-lymphotropic virus.  This is 
the same type of screening tests that were done prior to the time of kidney donation.  You will be informed of 
your test results.  You will be referred back to your primary care giver if any of the tests are positive for infection 
and need investigation or treatment. 
 
A study team member will review the information in this consent form with you at the transplant center.  You 
will be asked to sign the consent form before your blood is drawn.  You will not have blood drawn if the recipient 
is no longer eligible or willing to be in the study. 
 
Physical Examination 
A physical examination, including blood pressure and weight measurements, will be performed at the study visit. 
 
Cell Therapy Donor Interview 
Your medical records will be reviewed and you will be asked questions about your medical and social history to 
make sure you are eligible to donate cells for the manufacturing process.  The study investigator or member of 
the study staff will ask you these questions at the study visit.  The types of questions asked will be similar to 
those answered at the time of donation. 
 
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 4 of 9 
 
 
 
C. Risks and Discomforts 
Blood Draw 
The risks of blood draws are pain, bruising, infection, redness, and swelling at the sight of needle entry.  There is 
a chance you may feel dizzy while your blood is being drawn.   
 
Confidentiality 
• 
Infection Screening 
If any of the tests for infections turn out to be positive (not normal), there is a chance your recipient will know.  
The study team will not share personal information, including specific test results, without your permission.  
However, the recipient will be informed that he or she is no longer eligible to continue in the study. 
• 
Genetic Information 
Genetic information will be recorded from the specimen collected for this study.  However, the specimen will be 
used in experiments planned for this study only.  Your sample will not be looked at for other genetic conditions, 
genetic cloning, or paternity testing.  However, basic genetic information about you will be known to researchers 
(e.g. sex, racial or ethnic group).  We will keep all information recorded private as much as possible.  However, 
because genetic information is unique to you, complete confidentiality cannot be guaranteed.   
 
8. POTENTIAL BENEFITS    
There is no direct medical benefit to you for being in this study.  The information learned from this study might 
someday benefit future kidney transplant recipients. 
 
9. ALTERNATIVES TO PARTICIPATION 
Your doctor will talk with you about this study and other options available to you.  Your decision to participate in 
this study will not affect the care the recipient would otherwise receive.   
 
10. NEW FINDINGS  
Your doctor will tell you about any new findings from this or other research that may affect your willingness to 
allow testing and use of your cells for this study.  If new information is provided to you, we will ask for your 
consent to continue in the study. 
 
11. VOLUNTARY WITHDRAWAL FROM STUDY 
You will only have one study visit for the blood draw.  However, you may decide to withdraw consent for testing 
and use of your cells at any time during this study.  If you decide to withdraw, there is no penalty or loss of 
benefits in your routine medical care or any other benefit(s) that you or your recipient would otherwise be 
entitled to receive.  Your doctor or research staff will destroy your samples at your request.   
 
12. REASONS WHY YOU MAY BE TAKEN OFF STUDY WITHOUT YOUR CONSENT 
You may be removed from the study without your consent at any time.  Reasons why you may be removed from 
the study include, but are not limited to, the following:      
• 
Your study doctor determines that it is in your best interest not to take part.   
• 
You are unable to complete required study procedures. 
• 
The study is stopped by the Institution, the Sponsor, or health authorities. 
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 5 of 9 
 
 
 
13. COSTS TO THE SUBJECT (YOU) 
There will be no charge to you, your health insurance company, or your recipient’s insurance company for any 
costs which are done specifically for this study.    
  
14. PAYMENTS (REIMBURSEMENT) 
You will receive no payment for taking part in this study. 
  (or) 
 
You will receive $50 for coming into the hospital for a research visit if you travel within 100 miles for a study visit.  
If your travel distance is up to 300 miles, you will be reimbursed based on the current mileage reimbursement 
rate.   If your travel distance is 300 miles or more, you will be reimbursed for reasonable travel expenses (e.g. 
hotel, airfare, ground transportation, etc.) based on current government allowances (per diem rates).  You will 
need to give receipts and documentation of mileage to the study coordinator for reimbursement.  Government 
mileage and per diem rates are publicaly available here: https://www.gsa.gov/travel/plan-book/transportation-
airfare-rates-pov-rates-etc/privately-owned-vehicle-pov-mileage-reimbursement-rates 
 
(Payments are pro-rated and not held until completion of the study. If participant payments are tied to 
completion of specific study benchmarks, these stipulations should be noted in this section as well as other 
reimbursement specific to the study -i.e. parking, meal tickets, etc.    
 
15. RESEARCH-RELATED INJURY  
<Insert your IRB required language.> 
If are hurt or get sick from something that was done as part of this study, doctors at the clinic or hospital can 
arrange for emergency medical care. The Hospital does not offer financial compensation or payment for injuries 
due to participation in this research. No funds have been set aside to compensate you in the event of injury.  
Your insurance company will be billed for the costs of any care for injuries.  
  
In case of injury resulting from this study, you will not lose any legal rights by signing this form. 
 
16. CONFIDENTIALITY  
Your medical and research records will be confidential to the extent permitted by law.  Efforts will be made to 
keep personal information private.  However, we cannot guarantee complete confidentiality.   
 
As an NIH funded study, you are further protected through a policy that prevents the investigator from disclosing 
sensitive study information that would lead to your identity. This does not prevent you or a family member from 
voluntarily releasing information about yourself or your involvement in this research.                      
 
Your personal identifiers (for example, name and medical record number) will be used during the manufacturing 
process for the Treg infusion at the transplant center and at the UCSF laboratory (UCSF Human Islet and Cellular 
Transplantation Facility) where the Treg infusion is made.  Use of the identifiers is required to ensure the cells are 
tracked accurately through the manufacturing process.   
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 6 of 9 
 
 
 
In all other parts of the study, you will be identified by a code, and personal information from your records will 
not be released without your written permission.  You will not be identified in any publication or in the sharing of 
your data about this study.   
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. law.  This 
web site will not include information that can identify you.  At most, the Web site will include a summary of the 
results.  You can search this Web site at any time.  
 
Organizations that may look at and/or copy your medical records for research, quality assurance and data 
analysis include:   
• the National Institute of Allergy and Infectious Diseases, (NIAID) sponsor of the research  
• NIAID representatives, agents, employees, contractors, and other persons assisting in conducting, 
monitoring or analyzing the study   
• the U.S. Food and Drug Administration  
• other State and Local health authorities 
 
17. STORING INFORMATION/DATA FOR FUTURE STUDIES  
We will store information resulting from this study in a central data repository. Data is information organized for 
a reason, like this study.  A central data repository is a place that collects and stores data from many different 
studies.  The purpose of this collection is to make scientific information available for future studies which may 
help future patients. Your data may be stored indefinitely.  Any information released to a central data repository 
will not contain traditional information such as name, birthdate, address, etc. that is considered your personal 
information. 
 
18. PROBLEMS OR QUESTIONS   
If you ever have questions about this study or in case of research-related injuries, you should contact Dr. <Insert 
name> at <Telephone Number>, or if you have questions about research subjects’ rights you can call <Insert the 
name and title of the appropriate country/site-specific person> at <Insert the number>, or if you prefer, direct 
your questions to the following address: <Insert address of IRB patient representative here>. 
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 7 of 9 
 
 
 
 SIGNATURE PAGE  
 (Site may use the site-specific signature page if required) 
 
Please sign below if you agree to take part in this study.  
  
• 
you have read the informed consent and/or had it explained to you 
• 
you were given the opportunity to ask questions about the information, and 
• 
you voluntarily agree to take part in the study 
 
 
_________________ 
         
_____________________      
           ______________ 
Research Subject’s Name 
Research Subject’s Signature 
           Date 
(Typed or printed) 
 
 
Signature of person(s) explaining and obtaining the consent: 
 
 
____________________ 
____________________ 
 
_________________ 
Name and Title  
Signature 
 
 
 
Date 
(Typed or printed)   
 
____________________ 
____________________ 
 
_________________ 
Name and Title  
Signature 
 
 
 
Date 
(Typed or printed)   
 
  
(NOTE:  This consent form with the original signatures MUST be retained on file by the principal investigator.  A 
copy must be given to the research subject.  A copy should be placed in the research subject’s medical record, if 
applicable.)      
  


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 8 of 9 
 
 
 
19. STORAGE OF SAMPLES and/or INFORMATION FOR FUTURE USE  
(This consent is optional and agreement is not required for participation in the study) 
We are asking your permission to store samples of biological specimens (e.g., cells) collected for this study to be 
used in the future for tests that are not yet planned. These tests may or may not be related to the study of 
transplantation.     
 
Some information about a research specimen will always be linked to it (also see section 6c, Genetic 
Information).  For example, researchers might know a sample is from a kidney transplant recipient.  Because of 
this, allowing for specimens to be stored for future use also means allowing for the linked information to be 
stored and used when the specimens are needed. 
 
Genetic Testing on Stored Samples 
Your samples may be used to look at genetic information in relation to kidney transplantation (for example, the 
rejection process).  However, your samples could also be used in studies that are not related to transplantation 
(for example, the immune system as a whole).   
 
Confidentiality 
All samples will be labeled with a randomly assigned unique barcode.  They will not contain personal information 
like name, initials, or date of birth.  Reports on these stored samples and genetic tests will not be given to your 
study doctor and they will not be in your medical record.  Doctors will not know that your samples are being used 
for other studies and it will not affect your routine medical care. 
 
Benefits of Stored Material and Genetic Testing 
There is no benefit to you from the storage of samples and information.  However, the use of your samples and 
information may help researchers learn more about your disease or the genetics related to a specific condition.   
 
Samples will be stored at the core laboratories involved in this trial.  If you agree, your samples and information 
may be stored for an unknown length of time. 
 
Risks of Stored Material and Genetic Testing 
Although your stored research samples will not be sold, the information obtained from the research performed 
on your samples may in the future lead to the development of commercial products.  You will not receive any 
money from research using your stored samples and information 
There may be unknown risks associated with the storage of samples and information.    For example, if the future 
research involves genetic testing it is possible that it could be traced back to you because your genes are specific 
to you. We will make every attempt to protect your confidentiality and to make sure that your personal identity 
does not become known.    
 
Making a Decision for Stored Human Subject Material and Genetic Testing  
Your decision regarding the storage of samples or the information resulting from the analysis of your samples will 
not affect your ability to participate in this study.   
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Group 3 Donor Consent Template 
CTOT-21 Group 3 Donor Consent Template 
 
Version 8.0 / March 20, 2019 
 
 
Page 9 of 9 
 
 
 
 
Please indicate your response below: 
Choose yes or no to allow the storage and sharing of your sample (cells) and information for genetic tests.      
 
   Yes       
  No    
 
 
                                   _______________________ 
       
      Initials of Research Subject 
 
Choose yes or no to allow the storage and sharing for future use of remaining sample (cells) and information for 
other research tests not currently planned (not genetic).  
 
 
   Yes       
  No    
 
 
                                    _______________________ 
         
      Initials of Research Subject 
 
 
 
 
 
 
 
 
 
 

ICF_001.pdf: 
National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Recipient Consent Template 
CTOT-21 Recipient Consent Template 
 
Version 11.0/ January 11, 2022 
 
Page 1 of 15 
 
 
 
 
 
1. TITLE OF CLINICAL RESEARCH STUDY 
“Treg Adoptive Therapy in Subclinical Inflammation in Kidney Transplantation” (TASK) 
Protocol Number:   CTOT-21 
Short Title:  Tregs in Subclinical Inflammation 
 
2. YOUR PARTICIPATION IS VOLUNTARY 
We will explain this research study to you.  You may ask questions.   
Research is different from regular medical care.  When you receive standard medical care, your physician makes 
decisions based only on what is in your best interest. When you are in a research protocol, your physician is still 
bound to act in your best interest, but some medical decisions will be directed by the research study. 
• 
Taking part in this study is your decision. 
• 
You may change your mind about being in this study at any time. 
• 
You will be given a copy of this consent form for your records 
 
3. PRINCIPAL INVESTIGATOR 
(Insert site Principal Investigator as well as other investigators if desired.) 
 
4. KEY INFORMATION FOR THE STUDY 
This document contains information that will help you decide whether to take part in a research study.  We 
encourage you to read the entire document.  All the information is important, but here are some key points to 
help you understand the study.  Additional information is available in the document, in the sections noted in 
italics, below.  
 
• 
This research study is for people who received a kidney transplant between 3 and 7 months after 
transplant, and who recently had a kidney biopsy showing inflammation.  See Section 5, Introduction and 
Background  
• 
The study will test whether a new product called T-regulatory cells (Tregs) given one time through an 
intravenous line, is safe and will reduce inflammation in the kidney.  See Section 5, Introduction and 
Background; and Section 6, Purpose of the Study  
• 
The study requires participants to make 8 to 10 office or hospital visits over a one-year period.  See 
Section 7B, Study Overview/Visit Schedule 
• 
Study participants have procedures that are not part of routine care for kidney transplant recipients. 
These include: 
o Leukapheresis, in which some of their blood cells are collected by a machine, through 
intravenous lines.  
o An extra kidney biopsy, in which a piece of the transplanted kidney is removed with hollow 
needle 
o Extra urine and blood collections.  See Section 7C, Study Procedures 
• 
The risks of receiving Tregs are not known because they have only been given to a very small number of 
people.  The reason for giving them is that they might act on the immune system to prevent or decrease 
damage to the transplanted kidney. However, they might affect the immune system in other ways.  For 
example, the immune system might be less able to fight off infection or cancer. There is also a risk for an 
immediate bad reaction such as rash, swelling, or breathing problems when the Tregs product is given.  


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Recipient Consent Template 
CTOT-21 Recipient Consent Template 
 
Version 11.0/ January 11, 2022 
 
Page 2 of 15 
 
 
 
• 
There might be no direct medical benefit to you for being in this study.  The information learned from 
this study might someday benefit future kidney transplant recipients.  Some subjects may benefit if 
inflammation in the transplanted kidney can be decreased.  See Section 10, Potential Benefits 
 
5. INTRODUCTION/BACKGROUND 
This research study is for people who received a kidney transplant about 3 to 7 months after transplant, and who 
recently had a kidney biopsy showing inflammation.   
 
 
Inflammation occurs when the body’s defense system recognizes a foreign object (such as a transplanted kidney), 
and responds by sending white blood cells to attack the foreign object.  These cells and the substances they 
produce can damage the transplanted kidney.  There is currently no standard treatment for inflammation in the 
kidney; some transplant centers do not treat inflammation at all.  Rejection is a more severe form of 
inflammation and injury.  Both inflammation and rejection are diagnosed by looking at a piece of kidney (a kidney 
biopsy) under a microscope.  Kidneys that have inflammation and/or rejection do not work as well or last as long 
as kidneys without injury. 
 
People who have a transplant take immunosuppressive drugs (IS) to prevent inflammation and rejection.  
Although kidney transplant recipients usually do well in the first five years after transplant, transplant 
researchers are interested in finding ways to prevent inflammation and rejection without IS, or with lower doses 
of IS in order to avoid side effects.   
 
While some white blood cells cause inflammation, other types of white blood cells, called T regulatory cells 
(Tregs), can control inflammation.  Tregs may have an important role in controlling or preventing inflammation 
and rejection.  A person’s Tregs can be grown in the laboratory to increase their number.  These Tregs can be 
given back through a needle placed in a vein (IV).  When given to the recipient, Tregs might reduce inflammation 
in the transplanted kidney.  However, this effect has not yet been shown.   
 
One of the IS drugs used in kidney transplant is Everolimus.  Everolimus has been shown to help Tregs survive 
better than other types of IS drugs. 
 
6. PURPOSE OF THE STUDY 
The purpose of this study is:   
• 
To see if Tregs can reduce inflammation in a transplanted kidney.   
• 
To find out what effects, good or bad, Tregs will have in the kidney recipient. 
• 
To find out what effects, good or bad, taking everolimus after Tregs will have in the kidney recipient. 
 
Kidney transplant recipients who agree to participate in this study will be assigned to one of two groups.   
Group 1 will receive standard of care IS.  This means that Group 1 will receive the same IS drugs that are given to 
kidney transplant recipients who are not in the study.  Group 1 subjects will not receive Tregs.  Group 2 will 
receive Tregs.  Participants who are assigned to group 2 will have a kidney biopsy 2 weeks after receiving Tregs.  
If that biopsy shows inflammation is reduced, their IS drugs will be changed to include everolimus.  Subjects who 
do not have a decrease in inflammation on the biopsy will remain on their current IS medication. 
 


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Neither you nor your doctor can choose which group you are in.  The assignment is made using a computer 
program and will be based on whether your donor is available and how many people are in the study already.   
 
The study team will collect information about your medical history, laboratory test results, medication doses, and 
any problems that may arise in the study.  Blood, kidney tissue, and urine will be collected for research tests.   
 
7. STUDY COMPONENTS 
A. Number of Study Sites and Participants 
This study is sponsored by the National Institute of Allergy and Infectious Diseases (NIAID).  The lead study doctor 
is Dr. Flavio Vincenti at the University of California, San Francisco.  Dr. <insert site principal investigator name> is 
in charge of the study at <insert institution>.  Up to <insert expected number for your site> subjects will be 
enrolled at <insert institution>.  We plan to treat 14 kidney transplant recipients in this study at 6 transplant 
centers in the United States.   
 
B. Study Overview/Visit Schedule 
This study will last one year.   
• 
There are 8 study visits for subjects in Group 1. 
• 
There are 10 study visits for subjects in Group 2. 
 
Screening is when the study team checks to be sure you qualify and that it is safe for you to continue in the 
study.  Your eligibility to continue in the study might change, depending on laboratory test results.   The following 
table and list below summarizes the study.  Additional details about study procedures are in other sections of this 
document.  


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Study Visit / Study Procedure 
Group 1 
Group 2 
 
Screening Visit and Blood Draw/ Study Group Assignment 
X 
X 
 
 
 
 
 
16 Days before Infusion Study Visit/ WBC Collection 
 
X 
 
Day 41 (Group 1) / Infusion Day (Group 2) 
Study Visit and Blood Draw 
X 
X 
 
• 
Tregs Infusion 
 
X 
 
 
 
 
 
Day 1 after Infusion Study Visit and Blood Draw 
 
X 
 
Day 48 (Group 1) /Day 7 after Infusion (Group 2)  
Study Visit, Physical Exam, Blood Draw 
X 
X 
 
Day 55 (Group 1)/ Day 14 after Infusion (Group 2) 
Study Visit, Physical Exam, Blood Draw 
X 
X 
 
• 
Study Kidney Biopsy 
 
X 
 
Day 69 (Group 1)/ Day 28 after Infusion (Group 2) 
Study Visit, Physical Exam, Blood Draw 
X 
X 
 
Day 125 (Group 1)/ Day 84 after Infusion (Group 2)  
Study Visit, Physical Exam, Blood Draw 
X 
X 
 
Day 223 (Group 1)/ Day 182 after Infusion (Group 2) 
Study Visit, Physical Exam, Blood Draw 
X 
X 
 
• 
Standard of Care Kidney Biopsy 
X 
X 
 
Day 405 (Group 1)/ Day 364 after Infusion (Group 2)  
Study Visit, Physical Exam, Blood Draw  
X 
X 
 
Possible Unscheduled Visits  
X 
X 
 
All Groups 
• 
Study Screening and Group Assignment 
o Blood Draw – The study team will schedule an appointment with you at the transplant center.  
You will be asked to sign this consent form.  A blood draw will be done to see if you continue to 
qualify for the study.  Blood tests will include pregnancy test, if applicable, and kidney function 
tests.  If you are still eligible, you will be assigned to one of the two groups. 
 
You will have a transplant center visit for blood draw, physical examination, and general assessment at the 
following time points after study group assignment. 
• 
Day 41(Group 1) / Infusion Day (Group 2)  
For Group 2, see Treg Screening and Infusion below; no physical exam required at this visit 
• 
Day 48 (Group 1) /Day 7 after Infusion (Group 2) 
• 
Day 55 (Group 1)/ Day 14 after Infusion (Group 2)   
For Group 2, see Day 14 after Infusion below 
• 
Day 69 (Group 1)/ Day 28 after Infusion (Group 2) 
• 
Day 125 (Group 1)/ Day 84 after Infusion (Group 2) 
• 
Day 223 (Group 1)/ Day 182 after Infusion (Group 2) (approximately 1 year after transplant) 


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o This study visit will coincide with your standard of care biopsy.  Research specimens will be 
collected for the study. 
• 
Day 405 (Group 1)/ Day 364 after Infusion (Group 2) 
 
Group 2 Only 
Infusion Day and Day 14 after Infusion visits will be different for subjects in Group 2.  There are also 2 more visits 
than Group 1.  
• 
 WBC Collection 16 Days before Infusion.  The blood draw to collect cells for making Tregs will be done at 
this visit. 
• 
Treg Screening and Infusion (overnight hospital stay)  
o This visit will be an overnight stay for participants in Group 2.  Tregs (Group 2) infusion will take 
place approximately 41 days after study group assignment.  If you are a woman of child bearing 
potential, a negative pregnancy test is required before the Tregs infusion. 
 
You will have a transplant center visit for blood draw, physical examination, and general assessment at the 
following time points after Tregs infusion. 
• 
Day 1 after Infusion (no physical exam required at this visit) 
• 
Day 14 after infusion 
o You will have a kidney biopsy done at this visit, requiring a 6-8 hour observation period before 
leaving the hospital. 
o Switch to everolimus for subjects who are eligible 
Unscheduled Visits 
As described in the Kidney Biopsy section, you might have unplanned visits to the transplant center if your doctor 
thinks a kidney biopsy is needed. 
 
C. Study Procedures 
Blood Draw 
All transplant patients have blood drawn as part of their routine care.  Whenever possible, the study will use test 
results from your routine blood work for the study without drawing more blood.  In addition to routine blood 
draws, you will have blood drawn for research tests.  (See Study Schedule).   
 
Blood collected for research will be examined for the following: 
• 
T Cells – Different types of T cells will be identified in the blood. For subjects receiving Tregs, we will also 
try to find these Tregs in your blood over time. Different cell types will be compared amongst the 2 
groups. 
• 
Antibodies – Antibodies form when the immune system identifies something foreign, such as an organ 
transplant.  We will be looking for antibodies to your kidney donor.    
• 
Gene Expression – Researchers will look at when certain genes are turned on or off when the kidney is 
working normally, and when there is inflammation or rejection. 
• 
Biomarkers – Researchers will look for certain molecules in the blood to see if any correlate with the 
kidney’s health. 
 
You will not have more than 3 tablespoons (40 ml) of extra blood drawn for research tests at a single visit.   
 


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Collection of White Blood Cells by Leukapheresis or Blood Draw (Group 2)  
White blood cells (WBCs) will be collected by blood draw or leukapheresis.  Tregs will be taken from the WBCs 
and used to make the Tregs product that you will receive if you are in group 2.  Your study doctor will decide 
whether whole blood draw or leukapheresis is best for you based on lab values like hemoglobin (a part of your 
blood that carries oxygen).  For whole blood draw, you will have about 2 cups (450 ml; one unit) taken for 
manufacturing.  If your hemoglobin level and cell count in your blood is too low, you will have WBC collected by 
leukapheresis (see below), a process that removes only the WBCs and does not lower your hemoglobin.   
 
About 2 cups (450-500 ml) of blood is needed if WBC’s are collected by blood draw.  If there are not enough cells 
from the first blood draw, a second blood draw might be done.    
 
Leukapheresis is a procedure that collects WBCs from blood by using a machine that separates the WBCs from 
the red blood cells and other blood components.  Leukapheresis can be done using a central venous catheter 
(central line) or using two intravenous (IV) catheters, one in each arm.  If leukapheresis is needed, the study team 
will look at your veins to see which method of leukapheresis is best for you.   
 
A central line is a catheter (long tube) that is placed through the skin into one of the large veins near your heart.  
Inserting the central line will take approximately 1 hour. 
 
The leukapheresis procedure removes blood from a vein in one arm. The blood is then passed through a machine 
that selects and stores WBCs.  The rest of the blood is returned to you through the IV in your other arm. The 
equipment used is sterile and never re-used.  During the procedure, you will receive salt water and a drug to 
prevent blood clotting through your IV.  The procedure takes 2 to 3 hours. 
 
Kidney Biopsy 
A kidney biopsy is a procedure in which a small piece of kidney tissue is removed with a needle so that it can be 
examined under a microscope.    Two biopsy time points are considered standard of care at the study sites:   
• 
3 to 7 months after transplant    
• 
12 months after transplant   
Biopsies are done at these times even in people who are not in the study.  However, tissue samples and 
information from the biopsy will be collected for this study. An additional needle pass might be required during 
these two procedures to collect enough tissue for research tests. 
 
For Group 2 only, a research biopsy will be done about 14 days after the Treg infusion.  Up to two needle passes 
will be made to collect a tissue sample.   
 
In addition to these biopsies performed for research alone, you might have a ‘for-cause’ kidney biopsy done 
during the study.  For example, your doctor may perform a biopsy if kidney function tests are abnormal.  At the 
time of a for-cause biopsy, kidney tissue will also be collected for this study.  Up to one additional pass of the 
biopsy needle might be needed to collect a research specimen.  
 
For each procedure described above, kidney tissue about ¾ - 1 inch in length and the width of a pencil lead will 
be collected for research tests. 
 


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Kidney tissue collected for research will be examined for the following: 
• 
Changes in the number of cells or cell type seen.   
• 
Other evidence of a change in the health of the kidney. 
• 
For group 2, the study team will look to see if the Tregs can be found in the kidney.   
• 
Gene Expression – Researchers will look for evidence that certain genes are turned on or off  
 
Urine Collection 
You will be asked to collect 100 ml (less than ½ cup) of urine in a cup for study tests.  Researchers will look for 
molecules in the urine that might provide information about the health of the kidney. 
  
Tregs Infusion (Group 2) 
Tregs are given (infused) through an IV catheter placed in the arm.  Before the infusion, you will be given 
acetaminophen (Tylenol®) and diphenhydramine (Benadryl®).  The Treg infusion will be given over 20-30 
minutes.  Your vital signs (heart rate, blood pressure, temperature, and breathing rate) will be monitored during 
and after the infusion.  You will stay overnight at the hospital after the infusion to continue monitoring vital signs. 
 
IS Medications 
Tacrolimus, mycophenolate mofetil, and prednisone are IS medications that you would take after kidney 
transplant even if you were not in this study.   
 
Everolimus (Group 2 only) 
Everolimus is also an IS medication used after kidney transplantation.  However, everolimus is a newer drug in 
kidney transplantation and is not as widely used as tacrolimus, mycophenolate mofetil, and prednisone.  Patients 
usually take everolimus or tacrolimus, not both.  The study team will review the biopsy results performed two 
weeks after Tregs infusion.  If there is a decrease in inflammation on the biopsy after the infusion when 
compared to the screening biopsy, you will start taking everolimus.  The study team will tell you how to lower 
tacrolimus and increase everolimus over a 4 week period.  You may need additional blood draws (less than 1 
teaspoon, up to once a week) to make sure you have the right drug level.  Tacrolimus will be stopped when the 
everolimus target blood level is reached.  At the end of the study, your transplant doctor will discuss the best IS 
medication for you.    
 
8. RISKS AND DISCOMFORTS 
Blood Draw 
The risks of blood draws are pain, bruising, infection, redness, and swelling at the sight of needle entry.  There is 
a chance you may feel dizzy while your blood is being drawn.  Blood collection for research may be delayed or 
canceled if you have severe anemia (low red blood cell count).  Your doctor will treat your anemia if needed as 
normally would occur in your care as a kidney transplant recipient. 
 
Central Line Insertion (Group 2 only) 
The risks of getting a central line catheter include the following: failure to place the catheter, arterial puncture 
(where artery is entered instead of vein), catheter placed incorrectly, pneumothorax (punctured lung), 
hemothorax (punctured lung that bleeds), bruising, heart rhythm disturbance or stoppage, blood clot, bleeding, 
air embolism (air gets into your vein), infection, or pain at the catheter insertion site.  The catheter might leak or 
become accidentally dislodged.  The chance for these risks are low.   


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Leukapheresis (Group 2) 
The chance of a serious problem from leukapheresis is very low.  The risks and discomforts of leukapheresis 
include those described for blood draws.  In addition, an embolus (an air bubble in your blood stream), muscle 
cramping, numbness, chilliness, tingling sensations, anxiety, heart rhythm disturbances, and headaches may 
occur when blood is returned to your body. There is a very small chance of an infection when your blood is 
returned.  Your WBC count will be lower after the procedure. Your body and your WBC count will return to 
normal again in one or two days.  You may have difficulty fighting infections during this period.   
 
Kidney Biopsy 
Risks of a biopsy include bleeding in or around the kidney that can lead to a fall in blood pressure and rise in 
heart rate. This may require a blood transfusion, or very rarely, lead to a need for surgery or loss of the kidney. 
Additional risks include pain and bleeding at the site of biopsy, infection, discomfort and bloodstained urine. 
 
Urine Collection 
There are no known risks of urine collection. 
 
Treg Infusion (Group 2) 
Tregs have been given to adults after bone marrow and kidney transplantation, and in both adults and children 
with diabetes.  There might be risks that are not known.   
 
Risks of Treg infusion include transfusion reaction, infections, lymphoproliferative disease, cancer, and rejection. 
 
Deuterium:  The Tregs you will receive have a substance called deuterium in them.  Deuterium is also known as 
heavy hydrogen, and can be found naturally in ocean water or as a gas.  Deuterium is incorporated as a tracer 
when making the Tregs in the laboratory.  The deuterium tracer allows study doctors to detect infused cells in 
blood and in the kidney.  Deuterium is not radioactive and is not known to be toxic.  The use of deuterium in 
Tregs is not thought to be a risk to study participants.  
 
Transfusion Reaction:  Side effects may include fever, chills, and/or nausea.  You will be given acetaminophen 
(Tylenol®) and diphenhydramine (Benadryl®) prior to the Tregs infusion to prevent this reaction.  These 
medications may be repeated every six hours as needed. 
  
Infections: Tregs work by slowing or stopping the immune system response. After receiving Tregs, it is possible 
that some part(s) of the immune system might be temporarily weakened and not work normally. This can put 
you at higher risk for getting infections, typically until 3 months following treatment. These can be infections 
caused by bacteria, viruses, parasites or fungi.  
 
Study doctors will look closely for signs of infection.  You should avoid direct contact with anyone who has a 
known infection during this time (like the flu) but we expect that you can go back to usual daily life activities as 
soon as you are released from the hospital.  
 
Cancer: One type of T cell is in charge of finding and stopping the growth of tumors in your body.  Since Treg cells 
stop or slow the immune response, it is possible that tumors could grow unchecked by the immune system.  The 


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most common type of cancer after transplantation is PTLD (see below).  Although also very rare, the second most 
common type of cancer after transplantation is skin cancer.  It is not known whether giving Tregs increases the 
risk for these or other types of cancers.  Tregs have been shown to increase the growth of tumors in mice that 
are present when they receive Tregs.  People eligible for this study are unlikely to have any tumors.  However, it 
is not known what Tregs might do in people taking immunosuppressive drugs after kidney transplantation.  All 
participants who receive Tregs will be monitored for this or other complication for at least one year after the 
infusion as part of the study.  After the study, you will be monitored for cancer as part of routine care after 
transplant.        
 
Post-Transplant Lymphoproliferative Disease (PTLD):  PTLD is a type of cancer (tumor) that can develop in 
transplant recipients because they are required to take drugs that suppress the immune system over a long 
period of time.  PTLD occurs in less than 1% of adults who receive kidney transplants.  In lymphoproliferative 
disorders like PTLD, lymphocytes (a type of white blood cell) grow abnormally.  PTLD is almost always linked with 
a virus called Epstein Barr (EBV), the virus responsible for infectious mononucleosis.  The risk of PTLD is highest if 
a patient has his/her first EBV infection after kidney transplant and lower if a patient has had a previous EBV 
infection prior to kidney transplantation and has developed immunity.    This study only enrolls patients who 
have had previous EBV infection or if both the donor and recipient have never had an EBV infection.  
 
Rejection:  It is possible the Tregs could change after the infusion in such a way that they increase the risk of 
rejection.  Typically, rejection can be treated in kidney transplant recipients.  There is, however, no experience in 
treating rejection that results from Treg infusion.  Your kidney tests will be monitored very closely, especially 
after Treg infusion, to ensure early diagnosis of kidney rejection. 
 
There are times when the manufacturing process will not be successful.  For example, if there are not enough 
cells manufactured to meet the required dose or cells are deemed not suitable for infusion because of microbial 
growth.  After your blood draw or leukapheresis procedure for manufacturing is done, there is a chance you 
might not be able to receive the infusion as planned.   
 
While the cells grow in the laboratory, they are checked for contamination by microbes.  Microbes are small 
organisms such as viruses, bacteria, and fungi that can cause infections.  When a Treg product has successfully 
completed the manufacturing process, and all available tests show that the Treg product is free of microbes, the 
cells will be administered to you.  Some microbes grow very slowly, and final test results are not available for 
several weeks.  On very rare occasions, a final test will show signs of microbial growth after the infusion has been 
given to you.   If this were to happen, the study team will monitor you closely for infection and make sure you 
receive appropriate treatment, if any treatment is necessary. 
 
IS Medications 
The side effects of, tacrolimus, Mycophenolate mofetil, prednisone and everolimus are expected to be the same 
as if you were taking these IS medications without being in this study. 
 
Genetic Information 
Genetic information will be recorded from the specimen collected for this study.  However, the specimen will be 
used in experiments planned for this study only.  Your sample will not be looked at for other genetic conditions, 
genetic cloning, or paternity testing.  However, basic genetic information about you will be known to researchers 


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(e.g. sex, racial or ethnic group).  We will keep all information recorded private as much as possible.  However, 
because genetic information is unique to you, complete confidentiality cannot be guaranteed.   
 
9. Pregnancy 
You cannot take part in this study if:   
• 
You are pregnant or breastfeeding 
• 
You or your partner plan to get pregnant in the next 12 months. 
 
Study drugs and procedures involved in this research project may involve unexpected risks to your unborn or 
nursing child. If you are female of childbearing potential, a pregnancy test will be done before you receive study 
drugs.  
 
If you are female and take part in this study, you must agree to use birth control during the study. You and the 
study team will discuss acceptable methods of birth control.  Female subjects in this study must agree to practice 
birth control as outlined above.     
 
If you or your partner become pregnant while participating in this study, or if you think that you may be 
pregnant, you must contact the study team immediately. 
 
The study doctor will talk to you about your care or the care of your pregnant partner.    
 
 
10. POTENTIAL BENEFITS    
There might be no direct medical benefit to you for being in this study.  The information learned from this study 
might someday benefit future kidney transplant recipients. 
 
Some subjects may benefit if inflammation in the transplanted kidney can be decreased.   
 
11. ALTERNATIVES TO PARTICIPATION 
Your doctor will talk with you about this study and other options available to you.  Your decision to participate in 
this study will not affect the care you would otherwise receive.   
 
12. NEW FINDINGS  
Your doctor will tell you about any new findings from this or other research that may affect your willingness to 
allow testing and use of your cells for this study.  If new information is provided to you, we will ask for your 
consent to continue in the study. 
 
13. VOLUNTARY WITHDRAWAL FROM STUDY 
You may decide to withdraw consent at any time during this study.  If you decide to withdraw, there is no penalty 
or loss of benefits in your routine medical care or any other benefit(s) that you would otherwise be entitled to 
receive.  Your doctor or research staff will destroy your samples at your request.   
 
 
 


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14. REASONS WHY YOU MAY BE TAKEN OFF STUDY WITHOUT YOUR CONSENT 
You may be removed from the study without your consent at any time.  Reasons why you may be removed from 
the study include, but are not limited to, the following:      
• 
Your study doctor determines that it is in your best interest not to take part.   
• 
You are unable to complete required study procedures. 
• 
The study is stopped by the Institution, the Sponsor, or health authorities. 
 
15. COSTS TO THE SUBJECT (YOU) 
There will be no charge to you, your health insurance company, or your donor’s insurance company for any costs 
which are done specifically for this study.    
 
16. PAYMENTS (REIMBURSEMENT) 
You will receive no payment for taking part in this study. 
  (or) 
You will receive (insert amount) to cover the travel and parking cost for your visit to the transplant center to give 
your blood specimen. (Specify what test/procedures will result in compensation, as well as the amount. Explain 
the method of prorating the payment(s)) 
 
(Payments are pro-rated and not held until completion of the study. If participant payments are tied to 
completion of specific study benchmarks, these stipulations should be noted in this section as well as other 
reimbursement specific to the study -i.e. parking, meal tickets, etc.    
 
17. RESEARCH-RELATED INJURY  
<Insert your IRB required language.> 
If are hurt or get sick from something that was done as part of this study, doctors at the clinic or hospital can 
arrange for emergency medical care. The Hospital does not offer financial compensation or payment for injuries 
due to participation in this research. No funds have been set aside to compensate you in the event of injury.  
Your insurance company will be billed for the costs of any care for injuries.  
  
In case of injury resulting from this study, you will not lose any legal rights by signing this form. 
 
18. CONFIDENTIALITY  
Your medical and research records will be confidential to the extent permitted by law.  Efforts will be made to 
keep personal information private.  However, we cannot guarantee complete confidentiality.   
 
As an NIH funded study, you are further protected through a policy that prevents the investigator from disclosing 
sensitive study information that would lead to your identity. This does not prevent you or a family member from 
voluntarily releasing information about yourself or your involvement in this research.                      
 
Your personal identifiers (for example, name and medical record number) will be used in the manufacturing 
process for the Treg infusion at the transplant center and at the UCSF laboratory (UCSF Human Islet and Cellular 
Transplantation Facility) where the products are made.  Use of the identifiers is required to ensure the cells are 
tracked accurately through the manufacturing process.  In all other parts of the study, you will be identified by a 


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code, and personal information from your records will not be released without your written permission.  You will 
not be identified in any publication or in the sharing of your data about this study.   
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. law.  This 
web site will not include information that can identify you.  At most, the Web site will include a summary of the 
results.  You can search this Web site at any time.  
 
Organizations that may look at and/or copy your medical records for research, quality assurance and data 
analysis include:   
• the National Institute of Allergy and Infectious Diseases, (NIAID) sponsor of the research  
• NIAID representatives, agents, employees, contractors, and other persons assisting in conducting, 
monitoring or analyzing the study   
• the U.S. Food and Drug Administration  
• other State and Local health authorities 
 
19. STORING INFORMATION/DATA FOR FUTURE STUDIES  
As a NIH-sponsored clinical study, we are expected, as described in the Funding Opportunity Announcement 
and/or Terms of Award, to share research data with the scientific community.  
We will store information resulting from this study in a central data repository. Data is information organized for 
a reason, like this study.  A central data repository is a place that collects and stores data from many different 
studies.  The purpose of this collection is to make scientific information available for future studies which may 
help future patients. Your data may be stored indefinitely.  Any information released to a central data repository 
will not contain traditional information such as name, birthdate, address, etc. that is considered your personal 
information. 
 
20. PROBLEMS OR QUESTIONS   
If you ever have questions about this study or in case of research-related injuries, you should contact Dr. <Insert 
name> at <Telephone Number>, or if you have questions about research subjects’ rights you can call <Insert the 
name and title of the appropriate country/site-specific person> at <Insert the number>, or if you prefer, direct 
your questions to the following address: <Insert address of IRB patient representative here>. 


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SIGNATURE PAGE  
 (Site may use the site-specific signature page if required) 
 
Please sign below if you agree to take part in this study.  
  
• 
you have read the informed consent and/or had it explained to you 
• 
you were given the opportunity to ask questions about the information, and 
• 
you voluntarily agree to take part in the study 
 
 
_________________ 
         
_____________________      
           ______________ 
Research Subject’s Name 
Research Subject’s Signature 
           Date 
(Typed or printed) 
 
 
Signature of person(s) explaining and obtaining the consent: 
 
 
____________________ 
____________________ 
 
_________________ 
Name and Title  
Signature 
 
 
 
Date 
(Typed or printed)   
 
____________________ 
____________________ 
 
_________________ 
Name and Title  
Signature 
 
 
 
Date 
(Typed or printed)   
 
  
(NOTE:  This consent form with the original signatures MUST be retained on file by the principal investigator.  A 
copy must be given to the research subject.  A copy should be placed in the research subject’s medical record, if 
applicable.)       


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Recipient Consent Template 
CTOT-21 Recipient Consent Template 
 
Version 11.0/ January 11, 2022 
 
Page 14 of 15 
 
 
 
21. STORAGE OF SAMPLES and/or INFORMATION FOR FUTURE USE  
(This consent is optional and agreement is not required for participation in the study) 
We are asking your permission to store samples of biological specimens (e.g., cells) collected for this study to be 
used in the future for tests that are not yet planned. These tests may or may not be related to the study of 
transplantation.     
 
Some information about a research specimen will always be linked to it (also see section 6c, Genetic 
Information).  For example, researchers might know a sample is from a kidney transplant recipient.  Because of 
this, allowing for specimens to be stored for future use also means allowing for the linked information to be 
stored and used when the specimens are needed. 
 
Genetic Testing on Stored Samples 
Your samples may be used to look at genetic information in relation to kidney transplantation (for example, the 
rejection process).  However, your samples could also be used in studies that are not related to transplantation 
(for example, the immune system as a whole).   
 
Confidentiality 
All samples will be labeled with a randomly assigned unique barcode.  They will not contain personal information 
like name, initials, or date of birth.  Reports on these stored samples and genetic tests will not be given to your 
study doctor and they will not be in your medical record.  Doctors will not know that your samples are being used 
for other studies and it will not affect your routine medical care. 
 
Benefits of Stored Material and Genetic Testing 
There is no benefit to you from the storage of samples and information.  However, the use of your samples and 
information may help researchers learn more about your disease or the genetics related to a specific condition.   
 
Samples will be stored at the core laboratories involved in this trial.  If you agree, your samples and information 
may be stored for an unknown length of time. 
 
Risks of Stored Material and Genetic Testing 
Although your stored research samples will not be sold, the information obtained from the research performed 
on your samples may in the future lead to the development of commercial products.  You will not receive any 
money from research using your stored samples and information 
There may be unknown risks associated with the storage of samples and information.    For example, if the future 
research involves genetic testing it is possible that it could be traced back to you because your genes are specific 
to you. We will make every attempt to protect your confidentiality and to make sure that your personal identity 
does not become known.    
 
Making a Decision for Stored Human Subject Material and Genetic Testing  
Your decision regarding the storage of samples or the information resulting from the analysis of your samples will 
not affect your ability to participate in this study.   
 
 


National Institute of Allergy and Infectious Diseases (NIAID) 
Division of Allergy, Immunology and Transplantation (DAIT) 
Recipient Consent Template 
CTOT-21 Recipient Consent Template 
 
Version 11.0/ January 11, 2022 
 
Page 15 of 15 
 
 
 
Please indicate your response below: 
Choose yes or no to allow the storage and sharing of your sample (cells) and information for genetic tests.      
 
   Yes       
  No    
 
 
                                   _______________________ 
       
      Initials of Research Subject 
 
Choose yes or no to allow the storage and sharing for future use of remaining sample (cells) and information for 
other research tests not currently planned (not genetic).  
 
 
   Yes       
  No    
 
 
                                    _______________________ 
         
      Initials of Research Subject 
 
 
 
 
 
 
 
 
 
 

Prot_002.pdf: 
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                                               Version 11.0 / January 11, 2022 
 CLINICAL TRIALS IN ORGAN TRANSPLANTATION 
CTOT-21 
Treg Adoptive Therapy in Subclinical 
Inflammation in Kidney Transplantation 
 (TASK) 
Version 11.0 / January 11, 2022 
IND# 16626 
Study Sponsor(s): The National Institute of Allergy and Infectious Diseases (NIAID) 
NIAID Grant Number:  U01AI113362 
 
 
 
PRINCIPAL INVESTIGATOR 
Flavio Vincenti, MD 
Professor of Clinical Medicine 
Transplant Services 
University of California at San 
Francisco 
505 Parnassus Avenue, Rm #M884 
San Francisco, CA 94118-0780 
Phone: 415-353-1322 
E-mail: flavio.vincenti@ucsf.edu 
PROTOCOL CHAIR  
Sindhu Chandran, MD 
Assistant Professor of Medicine 
Division of Nephrology 
Department of Medicine 
University of California at San Francisco 
400 Parnassus Ave., Seventh Floor 
San Francisco, CA 94143 
Phone: 415-353-8374 
E-mail: sindhu.chandran@ucsf.edu 
MEDICAL MONITOR 
 
 
 
National Institute of Allergy and 
Infectious Diseases 
5601 Fishers Ln. 6B19 
Bethesda, MD 20892 
    
 
BIOSTATISTICIAN 
 
 
Rho, Inc. 
6330 Quadrangle Drive 
Chapel Hill, NC 27517 
 
 
 
PROJECT MANAGER 
 
 
 of Allergy and 
Infectious Diseases 
5601 Fishers Ln, 6B28 
Bethesda, MD 20892 Phone:   
  
REGULATORY OFFICER 
 
 
 
National Institute of Allergy and 
Infectious Diseases 
5601 Fishers Ln. 7B29 
Bethesda, MD 20892 
  
   
PROGRAM OFFICER 
 
 
 
NIAID 
5601 Fishers Ln, 6B35 
Bethesda, Maryland 20892 
 
 
 
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior permission of the Protocol Chair, or the Division of 
Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases of the National Institutes of Health. 
 


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INVESTIGATOR SIGNATURE PAGE 
Protocol: CTOT-21 (TASK) 
  
Version/Date:  
11.0 / January 11, 2022 
Title:   Treg Adoptive Therapy in Subclinical Inflammation in Kidney Transplantation 
  
Study Sponsor:  The National Institute of Allergy and Infectious Diseases (NIAID) 
 
 
INSTRUCTIONS: The site Principal Investigator should print, sign, and date at the indicated location 
below.  A copy should be kept for your records and the original signature page sent. After signature, 
please return the original of this form by surface mail to:  
DAIT Regulatory Management Center 
PPD, Inc. 
3900 Paramount Parkway 
Morrisville, NC 27560 
Phone: (919) 456-4229 
I confirm that I have read the above protocol in the latest version.  I understand it, and I will work 
according to the principles of Good Clinical Practice (GCP) as described in the United States Code of 
Federal Regulations (CFR) – 45 CFR part 46 and 21 CFR parts 50, 56, and 312, and in the International 
Conference on Harmonization (ICH) document Guidance for Industry: E6 Good Clinical Practice: 
Consolidated Guidance dated April 1996.  Further, I will conduct the study in keeping with local legal 
and regulatory requirements. 
As the site Principal Investigator, I agree to carry out the study by the criteria written in the protocol 
and understand that no changes can be made to this protocol without the written permission of the 
IRB and NIAID. 
 
____________________________________     
Site Principal Investigator (Print) 
 
____________________________________                                      _________________ 
Site Principal Investigator (Signature)                                                       Date 


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Protocol Synopsis 
Title 
Treg Adoptive Therapy in Subclinical Inflammation in Kidney 
Transplantation (TASK) 
Short Title 
Tregs in Subclinical Inflammation 
Clinicaltrials.gov Identifier 
NCT02711826 
Clinical Phase 
Phase I/ II 
Number of Sites 
Cleveland Clinic 
Northwestern University 
University of Alabama at Birmingham 
University of California at San Francisco 
University of Colorado 
University of Michigan 
IND Sponsor/Number   
NIH/NIAID/DAIT/ #16626 
Primary Safety Objective 
This study will evaluate the safety of polyTregs in adult kidney 
transplant recipients. 
Secondary Safety Objective 
Participants receiving polyTregs will be evaluated for the safety of 
converting from CNI-based maintenance therapy to mTOR inhibitors 
after Treg therapy. 
Primary Efficacy Objective 
This study will evaluate whether polyclonally expanded Tregs 
(polyTregs) reduce graft inflammation relative to their enrollment 
biopsy compared to those receiving CNI-based maintenance therapy 
with similar baseline biopsy findings. 
Secondary Efficacy Objective 
This study will evaluate whether polyTregs can reduce graft 
inflammation by 25% or more in the 2 weeks after polyTreg infusion 
relative to the enrollment biopsy. 
Mechanistic Objectives 
This study will evaluate the impact of polyTregs on the immunological 
profiles of kidney transplant recipients. 
 
 


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Study Design 
Multi-center, open-label, randomized, controlled trial with 2 cohorts:  
1. Maintenance CNI based immunosuppression therapy  
2. polyTregs 
The original study design included a third treatment arm (donor 
alloantigen reactive Tregs or darTregs).  One subject was treated with 
darTregs prior to protocol version 9.0 in which this treatment arm 
was eliminated due to excessive manufacturing failures. 
Primary Safety Endpoints  
The safety of polyTregs (Group 2) will be described in comparison 
with CNI-based maintenance IS therapy (Group 1) by:   
1. The timing and incidence of Banff 2A or higher acute cell-
mediated rejection and/or acute antibody mediated rejection  
2. The timing and incidence of study defined Grade 3 or higher 
infection  
Secondary Safety Endpoints for 
polyTregs  
The safety of PolyTregs will also be described in Groups 1 and 2 by: 
1. 
Timing, incidence, and severity of polyTreg infusion reactions  
2. 
Timing, incidence, and severity of culture-proven and 
clinically diagnosed infections after polyTreg infusion  
3. 
Timing, incidence, and severity of acute rejection using Banff 
grading  
4. 
Timing and incidence of BK viremia and CMV reactivation  
5. Timing and incidence of > 10% decrease in eGFR compared 
to baseline 
Secondary Safety Endpoint for 
mTOR Therapy 
The safety of mTOR therapy after Treg infusion will be assessed by 
the incidence and timing of acute rejection.  Subjects who receive 
Tregs and convert to mTOR therapy will be compared to subjects who 
did not convert to mTOR therapy, as well as subjects in Group 1. 
Primary Efficacy Endpoint  
The primary efficacy endpoint will be the change in inflammation as 
measured by the percentage area of cortex occupied by 
inflammatory cells using computer-assisted quantitative image 
analysis on the biopsy 7 months after group allocation, expressed as 
the percent change relative to the baseline biopsy.  
 
 


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Secondary Efficacy Endpoints 
The secondary efficacy endpoint will be the proportion of subjects 
exhibiting a relative decrease of 25% or more inflammation between 
the baseline kidney biopsy and the biopsy 2 weeks after polyTregs, 
measured as the percentage area of cortex occupied by inflammatory 
cells using computer-assisted quantitative image analysis. 
Additional secondary efficacy endpoints are the number of subjects 
who exhibit a relative decrease of 50% or more inflammation on 
kidney biopsy at 2 weeks after polyTregs; and the proportion of 
subjects who exhibit a relative decrease of 25% or more 
inflammation at 7 months after group allocation. 
Primary Mechanistic Endpoint 
The primary mechanistic endpoints are the immunologic profiles of 
kidney transplant recipients using graft CRM (common response 
module) gene expression of rejection and/or evidence of 
inflammation in biopsies at 2 weeks after infusion (Group 2) and 7 
months after group allocation (Groups 1 and 2). 
Secondary Mechanistic Endpoints 
The secondary mechanistic endpoints are the immunologic profiles of 
kidney transplant recipients using: 
 
Persistence of infused Tregs in blood and biopsies using 
deuterium labeling and T cell repertoire analysis (Group 2) 
 
Cytokine and CRM (common response module) mRNA and 
protein profiles in the urine as correlates of acute rejection 
and/or histologic evidence of inflammation and graft fibrosis 
(Groups 1 and 2) 
 
Peripheral blood kSORT (kidney solid organ response test) 
mRNA expression of rejection/increased immune response 
(Groups 1 and 2) 
Accrual Objective 
14 Subjects Total: 
7 subjects on CNI maintenance therapy  
7 subjects to receive polyTregs 
Study Duration 
6.5 Year Accrual, 12 Months Follow Up 
Treatment Description  
Subjects will be on maintenance IS therapy using CNI + MMF/MPA 
with or without steroids at the time of study entry.   
Eligible subjects will be randomized to receive: 
1. Standard CNI maintenance IS (no Tregs) 
2. 550 ± 450x 106 polyTregs 
After receiving at least 300 x 106 polyTregs infusion, eligible subjects 
will start mTOR inhibitor. 
 
 


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Inclusion Criteria 
Individuals who meet all of the following criteria are eligible for 
enrollment as study participants:  
1. Subject must be able to understand and provide informed 
consent 
2. Age ≥18 years of age at the time of study entry  
3. Recipients of non-HLA identical living or deceased donor renal 
transplants  
4. Protocol renal allograft biopsy at 5 months (± 8 weeks) after 
transplantation with Banff i1 and/or ti1 with concomitant t 
scores t0, t1, t2 or t3; Banff i2 and/or ti2 with concomitant t 
scores t0 or t1; and without v > 0, [ptc + g] ≥2, C4d >1 (by IF), or 
C4d > 0 (by IHC) confirmed by central pathologist. Subjects 
must not be treated for pathologic criteria (e.g., steroids).    
5. eGFR ≥30 ml/min at the time of study entry 
6. Maintenance immunosuppression consisting of tacrolimus, 
MMF/MPA ± prednisone (≤10 mg/day) 
7. Current immunizations including TdAP, pneumococcal and 
seasonal influenza vaccines prior to study treatment, 
completed prior to randomization and no less than 14 days 
prior to planned manufacturing collection  
8. Hepatitis B serologies must be: 
a.  Positive HB surface antibody, negative HB core antibody and 
negative HB surface antigen for recipients immune to 
hepatitis B  
b.  Negative HB surface antibody, negative HB core antibody 
and negative HB surface antigen for non-immune/ HBV 
naïve recipients provided donor had negative HB core 
antibody and negative HB surface antigen at the time of 
donation 
9.    Negative TB test (PPD, interferon-gamma release assay, ELISPOT 
testing) within 1 year prior to enrollment. Subjects with a history 
of TB (positive TB test without active infection) must have 
completed one of the latent TB infection treatment regimens 
endorsed by the CDC (Division of TB Elimination, 2016). 
Alternative regimens for latent TB infection eradication will be 
adjudicated by the site’s infectious disease specialist. 
10.  Female subjects with childbearing potential must have reviewed 
Mycophenolate REMS and have a negative pregnancy test upon 
study entry.  
11.  Female subjects with childbearing potential must agree to use 
FDA approved methods of birth control for the duration of the 
study; subjects must consult with their physician and determine 
the most suitable method(s) that are greater than 80% effective 
(http://www.fda.gov/birthcontrol) 
 
 


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Exclusion Criteria 
Individuals who meet any of these criteria are not eligible for enrollment 
as study participants: 
1. Inability or unwillingness of a participant to give written informed 
consent or comply with study protocol 
2. History of malignancy; except adequately treated basal cell 
carcinoma 
3. History of graft loss from acute rejection within 1 year after 
any previous transplant 
4. History of transplant renal artery stenosis 
5. History of cellular rejection prior to enrollment that did not 
respond to steroids and/or subsequent creatinine after 
treatment for rejection greater than 15% above baseline 
6. Known hypersensitivity to mTOR inhibitors or contraindication to 
everolimus (such as history of wound healing complications) 
7. Any chronic illness requiring uninterrupted anti-coagulation after 
kidney transplantation 
8. Post-transplant DSA >5000 MFI or post-transplant treatment with 
IVIg for DSA. Enrolled subjects with post-transplant DSA >2000 
MFI will not be eligible for mTOR conversion. 
9. Positive HIV 1 or HIV 2 serology prior to transplantation 
10. Known positive HBSAg, or HBcAb serology  
11. Proteinuria with urine pr/cr > 1.0 g/g 
12. Any condition requiring chronic use of corticosteroids >10mg/day 
at the time of study entry 
13. Subjects requiring treatment for pathologic findings on study 
eligibility biopsy (see inclusion 4). 
14. Active infection at the time of study entry 
15. History of active TB or latent TB without adequate treatment (see 
inclusion 10).  
16. Serum BK virus >1,000 copies/ml by PCR at the time of study 
entry 
17. Hematocrit <27%; ANC < 1,000/μL; lymphocytes <500/μL; at the 
time of study entry 
18. Participation in any other studies with investigational drugs or 
regimens in the preceding year 
19. Any condition or prior treatment which, in the opinion of the 
investigator, precludes study participation 
20. Unable to provide adequate biopsy specimen (paraffin embedded 
formalin fixed) from eligibility biopsy (3-7 months post-transplant) 
for quantitative analysis. 
21. EBV naïve recipient of a kidney from an EBV positive donor, 
historically EBV naïve recipient with primary EBV infection at the 
time of screening (primary anti-VCA IgM, without antibody to EBNA), 
positive EBV PCR 
22. Hepatitis C Virus AB positive subjects with negative HCV PCR are 
eligible if they have spontaneously cleared infection or are in 
sustained virologic remission for at least 12 weeks after treatment. 
23. Positive SARS-CoV2 testing by RT-PCR 
 


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Treg Infusion Inclusion Criteria 
1. Individuals randomized to group 2 who continue to meet all of 
the enrollment criteria are eligible for Treg infusion. 
2. Negative SARS-COV2 RTPCR testing within 1 week of Treg 
infusion 
Treg Infusion Exclusion Criteria 
1. Received any vaccination within 14 days prior to blood 
collection for Treg manufacture  
2. Unacceptable Treg product.  
3. Positive pregnancy test for women of childbearing potential. 
mTOR Conversion Inclusion 
Criteria 
1. Received at least 300 x 106 polyTreg infusion 
2. Resolution of inflammation on the 2-week post-infusion biopsy 
as compared to the baseline biopsy, confirmed by central 
pathologist 
mTOR Conversion Exclusion 
Criterion 
1. Post-transplant DSA >2000 MFI 
2. Any condition or clinical variable, which in the opinion of the 
site investigator, precludes conversion to mTOR. 
 
 
 


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Study Contacts: Participating Centers 
 
SITE PRINCIPAL INVESTIGATOR 
Flavio Vincenti, MD 
Professor of Clinical Medicine 
Transplant Services 
University of California at San Francisco 
505 Parnassus Avenue, Rm #M884 
San Francisco, CA 94118-0780 
Phone: 415-353-1322 
E-mail: flavio.vincenti@ucsf.edu 
SITE PRINCIPAL INVESTIGATOR 
Clifton E. Kew II, MD 
Medical Director, Kidney and Pancreas 
Transplantation 
Professor of Medicine, Division of 
Nephrology 
Profess of Surgery, Division of 
Transplantation 
University of Alabama at Birmingham 
1900 University Blvd, THT 643 
Birmingham, AL 35294-0006 
Phone: (205) 934-7220 
Email: ckew@uabmc.edu 
 
SITE PRINCIPAL INVESTIGATOR 
Emilio Poggio, MD 
Director, Clinical Research of Kidney 
Transplant Program 
Department of Nephrology & 
Hypertension 
Cleveland Clinic 
9500 Euclid Avenue 
Cleveland, OH 44195 
Phone: 216-444-6771 
Email: poggioe@ccf.org 
SITE PRINCIPAL INVESTIGATOR 
Yee Lu, MD 
Clinical Assistant Professor, Internal 
Medicine - Nephrology 
University of Michigan 
1500 E Medical Center Dr. SPC 5334 
Ann Arbor, MI 48109 
Phone: 734-936-5548 
Email:  yeel@med.umich.edu 
 
SITE PRINCIPAL INVESTIGATOR 
John Friedewald 
Professor of Medicine & Surgery 
Northwestern University 
Feinberg School of Medicine  
Arkes Family Pavilion Suite 1900 
676 N Saint Clair 
Chicago, IL 60611 
Phone: 312-695-8900 
Email:  John.Friedewald@nm.org 
SITE PRINCIPAL INVESTIGATOR 
Scott Davis, MD 
Assistant Professor, Medicine-Renal 
Med Diseases/Hypertension 
University of Colorado 
Anshutz Transplant Services 
1635 Aurora Ct 
7th Floor 
Phone: 720-848-0005 
Email: SCOTT.DAVIS@cuanschutz.edu  
 
  
 


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Study Contacts: Core Laboratories 
 
 
 
TREG MANUFACTURING,  
DEUTERIUM LABELING AND CELLULAR 
ASSAY CORE 
Qizhi Tang, PhD 
Associate Professor  
Director, Transplantation Research Lab 
University of California, San Francisco 
San Francisco, CA 94143-0780 
Phone: 415-476-1739 
Email: Qizhi.Tang@ucsf.edu 
DETECTION OF DEUTERIUM-
LABELED TREGS 
Marc Hellerstein, MD, PhD 
Professor  
Dept of Nutritional Science & 
Toxicology 
University of California, Berkeley 
54 Mulford Hall 
Berkely, CA 94720-3104 
Phone: 510-643-3104 
EMAIL: MARCH@BERKELEY.EDU 
HISTOPATHOLOGY CORE 
 
Zoltan Laszik, MD, PhD 
Professor of Clinical Pathology 
Department of Pathology, Box 0102 
University of California, San Francisco 
513 Parnassus Avenue, Room S566 
San Francisco, CA 94143-0102 
Phone: 415-502-8230 
Email: zoltan.laszik@ucsf.edu 
 
GENE EXPRESSION, PROTEIN AND 
ANTIBODY MONITORING CORE 
Minnie Sarwal, MD 
Professor 
Department of Surgery 
513 Parnassus Ave. Med Sci 
San Francisco, CA  94143 
Phone: 415-502-7921 
Email:  minnie.sarwal@ucsf.edu 
TUBULAR EPITHELIAL INJURY AND 
RENAL FIBROSIS BIOMARKERS  
Roslyn B. Mannon, MD 
University of Nebraska Medical 
Center 
MSB 5569 
601 S. Saddle Creek Road 
Omaha, NE 68106 
Phone: 402-559-9227 
Email: roslyn.mannon@unmc.edu 
 
 


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Table of Contents 
Table of Contents .................................................................................................................................................................. 11 
Glossary of Abbreviations ..................................................................................................................................................... 17 
Study Definitions Page .......................................................................................................................................................... 18 
 Study Hypotheses/Objectives ........................................................................................................................................... 19 
 
Hypotheses ............................................................................................................................................................ 19 
 
Primary Safety Objective ....................................................................................................................................... 19 
 
Secondary Safety Objective for mTOR Therapy .................................................................................................... 19 
 
Primary Efficacy Objective .................................................................................................................................... 19 
 
Secondary Efficacy Objective ................................................................................................................................ 19 
 
Mechanistic Objective ........................................................................................................................................... 19 
 Background and Rationale ................................................................................................................................................ 20 
 
Background and Scientific Rationale ..................................................................................................................... 20 
 
Chronic Allograft Dysfunction ....................................................................................................................... 20 
 
Graft Inflammation ....................................................................................................................................... 20 
 
Rationale for Selection of Investigational Product or Intervention ...................................................................... 22 
 
Treg Manufacturing ...................................................................................................................................... 22 
 
Preclinical Experience ........................................................................................................................................... 23 
 
Clinical Studies ...................................................................................................................................................... 24 
 
Rationale for Timing of Treg Infusion............................................................................................................ 24 
 
Rationale for Dose Selection ......................................................................................................................... 24 
 
Rationale for Everolimus ............................................................................................................................... 25 
 Study Design ...................................................................................................................................................................... 26 
 
Description of Study Design .................................................................................................................................. 26 
 
Primary Safety Endpoints ...................................................................................................................................... 27 
 
Secondary Safety Endpoints .................................................................................................................................. 27 
 
Secondary Safety Endpoints .......................................................................................................................... 27 
3.3.2 
Secondary Safety Endpoint for mTOR Therapy ............................................................................................. 27 
 
Primary Efficacy Endpoint ..................................................................................................................................... 27 
 
Secondary Efficacy Endpoints ............................................................................................................................... 28 
 
Primary Mechanistic Endpoints ............................................................................................................................ 28 
 
Secondary Mechanistic Endpoints ................................................................................................................ 28 
 
Stratification, Study Group Allocation, and Blinding/Masking ............................................................................. 28 


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 Rationale for Study Population ......................................................................................................................................... 29 
 
Enrollment Eligibility Criteria ................................................................................................................................ 29 
 
Inclusion Criteria ........................................................................................................................................... 29 
4.1.2 
Exclusion Criteria ........................................................................................................................................... 30 
 
Treg Infusion Eligibility Criteria ............................................................................................................................. 30 
 
Treg Infusion Inclusion Criteria .................................................................................................................. 30 
 
Treg Infusion Exclusion Criteria .................................................................................................................. 30 
 
mTOR Conversion Eligibility Criteria ................................................................................................................. 30 
 
mTOR Conversion Inclusion Criteria ......................................................................................................... 30 
4.3.2 
mTOR Conversion Exclusion Criterion ...................................................................................................... 31 
 
Selection of Clinical Sites ....................................................................................................................................... 32 
 Investigational Agent: PolyTregs ....................................................................................................................................... 33 
 
Formulation of PolyTregs ...................................................................................................................................... 33 
 
Packaging and Labeling of PolyTregs .................................................................................................................... 33 
 
Dosage, Preparation, and Administration ............................................................................................................. 33 
 
Drug Accountability ............................................................................................................................................... 34 
 
Toxicity Prevention and Management .................................................................................................................. 34 
 
Dose Escalation Plan ..................................................................................................................................... 34 
 
Waiting Period between Infusions ................................................................................................................ 34 
 
Premature Discontinuation of PolyTregs .............................................................................................................. 34 
 Investigational Immunosuppressive Regimen: Everolimus .............................................................................................. 35 
 
Zortress® (Everolimus) .......................................................................................................................................... 35 
 
Formulation, Packaging, and Labeling .......................................................................................................... 35 
 
Zortress® Prescribing Information (Per Package Insert) ............................................................................... 35 
 
Everolimus Study Dosing and Administration ............................................................................................... 35 
 
Assessment of Participant Compliance with mTOR and CNI Dosing .................................................................... 36 
 
Toxicity Prevention and Management .................................................................................................................. 36 
 
Rejection after Initiation of Everolimus ................................................................................................................ 36 
 Other Medications ............................................................................................................................................................ 37 
 
Immunosuppressive Medications ......................................................................................................................... 37 
 
Tacrolimus ..................................................................................................................................................... 37 
7.1.2 
Mycophenolate Mofetil/ Mycophenolic Acid ............................................................................................... 37 
7.1.3 
Prednisone .................................................................................................................................................... 37 


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7.1.4 
Other Immunosuppressive Medications ....................................................................................................... 37 
 
Anti-Infective Prophylactic Medications ............................................................................................................... 37 
 
Prohibited Medications ......................................................................................................................................... 37 
 
Non-Leukoreduced Blood Products .............................................................................................................. 37 
 
Vaccinations .................................................................................................................................................. 37 
 
Medication Interactions ................................................................................................................................ 37 
 
Treatment of Rejection ......................................................................................................................................... 38 
 Study Mandated Procedures ............................................................................................................................................ 39 
 
Blood Draws .......................................................................................................................................................... 39 
 
Leukapheresis ....................................................................................................................................................... 39 
 
Kidney Biopsy ........................................................................................................................................................ 39 
 Known and Potential Risks and Benefits to Participants .................................................................................................. 40 
 
Risks of PolyTregs .................................................................................................................................................. 40 
 
Potential Risks of Treg Administration .......................................................................................................... 41 
 
Risks of Zortress® (Everolimus) ............................................................................................................................. 42 
 
Risks of Tacrolimus and Mycophenolate Mofetil/Mycophenolic Acid ................................................................. 43 
 
Risks of Tacrolimus ........................................................................................................................................ 43 
 
Risks of Mycophenolate Mofetil/ Mycophenolic Acid .................................................................................. 44 
 
Risks of Study Procedures ..................................................................................................................................... 47 
 
Risks of Blood Draws ..................................................................................................................................... 47 
 
Risks of Leukapheresis .................................................................................................................................. 47 
 
Risks of Kidney Biopsy ................................................................................................................................... 47 
 
Potential Benefits .................................................................................................................................................. 47 
 
Decrease in Graft Inflammation .................................................................................................................... 47 
 
CNI Reduction ............................................................................................................................................... 47 
 Study Visits ...................................................................................................................................................................... 48 
 
Enrollment ......................................................................................................................................................... 48 
 
Study Eligibility and Study Group Allocation ..................................................................................................... 48 
 
Specific Visits for Group 2 ................................................................................................................................. 48 
 
Whole Blood Collection or Leukapheresis for Treg Manufacture (PBMC Collection Visit) .......................... 48 
 
Treg Infusion Visit.......................................................................................................................................... 48 
 
Follow Up Visits ................................................................................................................................................. 48 
10.4.1 
Provisions for COVID-19 ................................................................................................................................ 48 


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Unscheduled Visits/ Clinically Indicated Biopsies ............................................................................................. 49 
 
Visit Windows ................................................................................................................................................... 49 
 Mechanistic Assays ......................................................................................................................................................... 50 
 
Impact of Treg Infusion on Numbers of Circulating and Graft-Infiltrating Tregs .............................................. 50 
 
Detection of Infused Tregs Using Deuterium Labeling (Deu-bl) ................................................................... 50 
 
Detection of Infused Tregs in Graft Using Deuterium Labeling (Deu-biopsy) .............................................. 51 
 
Peripheral Blood Treg Number (MFC-Treg) .................................................................................................. 51 
 
Treg TruCount Analysis ................................................................................................................................. 51 
 
Impact of Treg Infusion on Graft Inflammation ................................................................................................ 51 
 
Detection of Intragraft Inflammation by Multiplexed Immunofluorescence and in situ Hybridization 
(mIF/ISH) 51 
 
Graft Common Response Module (CRM) Gene Expression .......................................................................... 52 
 
Urinary Biomarkers of Graft Inflammation and Injury .................................................................................. 55 
 
Impact of Treg Infusion on Peripheral Blood Biomarkers of Rejection and Graft Injury .................................. 56 
 
Peripheral Blood Leukocyte Population and Phenotype by Multiparameter Flow Cytometry (MFC) ......... 56 
 
Peripheral Blood Biomarkers of Acute Rejection (kSORT) ............................................................................ 56 
 
Alloimune Quiescence and Operational Tolerance (kSPOT) ......................................................................... 59 
 
Non-HLA Antibody Assays for Prediction of Chronic Allograft Injury (nHLA-Ab) .......................................... 59 
 
Markers of Tubular Epithelial Injury and Renal Fibrosis ............................................................................... 60 
 
Stored Plasma and Serum ................................................................................................................................. 60 
 Biospecimen Storage....................................................................................................................................................... 61 
 Criteria for Participant Completion and Premature Study Termination ......................................................................... 62 
 
Participant Stopping Rules and Withdrawal Criteria ........................................................................................ 62 
 
Participant Replacement ................................................................................................................................... 62 
 
Follow-up after Early Study Withdrawal ....................................................................................................... 62 
 
Study Stopping Rules......................................................................................................................................... 62 
 Safety Monitoring and Reporting ................................................................................................................................... 63 
 
Overview ........................................................................................................................................................... 63 
 
Definitions ......................................................................................................................................................... 63 
 
Adverse Event (AE) ........................................................................................................................................ 63 
 
Unexpected Adverse Event ........................................................................................................................... 63 
 
Serious Adverse Event (SAE) ......................................................................................................................... 63 
 
Other Significant Adverse Events ...................................................................................................................... 64 


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Grading and Attribution of Adverse Events ...................................................................................................... 64 
 
Grading Criteria ............................................................................................................................................. 64 
 
Attribution Definitions .................................................................................................................................. 65 
 
Collection and Recording of Adverse Events .................................................................................................... 66 
 
Collection Period ........................................................................................................................................... 66 
 
Collecting Adverse Events ............................................................................................................................. 66 
 
Recording Adverse Events ............................................................................................................................. 66 
 
Reporting of Serious Adverse Events and Adverse Events ............................................................................... 66 
 
Reporting of Serious Adverse Events to Sponsor .......................................................................................... 66 
14.5.2. Reporting to Health Authority .......................................................................................................................... 67 
 
Reporting of Adverse Events to IRBs/IECs .................................................................................................... 68 
 
Pregnancy Reporting ......................................................................................................................................... 68 
 
Reporting of Other Safety Information ............................................................................................................. 68 
 
Review of Safety Information ........................................................................................................................... 68 
 
Medical Monitor Review ............................................................................................................................... 68 
 
Planned DSMB Reviews ................................................................................................................................ 68 
 
Ad hoc DSMB Reviews................................................................................................................................... 69 
 Statistical Considerations and Analytical Plan ................................................................................................................ 70 
 
Overview ........................................................................................................................................................... 70 
 
Measures to Minimize Bias ............................................................................................................................... 70 
 
Analysis Plan ...................................................................................................................................................... 70 
 
Analysis Samples ........................................................................................................................................... 70 
 
Endpoint Assessments .................................................................................................................................. 70 
 
Descriptive Analyses ..................................................................................................................................... 73 
 
Interim Analyses ................................................................................................................................................ 74 
 
Sample Size Considerations .............................................................................................................................. 74 
 Identification and Access to Source Data ....................................................................................................................... 76 
 
Source Data ....................................................................................................................................................... 76 
 
Access to Source Data ....................................................................................................................................... 76 
 Protocol Deviations ......................................................................................................................................................... 77 
 
Protocol Deviation Definitions .......................................................................................................................... 77 
 
Reporting and Managing Protocol Deviations .................................................................................................. 77 
 Ethical Considerations and Compliance with Good Clinical Practice .............................................................................. 78 


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Statement of Compliance ................................................................................................................................. 78 
 
Informed Consent Process ................................................................................................................................ 78 
 
Privacy and Confidentiality ............................................................................................................................... 78 
 Publication Policy ............................................................................................................................................................ 79 
 References ...................................................................................................................................................................... 80 
Appendix 1.  Donor Assessments ........................................................................................................................................ 86 
Appendix 2.  Group 1 Recipient Schedule of Events ............................................................................................................. 87 
Appendix 3.  Group 2 Recipient Schedule of Events ............................................................................................................. 88 
Appendix 4.  Manufacturing Timeline ................................................................................................................................... 89 
 
 
 
 
 
 


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Glossary of Abbreviations 
CFR 
Code of Federal Regulations 
COVID-19 
Coronavirus Disease 2019 
CRF 
Case Report Form 
CRM 
Common Response Module 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAIT 
Division of Allergy, Immunology, and Transplantation 
darTregs 
Donor Alloantigen Reactive T Regulatory Cells 
DSMB 
Data Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonization 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
kSORT 
Kidney Solid Organ Response Test 
mTOR 
Mammalian Target of Rapamycin 
NIAID 
National Institute of Allergy and Infectious Diseases 
PI 
Principal Investigator 
polyTregs 
Polyclonal T Regulatory Cells 
Mycophenolate REMS 
Mycophenolate Risk Evaluation and Mitigation Strategy 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Suspected Adverse Reaction 
SARS-CoV2 
Severe Acute Respiratory Syndrome Coronavirus 2 
SCI 
Subclinical Inflammation 
SOP 
Standard Operating Procedure 
SUSAR 
Serious Unexpected Suspected Adverse Reaction 
 


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Study Definitions Page 
Acute Cell-mediated 
Rejection 
Banff 2007 Type 1A or higher and clinical treatment for acute rejection. 
Central reading will be utilized when accounting for study stopping rule and 
for safety endpoint.  
Severe Acute Cell-
mediated Rejection  
Banff 2007 Type 2A or higher and clinical treatment for acute rejection. 
Central reading will be utilized when accounting for study stopping rule and 
for safety endpoint. 
Antibody Mediated 
Rejection 
Diffusely positive staining for C4d, presence of circulating anti-donor 
antibodies, and morphologic evidence of acute tissue injury. 
Graft Inflammation 
Banff i1 and/or ti1+ t0, t1, t2 or-t3 or 
Banff i2 and/or ti2 + t0 or t1 
Graft Failure 
90 consecutive days of dialysis dependency 
Infection, Severe 
Study defined grade 3 or higher; culture-proven and clinically diagnosed 
Investigational Agent 
polyTregs, everolimus 
Protocol Mandated 
Procedures 
Any procedure or specimen collection performed solely for the purpose of 
this research study, not considered site specific standard of care. 
Randomized 
A subject who met eligibility criteria, signed informed consent document, 
and was randomly assigned to one of two treatment groups. 
Withdrawn from Study 
Therapy 
Subjects who prematurely discontinue the study after receiving polyTregs.   
Treatment for Rejection 
Treatment of rejection in this protocol constitutes use of IV steroids and/or 
Thymoglobulin.  Adjustment of maintenance immunosuppression 
medications will not be counted as treatment for rejection. 
Women of Childbearing 
Potential  
WOCBP includes any female who has experienced menarche and who has 
not undergone successful surgical sterilization (hysterectomy, bilateral tubal 
ligation, or bilateral oophorectomy) or is not postmenopausal (defined as 
amenorrhea ≥ 12 consecutive months; or women on hormone replacement 
therapy with documented serum follicle stimulating hormone level > 35 
mIU/mL). Even women who are using oral, implanted, or injectable 
contraceptive hormones or mechanical products such as an intrauterine 
device or barrier methods (diaphragm, condoms, spermicides) to prevent 
pregnancy or practicing abstinence or where the partner is sterile (e.g., 
vasectomy), should be considered to be of childbearing potential. 
 
 
 
 
 


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 Study Hypotheses/Objectives 
 Hypotheses 
This study will explore the following hypotheses: 
1.  PolyTreg infusions are safe and will increase the absolute number of Tregs in circulation and in the kidney allograft. 
2.  PolyTregs will suppress inflammation in the graft and reduce markers of inflammation in the graft and urine 
 Primary Safety Objective 
This study will evaluate the safety of polyTregs in adult kidney transplant recipients.  Incidence of death, graft loss, acute 
rejection and severe infections will be compared and overall safety events, including infusion reactions, will be 
described.  
 Secondary Safety Objective for mTOR Therapy 
Participants receiving polyTregs will be evaluated for the safety of converting from CNI-based maintenance therapy to 
mTOR inhibitors after Treg therapy. 
 Primary Efficacy Objective 
This study will evaluate whether polyTregs reduce graft inflammation relative to their enrollment biopsy compared to 
those receiving CNI-based maintenance therapy with similar baseline biopsy findings. 
 Secondary Efficacy Objective  
This study will evaluate whether polyTregs can reduce graft inflammation by 25% or more in the 2 weeks after the 
polyTreg infusion relative to the enrollment biopsy. 
 Mechanistic Objective 
This study will evaluate the impact of polyTregs on the immunological profiles of kidney transplant recipients. 
 
 
 
 
 
 
 
 
 


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 Background and Rationale  
 Background and Scientific Rationale 
 
Chronic Allograft Dysfunction 
Despite advances in transplantation reducing early acute rejection rates to <15% and improving 1-year graft survival to 
>90%, long-term graft attrition rates have remained unchanged at 4% loss per year (Meier-Kriesche HU, 2004). A major 
contributor to graft loss is progression of interstitial fibrosis and tubular atrophy (IF/TA) (Nankivell BJ F.-L. C., 2001) 
(Cosio FG, 2005) (Nankivell BJ B. R., 2003), which is the result of cumulative allograft damage of immunologic and 
nonimmunologic origin. Studies of protocol biopsies have provided important clues on how early changes in graft 
histology relate to long-term graft survival. Early sequential post-transplant protocol biopsies show a rapid increase in 
the prevalence of IF/TA (Cosio, 2005). When separated into two categories: IF/TA with and IF/TA without inflammation, 
it appears that is not IF/TA per se that is detrimental, but IF/TA associated with and/or secondary to inflammation that 
results in progression of renal dysfunction (Park, 2010) (Mannon, 2010). Chronic immune injury in the allograft is now 
recognized in the majority of patients with late graft loss (Mannon, 2010) (El-Zoghby ZM, 2009). 
 
Graft Inflammation 
Inflammation manifested as infiltration of mononuclear cells in the renal allograft appears to be detrimental to the graft 
in the long term, even when insufficient to meet Banff criteria for acute cellular rejection (types I, II or III). According to 
the Banff classification, interstitial inflammation (i1, i2 and i3) without tubulitis (t0) is considered “nonspecific”. When 
tubulitis is present but mild (t1) or accompanied by subthreshold inflammation (t2 with i0-i1), it is termed “borderline 
change” and deemed “suspicious for acute rejection” (Racusen LC, 1999). Such changes are common, reported in 11-
44% of surveillance biopsies within the first year (Gloor JM, 2002) (Heilman RL, 2010) (Thierry A, 2011). In one study, 
biopsies with borderline change had an intermediate level of expression of proinflammatory genes between that of 
histologically normal biopsies and those with acute rejection (Hoffmann SC, 2005). Another study found that a 
transcriptional profile indicative of activated Th1 cells, but not the magnitude or composition of the infiltrate, uniquely 
defined a functionally significant allograft rejection (Lipman ML, 1998). Thus, it appears that the immune response is 
qualitatively similar but quantitatively reduced in SCI and borderline change, indicating that these entities carry the 
same potential for alloimmune damage as clinical acute rejection. A study of 124 sequential protocol biopsies in 46 
patients who exhibited histologic evidence of chronic allograft nephropathy at 1 year found that the presence of 
subclinical rejection correlated with histologic progression of chronicity, a lower creatinine clearance at 5 years, and 
worse long-term graft survival (Shishido S, 2003). In another study of 435 allograft recipients, the combination of SCI 
injury and IF/TA on protocol biopsies was strongly associated with later graft failure, more so that IF/TA alone (Moreso 
F, 2006). Similarly, in 292 recipients both of living and deceased donor grafts, inflammation even with mild fibrosis 
predicted worsening graft function (Cosio FG, 2005). In contrast, the absence of acute inflammation at any time point 
was associated with minimal deterioration in renal function or progression of renal lesions. Finally, a study of 151 living-
donor recipients found that the combination of fibrosis and inflammation in 1-year protocol biopsies was associated 
with a rejection-like gene expression signature, reduced graft function, and shortened graft survival, even among 
tacrolimus + MPA-treated recipients without overt risk factors for poor outcomes (Park WD, 2010). In these patients, the 
iothalamate clearance at 3 years in those with inflammation and fibrosis was 52 ± 18 ml/min vs. 70 ± 23 ml/min in those 
with normal histology (p=0.01). Increased expression of innate and adaptive immune transcripts were seen in the group 
with inflammation and fibrosis, suggesting that early interventions aimed at altering rejection like inflammation may 
succeed in stabilizing graft function and extending graft survival. Also, while the previous discussion has highlighted the 
role of inflammation in areas of viable cortex of the kidney, recent studies demonstrate that inflammation in areas of 
fibrosis and atrophy, not usually scored by Banff criteria, are strongly associated with later graft loss (Mannon RB M. A., 


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2010)(Mengel M, 2009). Efforts to abrogate these infiltrates as a means of mitigating graft dysfunction have not been 
studied.  
2.1.2.1 Incidence and Impact of Sub-Clinical Inflammation 
A retrospective analysis of 380 consecutive 6-month surveillance kidney transplant biopsies at UCSF Medical Center 
between 7/2009-5/2011 scored by a single pathologist using the revised Banff 2007 classification (Solez K, 2008) found 
subclinical borderline change as defined by Banff, in 20.3%, and inflammation (Banff i1-i2) with minor or no tubulitis (t0 
or t1) in 10.9% of all biopsies without acute rejection (Table 1). 
Table 1. 6-month protocol biopsies* at UCSF sorted by inflammation (i) and tubulitis (t) scores 
Banff Category 
t0 
t1 
t2 
t3 
Total 
i0 
261 (68.7%) 
41 (10.8%) 
1 (0.3%) 
0 
303 (79.7%) 
i1 
9 (2.4%) 
23 (6.1%) 
2 (0.5%) 
0 
34 (8.9%) 
i2 
1 (0.3% 
8 (2.1%) 
0 
0 
8 (2.4%) 
i3 
0 
3 (0.8%) 
0 
0 
3 (0.8%) 
Total 
271 (71.3%) 
75 (9.7%) 
3 (0.8%) 
0 
349 (91.8%) 
*Note: Of a total of 380 six-month biopsies, 31 had acute rejection meeting Banff criteria. Numbers in parentheses are 
percentages of all 380 biopsies. 
In this analysis, chronic graft injury was significantly associated with the presence of inflammation. Banff ci-scores ≥1 
(representing interstitial fibrosis) and ct-scores ≥1 (representing tubular atrophy) were significantly more common in 
biopsies with inflammation than those without (62% vs. 14% for ci-score, p-value <0.0001; 78% vs. 53% for ct-score, p-
value <0.0001). Additionally, inflammation was associated with lower eGFR by the Modification of Diet in Renal Disease 
(MDRD) equation (Levey AS, 1999)at 6 months (60 vs. 67 ml/min/1.73 m2, p-value <0.0001) and at 12 months (58 vs. 68 
ml/min/1.73 m2, p-value <0.0001). Our findings are very similar to those seen in a study of 151 low risk, living-donor 
kidney transplant recipients on a CNI-based regimen (Park WD, 2010). In this study, 13.2% of 1-year protocol biopsies 
showed fibrosis with inflammation and these patients on follow-up exhibited a decline in GFR and reduced graft survival.  
In our cohort, a follow up biopsy was performed 6 months later in 23 recipients who were found to have subclinical 
rejection or borderline change on their 6-month protocol biopsy. At 12 months, persistent inflammation was seen in 
over 56.5% despite treatment, which included methylprednisolone pulse, Thymoglobulin and IVIg for subclinical 
rejection and oral prednisone pulse for those with borderline change. Therefore, it appears that SCI, which has been 
demonstrated to be damaging to the graft in the long-term, may persist despite conventional therapy and potentially 
lead to a long-term decline in graft function. 
2.1.2.2 Rationale and Experience with Multiplexed Immunofluorescence and in situ Hybridization 
for Detection of Graft Inflammation 
The Banff classification (Banff in brief), based on semiquantitative assessment of morphologic changes including 
inflammation, is considered the “gold standard” for evaluation of kidney transplant biopsies. However, the lack of 
qualitative information on the composition of the inflammation, imprecise quantification, and poor inter-observer 
reproducibility, make Banff a suboptimal tool for research. Since precise quantitative assessment of the inflammation in 
our study is essential to monitor treatment-related changes in follow-up biopsies, we have developed a quantitative 
immunofluorescence (IF) assay in formalin-fixed paraffin-embedded (FFPE) tissues combined with whole slide digital 
imaging and computer-assisted image analysis to measure the inflammatory load. The application of novel multiplex IF 
technologies also allows us to perform a detailed qualitative assessment of the inflammation in the biopsies.  


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Multiplex immunofluorescence (IF) assays to measure inflammation quantitatively and qualitatively: To take advantage 
of better morphology of FFPE tissues and easy multiplexing capabilities of IF, we developed various sets of IF stains on 
FFPE kidney biopsies using a combination of inflammatory cell markers to measure inflammation (1) quantitatively and 
(2) qualitatively. The total inflammatory load is measured by using leukocyte common antigen (LCA) as a marker while 
the composition of the inflammation is assessed via multiplexing including CD8, CD4, FoxP3, CD20, and CD68 markers, 
among others. Validation of the LCA assay revealed excellent reproducibility and repeatability. Twenty-eight serially cut 
sections of transplant kidney biopsies were stained with LCA. Whole slide digital images were generated from each 
section and the signals were quantitated in each section. The average values from the corresponding consecutive 
sections stained by one operator versus those stained by another operator were statistically analyzed. No significant 
differences in LCA expression were noted in any of the corresponding sections. 
Multiplex IF and in situ hybridization (miFish) stains on FFPE tissues: Immunohistochemistry (IH)/IF is often hampered by 
subpar sensitivity and specificity of certain antibodies that are difficult to validate. In some cases, the low-level 
expression of antigens, such as seen with certain cytokines, makes immunohistochemical detection of these antigens 
difficult or impossible. As an alternative to IH/IF, we have adapted the RNAScope® in situ hybridization (ISH) platform to 
analyze FFPE biopsies for mRNA expression. This is a novel and highly sensitive ISH technology with very high specificity. 
To determine the cellular source of mRNA (IL-6) expression, we applied multiplex IF stains, similar to that described 
above, to the same sections that the ISH was performed on. We conclude that multiplex IF stains can be performed 
following ISH hybridization. The quality of both the ISH and multiplex IF signals is sufficient to apply computer-assisted 
image analysis tools for quantitation 
 Rationale for Selection of Investigational Product or Intervention 
 
Treg Manufacturing 
UCSF has developed a GMP-compliant process for producing polyTregs (Putnam, 2009).  This process starts by purifying 
CD4+CD25+CD127lo Tregs from peripheral blood or leukapheresis product using single–step fluorescence activated cell 
sorting (FACS) for CD4+CD25+CD127lo cells or sequential magnetic activated cell sorting (MACS) to enrich for CD25+ cells 
followed by FACS purification of CD4+CD127lo cells.  The purified Tregs are stimulated twice with anti-CD3 and anti-CD28 
beads on day 0 and day 9, leading to 300 to 3000-fold expansions in a 14-day period. Up to 5 billion polyTregs can be 
produced from one unit of blood. The expanded Tregs retain their phenotype and are highly suppressive in vitro. Tregs 
manufactured using this protocol have been infused into 14 type 1 diabetes patients in an UCSF-led trial. To determine if 
this process can be used to expand Tregs from immunosuppressed kidney transplant patients, we have recently 
expanded Tregs isolated from transplant recipients receiving tacrolimus and MPA. As shown in Figure 1, Tregs from this 
immunosuppressed patient expanded comparably to Tregs from non-transplant patients who were not receiving 
immunosuppressive therapy. Post expansion flow cytometric analysis shows that the expanded CD4+ T cells are 
uniformly CD25+ and FOXP3+. Thus, it is feasible to expand polyTregs from patients on immunosuppression.  
 


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Figure 1.  Expansion of Tregs isolated from a transplant recipient on IS 
 Preclinical Experience 
Tregs are a small subset of CD4+ T cells that depend on the FOXP3 transcription factor for their lineage differentiation 
and function. They function by preventing the initiation of unwanted immune activation and by suppressing ongoing 
immune responses to limit bystander tissue destruction. Thus, Tregs are a natural part of an immune response, essential 
for resolving the associated inflammation. In kidney transplantation, acute rejections as well as borderline change are 
often associated with an increase of Tregs in the graft (Muthukumar T, 2005) (Taflin C N. D., 2010) and it has been 
suggested that Treg recruitment at the acute phase of the allogeneic response can diminish the interstitial inflammation 
and reverse the rejection. Thus, infusion of Tregs before extensive graft damage may improve long-term graft outcomes 
(Bluestone, 2004) (Kang, 2007) (Long, 2009) (Sagoo, 2008) (Waldmann, 2008) (Walsh, 2004) (Wood, 2003). Unlike 
generalized immunosuppressive regimens, Tregs are long-lived and can function in a dominant and antigen specific 
manner. Thus, therapeutic infusion of Tregs has the potential to induce long-term donor-specific tolerance without 
impeding desired immune responses to pathogens and tumors in transplant patients.   
Preclinical studies have demonstrated that Tregs can be used to control alloimmune responses in graft-versus host-
disease (GvHD) as well as organ and cell transplantation models. Treg reactivity, dosing, adjunct immunosuppression, 
and timing of Treg infusion critically impact the efficacy of Treg therapy in organ transplantation (Wood, 2003). Tregs 
enriched for donor alloantigen reactivity are more effective than polyclonal Tregs. Most mouse experiments have 
evaluated Treg therapy without adjunct immunosuppression and administered Tregs at the time of transplantation. 
However, the peri-transplant period is not the ideal time for Treg therapy because the high intensity 
immunosuppression used at this time may antagonize Tregs and inflammation triggered by surgical trauma and ischemia 
reperfusion may destabilize Tregs. In addition, the complexity of the peri-transplant period may render it difficult to 
assess the safety profile of Treg therapy. Therefore, we favor the use of Tregs when patients are more stable at least 
several months after transplant. Results from mouse models show that delayed administration of Tregs is effective at 
inducing long-term drug-free graft survival when the recipients are initially treated with depleting agents or mTOR 
inhibitors (Fan Z, 2010) (Raimondi, 2010). 
 


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 Clinical Studies 
A key advance for Treg therapy in humans is the finding that human Tregs can be isolated and expanded in vitro while 
maintaining their immunoregulatory function. As of mid-2013, 1 case study and 2 clinical trials evaluating the safety and 
efficacy of Tregs in treating GvHD have been reported, all demonstrating promising safety and potential efficacy profiles 
(Brunstein, 2011) (Di lanni, 2011) (Trzonkowski, 2009). These studies treated bone marrow transplant recipients with 
Tregs obtained from the bone marrow or third-party donors. A total of 53 patients were treated with Treg doses ranging 
from 3 to 6 x 106 Tregs/kg body weight. A more recent trial infused 10-20 x 106/kg ex vivo expanded 
CD3+CD4+CD25hiCD127- Tregs in children with recent onset type 1 diabetes and found the treatment well tolerated and 
associated with preservation of C-peptide 6 months after the infusion (Marek-Trzonkowska N, 2012). In a study in type 1 
diabetes at UCSF, Bluestone and colleagues have performed a dose escalation study treating 14 patients. Patients 
showed no signs of a serious safety signal. Persistence of infused Tregs at 6 months after infusion; and preservation of C-
peptide as long as 2 years after infusion have been observed. 
 
Rationale for Timing of Treg Infusion 
The choice of introducing the Tregs 3-7 months after transplantation offers multiple advantages over therapy at the time 
of surgery or early after transplantation. By delaying the administration of Tregs, we avoid the effect of induction 
therapy (especially the clearly detrimental effect of the anti-IL-2R antibodies) and the high exposure of CNIs that is 
required early post-transplant. The choice of using patients with inflammation will allow direct assessment of impact of 
Tregs on the graft in the follow up biopsy at 2 weeks and at 6 months after the therapy. In addition, it is safer to convert 
to an mTOR inhibitor to provide Tregs the optimum environment for in vivo expansion and durability later post-
transplant.  
 
Rationale for Dose Selection 
The Treg dose was chosen by considering three primary factors: estimated effective dose, previous experiences in 
human patients, and manufacturing capacity.  
 
The effective dose of Treg therapy for reversing inflammation in allografts is currently unknown.  Based on preclinical 
studies of Treg therapy in transplantation, we estimate that inducing transplant tolerance would require more than 50 x 
109 of polyTregs. This dose may be reduced by an order of magnitude by depleting T cells, using Treg-supportive 
immunosuppression, or the avoidance of the highly inflammatory conditions during the period immediately following 
the transplant (Tang Q, 2012). Therefore, we estimate that 5 x 109 polyTregs will likely to have an impact of the anti-
graft response.  
 
Among all the published studies of Treg therapy in humans, the highest dose infused is 6 x 106/kg polyTregs in adults for 
GvHD prevention (Di lanni, 2011) and 20 x 106/Kg polyTregs in children with type 1 diabetes (Marek-Trzonkowska N, 
2012).  A phase 1 polyTreg therapy trial completed enrollment and infusion in October 2013 at UCSF.  A total of 14 
patients with recent onset of type 1 diabetes were infused in the highest dosing cohort receiving 2.6 x 109 total Tregs 
(equivalent to 37 x 106/kg).   
 
As of January 2017, three kidney transplant recipients with subclinical inflammation 6 to 7 months after transplant were 
enrolled in a pilot trial and received 320 x 106 polyTregs at UCSF.  All patients have completed 1 year of follow up after 
the infusion of polyTregs.  None of the patients had an infusion reaction.  The first patient experienced transient 50% 
reduction of all leukocyte counts that began 4 days after Treg infusion and resolved by 28 days after infusion.  The 
patient was completely asymptomatic during this period.  The protocol biopsy taken 2 weeks after Treg infusion showed 
complete resolution of the subclinical inflammation.  It is unclear whether leukopenia and clearance of inflammation is 


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related to polyTregs infusion in this patient.  Leukopenia was not observed in any of the type 1 diabetes patients that 
received up to 2.6 x 109 polyTregs manufactured in the same facility.  However, it is possible that 320 x 106 polyTregs of 
polyTregs may have a biological impact when given to patients who are also on triple immunosuppression regimen 
consisted of CNI, MMF, and steroids.  Of note, leukopenia was not seen in the 2nd and 3rd patients in this trial.  None of 
the patients experienced any treatment related serious AEs. 
We plan to infuse 550 ± 450 x 106 of polyTregs to patients enrolled in this trial.  This dose is higher than targeted earlier 
in this protocol.  At the time when this protocol was first implemented, we planned to compare the efficacy of 
polyclonal vs donor alloreactive Tregs and wished to keep Treg dose the same between the two arms in order to make a 
rigorous comparison.  Given that the donor alloreactive Treg arm has been discontinued, we do not have any reason to 
limit the target dose in the polyTreg arm; especially since a higher dose is expected to have more biologic activity 
(Battaglia M., 2005).  We have chosen a target dose of 550 ± 450 x 106 polyTregs because it is within the range that has 
been safely infused in humans (Bluestone J., 2015), is highly feasible to produce, and provides the best chance of 
efficacy. 
 
Rationale for Everolimus 
The mechanistic target of rapamycin (mTOR) pathway integrates diverse environmental inputs, including immune signals 
and metabolic cues, to direct T cell fate decisions (H, 2012). In the Treg compartment, the Akt-mTOR axis is widely 
acknowledged as a crucial negative regulator of Treg de novo differentiation  (Haxhinasto S, 2008) (Sauer S, 2008) (Liu G, 
2009) (Liu G Y. K., 2010) and population expansion (Battaglia M, 2005). In contrast to CNIs, sirolimus, an mTOR inhibitor, 
has been shown to favor Treg expansion and survival both in vitro (Strauss L, 2009 ) (Gallon L, 2015 ) (Bocian K, 2010) 
(Levitsky J, 2011)and in vivo (Battaglia M S. A.-H., 2006 ) (Akimova T, 2012) 
 
Further, it has been shown that conversion from a CNI to an mTOR inhibitor in transplant recipients is associated with an 
increase in Treg number and activity (Carroll RP, 2013 ) (Gallon L T. O.-R., 2015). Animal studies of tolerance induction 
using Treg infusions have used shown prolonged survival of infused Tregs  (Singh K, 2014 )and graft acceptance with 
concomitant sirolimus therapy  (Ma A, 2011) (Ma A Q. S., 2009). Therefore, as an added strategy to improve the survival 
of the infused Tregs, we propose to convert the maintenance immunosuppression from a CNI-based regimen to an 
everolimus-based regimen in eligible subjects who have safely received polyTregs infusions and have demonstrated a 
reduction in inflammation on their follow up biopsies at 2 weeks. The previous experiences at UCSF in patients with type 
1 diabetes mellitus as well as in a pilot study involving kidney transplant recipients found that infused Tregs reach a peak 
in the circulation at 2 weeks post-infusion and are still detectable in the circulation at 6 months and 1 year. In animal 
models, trafficking of infused Tregs in inflamed tissue and draining lymph nodes can be seen within a few hours after 
infusion.  The reduction of inflammatory cell load can be detected in three days, but is typically more pronounced 1 to 2 
weeks after the Treg infusion (Lee K, 2014) (Mahne AE, 2015). This delay is likely because Tregs need to accumulate, 
become activated, and proliferate locally before exerting their suppressive effects.  In the clinical setting when we 
cannot frequently sample the graft, we think it is most likely that we will observe an improvement in graft inflammation 
at 2 weeks and this will be our signal to initiate a conversion from a CNI to everolimus. Previous studies in kidney 
transplant recipients have adequately demonstrated the safety of conversion from CNI to everolimus at > 6 months 
post-transplant (Budde K & Investigators., 2012) (Chhabra D, 2013). 


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 Study Design 
 Description of Study Design  
This is an open-label trial to determine the safety and efficacy of a single dose of autologous polyTregs in renal 
transplant recipients with SCI in the 3-7 months post-transplant allograft biopsy compared to control patients treated 
with CNI-based immunosuppression. The efficacy of the Treg therapy will be assessed by the reduction of graft 
inflammation on biopsies performed at 7 months after study group allocation compared to the eligibility biopsy.  The 
original study design included an additional treatment arm with a single dose of darTregs.  However, due to the inability 
to manufacture an adequate number of cells for infusion, this treatment arm was removed from the study in protocol 
version 9.0.  One subject was treated in the darTreg arm and completed follow-up prior to the arm being removed from 
the protocol.  The accrual goal for the study was reduced due to the removal of this arm as well as challenges associated 
with recruitment of participants in the setting of the COVID-19 pandemic. Given that this is primarily a pilot, proof-of-
concept, we believe that the target of 7 evaluable participants in each arm is sufficient to provide the necessary clinical 
and mechanistic data that will allow us to assess the impact of polyTregs on graft inflammation.  
The study will also describe the safety of mTOR therapy after polyTregs.  The secondary efficacy endpoint, as well as 
safety and mechanistic endpoints relative to both Tregs and mTOR therapy are described below. 
 


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Figure 2.  Study Design Figure 
 
 
Figure 3.  Study Flow/ Timeline Diagram 
 Primary Safety Endpoints 
The safety of polyTregs (Group 2) will be described in comparison with CNI-based maintenance IS therapy (Group 1) by:   
1. The timing and incidence of Banff 2A or higher acute cell-mediated rejection and/or acute antibody mediated 
rejection  
2. The timing and incidence of study defined Grade 3 or higher infection 
 Secondary Safety Endpoints 
 
Secondary Safety Endpoints  
The safety of PolyTregs (Group 2) will be described in comparison with CNI-based maintenance IS therapy (Group 1) by: 
1. Timing, incidence, and severity of polyTregs infusion reaction  
2. Timing, incidence, and severity of culture-proven and clinically diagnosed infection after PolyTreg infusion  
3. Timing, incidence, and severity of acute rejection using Banff grading  
4. Timing and incidence of BK viremia and CMV reactivation  
5. Timing and incidence of > 10% decrease in eGFR compared to baseline  
3.3.2 
Secondary Safety Endpoint for mTOR Therapy 
The safety of mTOR therapy after Treg infusion will be assessed by the timing and incidence of acute rejection in Group 
2.  Subjects who receive Tregs and convert to mTOR therapy will be compared to subjects who did not convert to mTOR 
therapy; as well as subjects in group 1. 
 Primary Efficacy Endpoint 
The primary efficacy endpoint will be the change in inflammation as measured by the percentage area of cortex 
occupied by inflammatory cells using computer-assisted quantitative image analysis on the biopsy 7 months after study 
group allocation, expressed as the percent change relative to the baseline biopsy. 
Kidney 
Transplant
Biopsy for 
Primary 
Endpoint 
Assessment 
7 months after 
randomization
3-7 
Months 
after 
Transpant
Day 0
16 days 
before 
Infusion
Screen for 
Study 
Eligibility, 
Including 
Biopsy 
Infusion
14 Days 
Post 
Infusion
28 Days 
Post 
Infusion
182 Days 
Post 
Infusion
Biopsy    
14 days 
after 
infusion
Everolimus 
Conversion, 
if eligible
Group 1 
(SOC)
Remain on 
SOC
Group 2 
(PolyTreg)
PBMC 
Collection for 
Manufacturing
Infuse 
PolyTreg


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 Secondary Efficacy Endpoints 
The secondary efficacy endpoint will be the proportion of subjects exhibiting a relative decrease of 25% or more 
inflammation between the baseline kidney biopsy and the biopsy 2 weeks after polyTregs, measured as the percentage 
area of cortex occupied by inflammatory cells using computer-assisted quantitative image analysis. 
Additional secondary efficacy endpoints are the proportion of subjects who exhibit a relative decrease of 50% or more 
inflammation on kidney biopsy at 2 weeks after polyTregs; and the proportion of subjects who exhibit a relative 
decrease of 25% or more inflammation at 7 months after study group allocation. 
 Primary Mechanistic Endpoints 
The primary mechanistic endpoints are the immunologic profiles of kidney transplant recipients using the common 
response module (CRM) graft gene expression of rejection and/or histologic evidence of inflammation in biopsies at 2 
weeks after infusion (Group 2) and 7 months after study group allocation (Groups 1 and 2). 
 
Secondary Mechanistic Endpoints 
The secondary mechanistic endpoints are the immunologic profiles of kidney transplant recipients using: 
 
Persistence of infused Tregs in blood and biopsies using deuterium labeling and T cell repertoire analysis (Group 2) 
2. Cytokine and CRM mRNA and protein profiles in the urine as correlates of acute rejection and/or histologic evidence 
of inflammation and graft fibrosis (Groups 1 and 2)  
3. Peripheral blood (kidney solid organ response test) kSORT mRNA expression of rejection/ increased immune 
response (Groups 1 and 2) 
 Stratification, Study Group Allocation, and Blinding/Masking 
This is an open label, parallel group study in which subjects are assigned to treatment alternatives in an unblinded 
fashion using a variation of the Pocock and Simon adaptive randomization strategy.  A web-based randomization system 
will be used in order to minimize the possibility of bias entering into those assignments and to maintain approximate 
equality in the group sizes.  The initial treatment allocation will weight each treatment equally.  In subsequent 
allocations, the less-populated arm will be weighted more in order to restore the balance between the groups.  If a 
recipient is assigned to polyTreg and the recipient does not receive intended polyTreg infusion, that recipient will be re-
assigned to Group 1.  The allocation schedule algorithm/scheme will not incorporate stratification variables.    
 


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 Rationale for Study Population 
Adult recipients of kidney transplants with stable graft function who are found to have inflammation with or without 
minor tubulitis on a 6-month surveillance biopsy will be eligible for the trial. Inflammation in the renal allograft is well-
recognized as a predictor of long-term graft dysfunction and graft loss ( (Park WD, 2010); (Mannon RB M. A., 2010)). 
Patients who are found to have inflammation on the 6-month surveillance biopsy have already received maximal 
treatment with induction therapy and are maintained on dual or triple immunosuppression, so more intense therapy 
may not control the inflammation and is often associated with drug-induced toxicities. Currently, these patients do not 
receive any specific intervention to treat the inflammation despite the future risk of graft dysfunction. These patients 
are therefore excellent candidates for immunomodulatory therapy that can alter the balance of the immune system 
towards a more regulatory phenotype and lead to better long-term function.      
 Enrollment Eligibility Criteria 
 
Inclusion Criteria 
Individuals who meet all of the following criteria are eligible for enrollment as study participants:  
 
Subject must be able to understand and provide informed consent 
 
Age ≥18 years of age at the time of study entry  
 
Recipients of non-HLA identical living or deceased donor renal transplants  
 
Protocol renal allograft biopsy at 5 months (± 8 weeks) after transplantation with Banff i1 and/or ti1 with 
concomitant t scores t0, t1, t2 or t3; Banff i2 and/or ti2 with concomitant t scores t0 or t1; and without v > 0, 
[ptc + g] ≥2, C4d >1 (by IF), or C4d > 0 (by IHC); confirmed by central pathologist.  Subjects must not be treated 
for pathologic criteria (e.g. steroids).   
 
eGFR ≥30 ml/min at the time of study entry 
 
Maintenance immunosuppression consisting of tacrolimus, MMF/MPA ± prednisone (≤10 mg/day) 
 
Current immunizations including TdAP, pneumococcal and seasonal influenza vaccines prior to study treatment, 
completed prior to randomization and no less than 14 days prior to planned manufacturing collection. 
 
Hepatitis B serologies must be the following: 
a. Positive HB surface antibody, negative HB core antibody and negative HB surface antigen for 
recipients immune to hepatitis B  
b. Negative HB surface antibody, negative HB core antibody and negative HB surface antigen for non-
immune/ HBV naïve recipients provided donor had negative HB core antibody and negative HB 
surface antigen at the time of donation 
 
Negative TB test (PPD, interferon-gamma release assay, ELISPOT) within 1 year prior to enrollment.  Subjects 
with a history of latent TB (positive TB test without active infection) must have completed one of the current 
latent TB infection treatment regimens endorsed by the CDC (Division of TB Elimination, 2016). 
 Women of childbearing potential must have reviewed Mycophenolate REMS and have a negative 
pregnancy test upon study entry 
 Female subjects with child-bearing potential, must agree to use FDA approved methods of birth 
control for the duration of the study; subjects must consult with their physician and determine the most 
suitable method(s) that are greater than 80% effective (http://www.fda.gov/birthcontrol) 
 
 
 
 


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4.1.2 
Exclusion Criteria 
Individuals who meet any of these criteria are not eligible for enrollment as study participants:  
 
Inability or unwillingness of a participant to give written informed consent or comply with study protocol 
 
History of malignancy; except adequately treated basal cell carcinoma 
 
History of graft loss from acute rejection within 1 year after any previous transplant 
 
History of transplant renal artery stenosis 
 
History of cellular rejection prior to enrollment that did not respond to steroids and/or subsequent 
creatinine after treatment for rejection greater than 15% above baseline 
 
Known hypersensitivity to mTOR inhibitors or contraindication to everolimus (including history of 
wound healing complications) 
 
Any chronic illness requiring uninterrupted anti-coagulation after kidney transplantation 
 
Post-transplant DSA >5000 MFI or post-transplant treatment with IVIg for DSA. Enrolled subjects with 
post-transplant DSA >2000 MFI will not be eligible for mTOR conversion. 
 
Positive HIV 1 or HIV 2 serology prior to transplantation 
 Known positive HBSAg, or HBcAB serology  
 Proteinuria with urine pr/cr > 1.0 g/g 
 Any condition requiring chronic use of corticosteroids >10mg/day at the time of study entry 
 Subjects requiring treatment for pathologic findings on study eligibility biopsy (see inclusion 4). 
 Active infection at the time of study entry 
 History of active TB or latent TB without adequate treatment (see inclusion 10) 
 Serum BK virus >1,000 copies/ml by PCR at the time of study entry 
 Hematocrit <27%; ANC < 1,000/μL; lymphocytes <500/μL at the time of study entry 
 Participation in any other studies with investigational drugs or regimens in the preceding year 
 Any condition or prior treatment which, in the opinion of the investigator, precludes study 
participation 
 Unable to provide adequate biopsy specimen (paraffin embedded formalin fixed) from eligibility biopsy (3-7 
months post-transplant) for quantitative analysis. 
 EBV naïve recipient of a kidney from an EBV positive donor, historically EBV naïve recipient with primary EBV 
infection at the time of screening (primary anti-VCA IgM, without antibody to EBNA), positive EBV PCR 
 Hepatitis C Virus Ab positive subjects with negative HCV PCR are eligible if they have spontaneously cleared 
infection or are in sustained virologic remission for at least 12 weeks after treatment. 
 Positive SARS-CoV2 testing by RT- PCR   
 Treg Infusion Eligibility Criteria 
 
Treg Infusion Inclusion Criteria 
1. Individuals randomized to group 2 who continue to meet all of the enrollment criteria.  
2. Negative SARS-COV2 RTPCR within 1 week of Treg infusion 
 
Treg Infusion Exclusion Criteria 
Individuals randomized to group 2 who meet any of these criteria are not eligible for Treg infusion: 
 Received any vaccination within 14 days prior to blood collection for Treg manufacture 
 Unacceptable Treg product 
 Positive pregnancy test for women of childbearing potential 
 mTOR Conversion Eligibility Criteria 
 
mTOR Conversion Inclusion Criteria 
Individuals who meet all of these criteria are eligible for mTOR conversion: 


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1. Received at least 300 x 106 polyTreg infusion 
2. Resolution of inflammation on the 2-week post-infusion biopsy as compared to the baseline biopsy, 
confirmed by central pathologist 
4.3.2 mTOR Conversion Exclusion Criterion 
Individuals who meet any of these criteria are not eligible for mTOR conversion: 
1. Post-transplant DSA >2000 MFI 
2. Any condition or clinical variable, which in the opinion of the site investigator, precludes conversion to mTOR. 
 
 


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 Selection of Clinical Sites 
All participating centers use immunosuppressive regimens consisting of tacrolimus, MMF/MPA ± steroids. Based on 
preliminary data on SCI, 10.9% of those who undergo a protocol kidney biopsy at 6 months will meet histologic criteria 
for inclusion in the TASK trial.  The combined annual volume of both living and deceased kidney transplants at the 
clinical sites is about 1,927. We are planning to enroll a total of 14 patients, thus requiring a consent rate of 
approximately 30% which is attainable for the patient population at these clinical centers. 


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 Investigational Agent: PolyTregs 
 Formulation of PolyTregs 
Collection of Recipient T cells  
Peripheral blood leukocytes will be collected from eligible participants after eligibility is confirmed.  The whole blood or 
leukapheresed cells will be immediately transported to the manufacturing facility.  If the whole blood or leukapheresis 
product does not produce sufficient numbers of Tregs for infusion, the leukapheresis or phlebotomy may be repeated to 
ensure an adequate initial cell number for product manufacture.  
PolyTregs Expansion 
Treg isolated from autologous starting material will be expanded with commercially available anti-CD3 and anti-CD28 
magnetic bead for 14 days in cultures containing medium supplemented with deuterated glucose.  Blood or 
leukapheresis product from recipients participating at a clinical site other than UCSF will be shipped at room 
temperature via next-day service to UCSF for further processing.  
 Packaging and Labeling of PolyTregs  
At the end of the 14-day expansion of polyTregs, the anti-CD3 and anti-CD28 beads will be removed by magnetic 
separation.   The expanded Tregs will be re-suspended in 100 ml of a solution of 49.02% (v/v) PlasmaLyte-A, 49.02% (v/v) 
Dextrose 5%, 0.45% NaCl, and 1.96% (v/v) 25% human serum albumin and filled in a sterile infusion bag.  The filled 
product will be refrigerated and quarantined until release.  
Prior to release, a product label will be affixed on the infusion bag.  The label will provide:  the unique product identifier, 
recommended storage temperature, date of cell harvest, expiration date and time, and clearly indicate the name and 
unique identifier of the intended recipient.  A Certificate of Analysis documenting the test results of release assays will 
accompany the final product during delivery to the bedside. 
The Tregs products manufactured for subjects at an off-site location will be shipped from UCSF to the clinical site via 
next-day service using validated conditions and containers.  Qualified staff at the remote site will receive the polyTregs 
and verify the recorded shipment temperatures were maintained within the validated range according to UCSF 
established SOPs. 
 Dosage, Preparation, and Administration 
Subjects randomized to Group 2 will receive a single infusion of 550 ±450 x 106 polyTregs.  Preparations less than 100 x 
106 will not be infused.  Cells not infused will be used for research. 
Pre-medications will be administered 30-60 minutes prior to infusion.  Pre-medications will include 650 mg 
acetaminophen and 25-50 mg diphenhydramine intravenously or by mouth.   
After double verification of cell therapy product and recipient identification, the product will be infused via a peripheral 
intravenous (IV) line primed with saline by gravity in approximately 20 to 30 minutes. Following administration of the 
Treg product, product bag, tubing and peripheral IV line will be flushed with normal saline to ensure the complete dose 
is infused. 
Vitals signs will be monitored before, during, and after the infusion. Emergency medical equipment will be available 
during the infusion in case the subject has an allergic response or an infusion reaction that can result in a CRS. The IV line 


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will be maintained after the infusion and the subject will be asked to remain in the clinical research unit for a minimum 
of 24 hours which will allow ongoing monitoring for any infusion-related signs and symptoms. 
 Drug Accountability 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62) the investigator will maintain adequate records of the 
disposition of the investigational agent, including the date and quantity of the drug received, to whom the drug was 
dispensed (participant-by-participant accounting), and a detailed accounting of any drug accidentally or deliberately 
destroyed. 
Records for receipt, storage, use, and disposition will be maintained by the study site.  A drug-dispensing log will be kept 
current for each participant.  This log will contain the identification of each participant and the date and quantity of drug 
dispensed. 
All records regarding the disposition of the investigational product will be available for inspection. 
 Toxicity Prevention and Management 
 
Dose Escalation Plan 
There is no plan for dose escalation in this trial. 
 
Waiting Period between Infusions 
Due to manufacturing constraints, there will be a naturally imposed waiting period of at least 1 week in between 
infusions.  This will provide a window to delay or cancel the next infusion in case a stopping rule has been met.  Other 
than the 1-week manufacturing delay, there will be no mandated waiting period between infusions in the polyTregs 
treatment arm (Group 2), since the proposed starting dose has been infused safely previously in kidney transplant 
recipients. 
 Premature Discontinuation of PolyTregs 
PolyTregs will be stopped and will not be restarted if there is a hypersensitivity reaction, a CTCAE ≥ 3 infusion-related 
reaction, a CTCAE Grade ≥ 3 CRS, or any other infusion related serious adverse event. 


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 Investigational Immunosuppressive Regimen: Everolimus  
At the time of Treg infusion, the maintenance immunosuppression will consist of tacrolimus with target levels of 4-11 
µg/L, MMF/MPA ± steroids. On the follow-up biopsy 2 weeks after the polyTreg infusion, the inflammatory load will be 
assessed by computer assisted image analysis calculating the number of infiltrating cells per square mm as well as the 
percentage of renal cortex infiltrated with lymphocytes. If the inflammation has resolved on the 2 week follow up biopsy 
as compared to the baseline biopsy, everolimus will be started at 1.5 mg bid and the dose of tacrolimus reduced by 50% 
for 4 weeks and then discontinued.  Target everolimus trough levels will be 3-8 µg/L with concomitant tacrolimus 
therapy, and 6-10 µg/L once tacrolimus is discontinued.  These patients will remain on everolimus, MPA ± prednisone 
through the end of the trial and the follow-up period. If on the 2-week follow up biopsy, inflammation is still present 
(even if reduced in intensity compared to the baseline biopsy), no change will be made to the maintenance 
immunosuppressive regimen. 
 Zortress® (Everolimus) 
Zortress® is an mTOR inhibitor immunosuppressant manufactured by Novartis Pharmaceuticals Corporation and is 
commercially available.  Zortress® is FDA licensed for the prophylaxis of kidney transplant rejection in adults with low to 
moderate immunologic risk.     
 
Formulation, Packaging, and Labeling  
Everolimus is a macrolide immunosuppressant.  Everolimus inhibits antigenic and interleukin (IL-2 and IIL-15) stimulated 
activation and proliferation of T and B lymphocytes.  Everolimus is supplied as tablets for oral administration containing 
0.25 mg, 0.5 mg, and 0.75 mg of everolimus together with butylated hydroxytoluene, magnesium sterate, lactose 
monohydrate, hypromellose, crospovidone, and lactose anhydrous as inactive ingredients. 
Zortress® tablets are packed in child-resistant blisters.  Each strength is available in boxes of 60 tablets (6 blister strips of 
10 tablets each).  Tablets are white to yellowish, marbled, round, and flat with beveled edges.   Tablets should be stored 
at 25°C (77°F) with excursions permitted to 15-30°C (50-86°F).  Tablets should be protected from light and moisture. 
 
Zortress® Prescribing Information (Per Package Insert) 
According to the package insert, Zortress® is to be used in combination with basilixamab induction and concurrently 
with reduced doses of cyclosporine and corticosteroids.  An initial everolimus dose of 0.75 mg orally twice daily (1.5 
mg/day) is recommended for adult kidney transplant patients in combination with reduced dose cyclosporine, 
administered as soon as possible after transplantation.  Pharmacokinetic studies in kidney transplant patients show that 
steady-state of everolimus is reached by Day 4.  The recommended therapeutic range for everolimus is 3 to 8 ng/mL.  
Recommended cyclosporine blood concentrations are recommended as follows:  100-200 ng/mL through 1 month after 
transplant, 75-150 ng/mL at 2 and 3 months after transplant, 50-100 ng/mL at 4 months after transplant, and 25-50 
ng/mL from 6 months to 12 months after transplant.  Dose adjustments based on everolimus blood concentrations can 
be made at 4-5 day intervals.   
 
Everolimus Study Dosing and Administration 
The use of everolimus in this trial will deviate from package instructions.  Subjects in this trial are enrolled between 3 
and 7 months after transplantation and induction therapy is site-specific (not study mandated).  Furthermore, subjects 
will be on tacrolimus at study entry, rather than cyclosporine.  In eligible patients, everolimus will be initiated within 2 
weeks after kidney biopsy showing resolution of inflammation following Treg infusion. Everolimus will be initiated at a 
dose of 1.5 mg orally twice daily and titrated, as needed.  Subjects will begin everolimus with target trough levels of 3-8 


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µg/L for 4 weeks while still taking tacrolimus.  Everolimus target trough levels will be 6-10 µg/L when tacrolimus is 
discontinued. 
 Assessment of Participant Compliance with mTOR and CNI Dosing  
Study subjects will have, at a minimum, weekly monitoring of trough levels. 
 Toxicity Prevention and Management 
In subjects who develop any of the following conditions after initiation of everolimus therapy, everolimus will be 
discontinued and tacrolimus will be restarted. Target tacrolimus trough levels will be 4-11 mcg/L. 
1. Worsening of proteinuria with urine pr/cr >1 g/g 
2. Severe hypercholesterolemia (LDL>190 mg/dL or triglycerides >500 mg/dL) not responsive to therapy with lipid 
lowering agents 
3. Severe edema or fluid accumulations such as pleural or pericardial effusions 
4. Interstitial pneumonitis 
5. Severe mouth ulcers 
6. Severe cytopenia (CTCAE grade 3 or higher) not responsive to dose adjustment of everolimus or MMF/ MPA 
7. Any condition which in the determination of the investigator requires discontinuation of everolimus therapy 
such as planned surgery.     
 Rejection after Initiation of Everolimus 
Any subject with biopsy proven or treated rejection after the start of study mandated everolimus will discontinue 
everolimus and resume tacrolimus.  Target tacrolimus trough levels will be 4-11 mcg/L. 
 
 


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 Other Medications 
 Immunosuppressive Medications 
 
Tacrolimus 
Target tacrolimus trough levels for this study prior to conversion to everolimus are 4-11 µg/dl, which falls within 
standard of care.  For eligible subjects in Group 2, the tacrolimus dose will be reduced by 50% when everolimus is 
initiated.  Tacrolimus will be discontinued 4 weeks after initiation of everolimus therapy.    
7.1.2 
Mycophenolate Mofetil/ Mycophenolic Acid 
All enrolled subjects will be on MMF/MPA at the time of study entry at a minimum dose of 1000 mg/720 mg per day. 
Once subjects are enrolled in the study, doses may be adjusted at the discretion of the study investigator for 
gastrointestinal intolerance, cytopenias, infections or other conditions that require dose adjustment.  All mycophenolate 
prescribers in the study will be required to enroll in the FDA mycophenolate REMS (risk evaluation and mitigation 
strategy) program.        
7.1.3 
Prednisone 
Use of corticosteroids is not prohibited in this trial.  However, subjects requiring use of corticosteroids >10 mg per day 
for treatment of long-term conditions (e.g., lupus) will not be enrolled.  
7.1.4 
Other Immunosuppressive Medications 
Subjects requiring the use of cyclosporine and/or azathioprine will not be enrolled in this study. 
 Anti-Infective Prophylactic Medications 
Anti-infective prophylaxis medications will be per standard of care at each clinical center. 
 Prohibited Medications 
 
Non-Leukoreduced Blood Products  
For subjects requiring treatment, leukoreduced blood products should be used whenever possible. 
 
Vaccinations 
The use of live vaccines will be proscribed during trial participation, as per standard of care for kidney transplant 
recipients. Examples include (but are not limited to) the following: intranasal influenza, measles, mumps, rubella, oral 
polio, BCG, yellow fever, varicella, and TY21a typhoid vaccines. 
Subjects should not receive any vaccination (live or inactivated) within 14 days prior to blood collection for Treg 
manufacturing and 28 days after the date of Treg infusion.  These requirements are in place to minimize the chance of 
having enrichment of vaccine specific Tregs in the product and ensure that Treg infusion does not negatively impact 
immunity elicited by the vaccine. 
 
Medication Interactions 
Grapefruit and grapefruit juice inhibit cytochrome P450 3A4 and P-gp activity and should therefore be avoided with 
concomitant use of EVR. Patients with rare hereditary problems of galactose intolerance, the Lapp lactase deficiency or 
glucose-galactose mal absorption should not take EVR as this may result in diarrhea and malabsorption. 


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Administration of medications known to interact with tacrolimus and/or EVR is allowed but tacrolimus and EVR levels 
should be carefully monitored and dosing titrated to maintain the target levels to minimize toxicity while maintaining 
efficacy. 
 Treatment of Rejection 
Patients who are clinically suspected to have a rejection will undergo a kidney biopsy. Biopsies will be read locally 
according to the Banff criteria and if acute rejection is diagnosed, it will be treated according to the current standard of 
care. Samples of blood, urine and kidney tissue will be obtained for mechanistic analysis prior to the treatment. Digital 
images of the biopsies will also be reviewed later by the central pathology core. 


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 Study Mandated Procedures 
 Blood Draws 
Blood draws are necessary after kidney transplantation to monitor allograft function.  Whenever possible, study 
participants will have additional blood drawn for research purposes at time points when standard of care testing is done.  
Some blood tests will be performed at the local hospital laboratory.  Blood for mechanistic assays described in this 
protocol will also be collected and shipped to core laboratories.   
A blood collection of 450-500 mls will be drawn for Treg isolation and manufacture for subjects with hematocrit 30% or 
higher once the subject is determined to be eligible and randomized to Group 2.  Blood collection for this purpose can 
be repeated once, if necessary, for manufacture of the Treg product (see 8.2 Leukapheresis).   
 Leukapheresis  
Subjects with hematocrit less than 30% will have leukapheresis performed for PBMC collection.  Subjects with 
hematocrit greater than 30% may undergo leukapheresis or have whole blood collection.  The study team will make a 
decision in the best interest of a particular subject based on the current clinical picture.      
 Kidney Biopsy 
Study participants will undergo surveillance biopsies as standard of care between 3 and 7 months after transplantation 
and approximately one year after transplantation.  Research specimens will be collected during these procedures and 
digital images will be sent to the central pathologist for scoring.  The biopsy obtained 3-7 months after transplantation 
will be used to determine study eligibility and local read will be confirmed by central pathologist.  The one-year biopsy 
will fall 7 months after study group assignment and will be used to assess the efficacy endpoint. The one-year biopsy and 
all for-cause biopsies will be reviewed by the central pathologist.     
Additionally, a protocol kidney biopsy will be performed 2 weeks after polyTreg infusion (Group 2).  This biopsy is for 
research purposes only and will be used for mechanistic assays and to confirm eligibility for mTOR conversion. This 
biopsy will be reviewed by the central pathologist.  
 


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 Known and Potential Risks and Benefits to Participants 
 Risks of PolyTregs  
Although Treg infusion has been innocuous in animal models, there is currently scant experience in humans. Three Treg 
therapy trials in GvHD have been reported. The first-in-man trial by Trzonkowski et al involved two patients 
(Trzonkowski, 2009). The first patient had chronic GvHD two years after transplantation. After receiving 0.1 x 106/Kg 
flow sorted ex vivo expanded Tregs from the donor, the symptoms subsided, and the patient was successfully 
withdrawn from IS. The second patient had acute GvHD disease that progressed despite three infusions with an 
accumulative dose of 3 x 106/Kg expanded donor Tregs. A larger scale phase I trial led by Brunstein et al (Brunstein, 
2010) enrolled twenty- three patients with advanced hematologic malignancy. The patients were treated with two units 
of umbilical cord blood as source of stem cells and effector T cells. Tregs were isolated using anti-CD25 immunomagnetic 
bead selection from third-party cord blood samples that had 4 to 6 HLA match with the recipient. Up to 6 x 106/Kg Tregs 
were infused after ex vivo expansion using anti-CD3 and anti-CD28 conjugated beads.  The infused Tregs were detectible 
in circulation for up to 7 days. During the one-year period after Treg infusion, the investigators observed no DLTs and 
AEs when compared to historical controls. Incidences of acute and chronic GvHD were reduced in patients received Treg 
therapy. The third trial enrolled 28 patients with high-risk hematological malignancies (Di lanni, 2011). Patients received 
anti-CD25 immunomagnetic bead enriched donor Tregs without ex vivo expansion four days before receiving one haplo-
mismatched hematopoietic stem cell and Tconv transplants from the same donors. A majority of the patients received 2 
x 106/Kg Tregs with 1 x 106/Kg Tconvs and no adjunct IS was given after transplant. Patients demonstrated accelerated 
immune reconstitution, reduced CMV reactivation, low incidence of tumor relapse and GvHD. Collectively, these studies 
show that Treg therapy has minimal toxicity in the setting of GvHD. 
Recently, a phase I/II study applying polyclonally expanded FACS purified Tregs to type diabetic patients has been 
reported by the Trzonkowski group (Marek-Trzonkowska N, 2012). The study enrolled 10 type 1 diabetic children (aged 
8-16 years) within 2 months after diagnosis. Four patients received 10 × 106 Tregs/kg body wt and the remaining 6 
patients received 20 × 106 Tregs/kg body weight. The patients were followed for 4-5 months after Treg infusion, and no 
toxicity of the therapy was noted. The authors concluded that Treg therapy was safe and well tolerated in children. 
A phase I Treg dose-escalating trial evaluating safety of Treg therapy in type 1 diabetic patients completed enrollment 
and infusion in October 2013.  Fourteen enrolled subjects received a single infusion of Tregs. Three subjects were 
treated in each of the first and second dosing cohorts with 0.05x108 and 0.4x108 cells, respectively, and four subjects 
were treated in the third dosing cohort with 3.2x108 cells. Notably, three of the subjects in cohort 3 and 4 were enrolled 
at treated at Yale (New Haven, Connecticut).  As of August 2014, 115 adverse events had been reported in 15 subjects 
since the beginning of the trial.  All 14 treated subjects have reported at least 1 adverse event.  One subject who 
underwent phlebotomy but was withdrawn before treated reported 1 adverse event before withdrawal from the trial. 
Seventy-one events were judged as mild in severity, 35 were judged as moderate, and nine were judged as severe.  Four 
events were judged to be serious. Thirty events were judged to be possibly related, 21 unlikely related and 64 unrelated 
to study therapy.  There were no grade 4 or 5 adverse events. Four serious adverse events (SAE) have been reported 
since the beginning of the trial. Three severe (grade 3) hypoglycemic SAEs, one judged unlikely related and two judged 
unrelated to the investigational product have been reported.  One severe (grade 3) hyperglycemic SAE judged unrelated 
to the investigational product has been reported. 
 
 


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Potential Risks of Treg Administration 
T cell suppression 
Tregs are known to suppress naïve T cell responses to a variety of antigens. Less is known about ongoing immune 
responses especially to viruses and bacteria. It is not known whether Tregs will alter protective immunity. Only 
recipients with current immunizations including TdAP, pneumococcal and seasonal influenza vaccines will be enrolled in 
the study. During the flu season, seasonal influenza vaccine will be administered prior to the qualifying biopsy for all 
transplant recipients and at least 28 days prior to Treg isolation.  Subjects immune to hepatitis B can be enrolled in the 
study; or if non-immune/ HBV naïve, can be enrolled only if their kidney donor has negative HB core antibody and 
negative HB surface antigen. 
Infusion reaction 
Side effects reported from previous human trials involving T cell infusions include transient fever, chills, and/or nausea. 
Subjects will be pre-medicated with acetaminophen 650 mg by mouth and diphenhydramine hydrochloride 25-50 mg by 
mouth or IV, prior to the infusion of Tregs. These medications may be repeated every six hours as needed. Patients will 
not receive systemic corticosteroids such as hydrocortisone, prednisone, prednisolone (Solu-Medrol) or dexamethasone 
(Decadron) at any time, except in the case of a life-threatening emergency, since this may have an adverse effect on T 
cells. If corticosteroids are required for an acute infusion reaction, an initial dose of hydrocortisone 100 mg may be used. 
Infection 
As with any therapy that suppresses the immune system, there is a risk of developing infections. It should be noted that 
on a theoretical basis, this risk is minimal, since the total input of Tregs is approximately 3-5% of the endogenous 
resident Treg population. We will perform plasma CMV DNA quantitative PCR in all recipients prior to enrollment and 
exclude those who are positive. Study subjects will be monitored for CMV reactivation by plasma CMV DNA quantitative 
PCR. 
Lymphoproliferative disease 
Treg immunosuppression has been shown to enhance tumor growth in some small animal model systems. Thus, 
complications such as lymphoproliferative disease are possible on a theoretical basis. Clinical experience in transplant 
recipients suggests that the risk of lymphoproliferative disease is highest in those who develop a primary EBV infection 
while immunosuppressed. Thus, individuals without prior exposure to EBV are excluded from this study unless the 
kidney donor is EBV naive.  In previously exposed individuals, EBV reactivation is associated with a degree of 
immunosuppression higher than that likely to be observed in this study since the total input of Tregs is approximately 3-
5% of the endogenous resident Treg population. Furthermore, the previously observed rate of PTLD in kidney recipients 
is relatively low at 0.68% (Smith JM, 2006).  Nonetheless, careful attention will be paid to the potential for this 
complication and we will perform EBV quantitative DNA PCR to screen patients prior to enrollment and monitor for 
reactivation during the study.   
Loss of tumor surveillance 
T lymphocytes are one major component of tumor surveillance, and it is possible that cells that inhibit T lymphocytes 
could impair this function. There has not been evidence of tumors in preclinical models. Nonetheless, the long term 
follow up of all treated patients will determine whether there is evidence of an increase in the frequency of tumors. 
Reproductive risks 
There may be an unexpected risk to an unborn or nursing child. Pregnant and breastfeeding women will be excluded 
from participation in the study. Females must have a negative pregnancy test prior to enrolling in the study. Female 
participants must agree to not participate in a conception process (e.g., active attempt to become pregnant or in vitro 


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fertilization) for up to one year after Treg dosing. Females must agree to use a reliable and effective form of birth control 
for two years after Treg dosing. This includes oral contraceptives, barrier methods, or abstinence.  Female subjects will 
also be required to undergo a urine pregnancy test prior to cell administration. A positive pregnancy test will result in 
holding of scheduled cell administration. 
 Risks of Zortress® (Everolimus) 
Boxed warnings for everolimus include increased susceptibility to malignancies and serious infections from 
immunosuppression; increased risk of kidney arterial and venous thrombosis resulting in graft loss within 30 days post-
transplantation; increased nephrotoxicity if used with standard doses of cyclosporine; and increased mortality 
(associated with serious infections) within the first three months of heart transplantation.    
In this study, the period of overlap between everolimus and a CNI (tacrolimus) will be 4 weeks; and during this period 
tacrolimus will be administered at a reduced dose. The risks that have been noted previously with combination therapy 
of cyclosporine and everolimus are therefore not anticipated in this trial.  Risks applicable to the use of everolimus in 
this study include:    
Angioedema 
Everolimus has been associated with the development of angioedema. The concomitant use of everolimus with other 
drugs known to cause angioedema, such as angiotensin converting enzyme (ACE) inhibitors may increase the risk. 
Wound Healing and Fluid Accumulation 
Everolimus delays wound healing and increases the occurrence of wound-related complications like wound dehiscence, 
wound infection, incisional hernia, lymphocele and seroma. These wound-related complications may require more 
surgical intervention. Generalized fluid accumulation, including peripheral edema (e.g., lymphoedema) and other types 
of localized fluid collection, such as pericardial and pleural effusions and ascites have also been reported. 
Hyperlipidemia 
Increased serum cholesterol and triglycerides, requiring the need for anti-lipid therapy, have been reported to occur 
following initiation of everolimus and the risk of hyperlipidemia is increased with higher everolimus whole blood trough 
concentrations.  Use of anti-lipid therapy may not normalize lipid levels in patients receiving everolimus. 
Due to an interaction with cyclosporine, clinical trials of everolimus and cyclosporine in kidney transplant patients 
strongly discouraged patients from receiving the HMG-CoA reductase inhibitors simvastatin and lovastatin. During 
everolimus therapy with cyclosporine, patients administered an HMG-CoA reductase inhibitor and/or fibrate should be 
monitored for the possible development of rhabdomyolysis and other adverse effects, as described in the respective 
labeling for these agents.  
Proteinuria 
The use of everolimus with cyclosporine in transplant patients has been associated with increased proteinuria. The risk 
of proteinuria increased with higher everolimus whole blood trough concentrations.  
Polyoma Virus Infections 
Patients receiving immunosuppressants, including everolimus, are at increased risk for opportunistic infections; 
including polyoma virus infections.  Polyoma virus infections in transplant patients may have serious, and sometimes 
fatal, outcomes.  These include polyoma virus-associated nephropathy (PVAN), mostly due to BK virus infection, and JC 
virus associated progressive multiple leukoencephalopathy (PML). PVAN has been observed in patients receiving 
immunosuppressants, including everolimus.   


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Non-Infectious Pneumonitis 
A diagnosis of non-infectious pneumonitis should be considered in patients presenting with symptoms consistent with 
infectious pneumonia or radiologic changes in whom infectious, neoplastic and other non-drug causes have been ruled 
out through appropriate investigations. Fatal cases have been reported. Non-infectious pneumonitis may respond to 
drug interruption with or without glucocorticoid therapy.  
Thrombotic Microangiopathy/ Thrombotic Thrombocytopenic Purpura/ Hemolytic Uremic Syndrome (TMA/TTP/HUS) 
The concomitant use of everolimus with cyclosporine may increase the risk of thrombotic microangiopathy/thrombotic 
thrombocytopenic purpura/hemolytic uremic syndrome.  
New Onset Diabetes after Transplant 
Everolimus has been shown to increase the risk of new onset diabetes mellitus after transplant.  
Male Infertility 
Azospermia or oligospermia may be observed.  Everolimus affects rapidly dividing cells like the germ cells which can lead 
to reduced male fertility.  These effects are reversible with discontinuation of therapy.  
Pregnancy and Nursing 
Everolimus is a pregnancy category C drug. In animal studies, everolimus crossed the placenta and was toxic to the 
conceptus. The potential risk for humans is unknown. Women of childbearing potential should be advised to use 
effective contraception methods while they are receiving everolimus and up to 8 weeks after treatment has been 
stopped. Because many drugs are excreted in human milk and because of the potential for serious adverse reactions in 
nursing infants from everolimus, women should avoid breast-feeding during treatment with everolimus. 
 Risks of Tacrolimus and Mycophenolate Mofetil/Mycophenolic Acid 
Subjects will be on CNI and mycophenolate at the time of study entry. Potential risks applicable to their use in this study 
are listed below. 
 
Risks of Tacrolimus 
Post-Transplant Diabetes Mellitus  
Insulin-dependent post-transplant diabetes mellitus (PTDM) was reported in 20% of Prograf-treated kidney transplant 
patients without pretransplant history of diabetes mellitus in the Phase III study.  The median time to onset of PTDM 
was 68 days.  Insulin dependence was reversible in 15% of these PTDM patients at one year and in 50% at 2 years post- 
transplant.  Black and Hispanic kidney transplant patients were at an increased risk of development of PTDM. 
Nephrotoxicity  
Tacrolimus can cause nephrotoxicity, particularly when used in high doses.  Nephrotoxicity was reported in 
approximately 52% of kidney transplantation patients and in 40% and 36% of liver transplantation patients receiving 
tacrolimus in the U.S. and European randomized trials, respectively, and in 59% of heart transplantation patients in a 
European randomized trial.  In patients with persistent elevations of serum creatinine who are unresponsive to dosage 
adjustments, consideration should be given to changing to another immunosuppressive therapy.  Care should be taken 
in using tacrolimus with other nephrotoxic drugs.   
Hyperkalemia  
Mild to severe hyperkalemia was reported in 31% of kidney transplant recipients and in 45% and 13% of liver transplant 
recipients treated with tacrolimus in the U.S. and European randomized trials, respectively, and in 8% of heart transplant 


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recipients in a European randomized trial and may require treatment.  Serum potassium levels should be monitored and 
potassium-sparing diuretics should not be used during tacrolimus therapy. 
Neurotoxicity  
Tacrolimus can cause neurotoxicity, particularly when used in high doses.  Neurotoxicity, including tremor, headache, 
and other changes in motor function, mental status, and sensory function were reported in approximately 55% of liver 
transplant recipients in the two randomized studies.  Tremor occurred more often in tacrolimus-treated kidney 
transplant patients (54%) and heart transplant patients (15%) compared to cyclosporine-treated patients.  The incidence 
of other neurological events in kidney transplant and heart transplant patients was similar in the two treatment groups. 
Tremor and headache have been associated with high whole-blood concentrations of tacrolimus and may respond to 
dosage adjustment. Seizures have occurred in adult and pediatric patients receiving tacrolimus.  Coma and delirium also 
have been associated with high plasma concentrations of tacrolimus.  Patients treated with tacrolimus have been 
reported to develop posterior reversible encephalopathy syndrome (PRES).  Symptoms indicating PRES include 
headache, altered mental status, seizures, visual disturbances and hypertension. Diagnosis may be confirmed by 
radiological procedure. If PRES is suspected or diagnosed, blood pressure control should be maintained and immediate 
reduction of immunosuppression is advised.  This syndrome is characterized by reversal of symptoms upon reduction or 
discontinuation of immunosuppression. 
Malignancy and Lymphoproliferative Disorders 
As in patients receiving other immunosuppressants, patients receiving tacrolimus are at increased risk of developing 
lymphomas and other malignancies, particularly of the skin.  The risk appears to be related to the intensity and duration 
of immunosuppression rather than to the use of any specific agent.  A lymphoproliferative disorder (LPD) related to 
Epstein-Barr Virus (EBV) infection has been reported in immunosuppressed organ transplant recipients.  The risk of LPD 
appears greatest in young children who are at risk for primary EBV infection while immunosuppressed or who are 
switched to tacrolimus following long-term immunosuppression therapy.  Because of the danger of over suppression of 
the immune system which can increase susceptibility to infection, combination immunosuppressant therapy should be 
used with caution. 
Latent Viral Infections  
Immunosuppressed patients are at increased risk for opportunistic infections, including activation of latent viral 
infections.  These include BK virus associated nephropathy and JC virus associated progressive multifocal 
leukoencephalopathy (PML) which have been observed in patients receiving tacrolimus.  These infections may lead to 
serious, including fatal, outcomes. 
Tacrolimus in Combination with Sirolimus  
The use of full-dose tacrolimus with sirolimus (2 mg per day) in heart transplant recipients was associated with increased 
risk of wound healing complications, renal function impairment, and insulin-dependent post-transplant diabetes 
mellitus, and is not recommended.  Study subjects will not be prescribed full-dose tacrolimus in this trial.  Rather, 
subjects will be weaned off tacrolimus once target everolimus levels have been reached.   
 
Risks of Mycophenolate Mofetil/ Mycophenolic Acid 
Embryofetal Toxicity 
Mycophenolate mofetil (MMF) can cause fetal harm when administered to a pregnant female.  Use of MMF during 
pregnancy is associated with an increased risk of first trimester pregnancy loss and an increased risk of congenital 
malformations, especially external ear and other facial abnormalities including cleft lip and palate, and anomalies of the 
distal limbs, heart, esophagus, kidney and nervous system. 


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All prescribers participating in the study will be required to enroll in the FDA mycophenolate REMS program.  Of note, all 
study participants will already have been on a maintenance regimen containing MMF/MPA and will not be started on it 
as part of the study.  All females must be willing to use FDA approved methods of birth control acceptable during the 
entire period of the study. Urine pregnancy test is part of the screening laboratory tests and will be repeated prior to 
polyTreg infusion.   
For those females who are discovered to be pregnant either at study screening or enrollment or during the study and 
who are on MMF/MPA or within 6 weeks of discontinuing therapy, the study investigators will report the pregnancy to 
the Mycophenolate Pregnancy registry (1-800-617-8191) and strongly encourage the patient to enroll in the pregnancy 
registry. When appropriate, pregnant patients will be switched to alternative immunosuppression with less potential for 
embryo-fetal toxicity after a discussion of maternal and fetal risks and benefits. 
Lymphoma and Malignancy  
Patients receiving immunosuppressive regimens involving combinations of drugs, including MMF, as part of an 
immunosuppressive regimen are at increased risk of developing lymphomas and other malignancies, particularly of the 
skin.  The risk appears to be related to the intensity and duration of immunosuppression rather than to the use of any 
specific agent.  As usual for patients with increased risk for skin cancer, exposure to sunlight and UV light should be 
limited by wearing protective clothing and using a sunscreen with a high protection factor.  Lymphoproliferative disease 
or lymphoma developed in 0.4% to 1% of patients receiving MMF (2 g or 3 g) with other immunosuppressive agents in 
controlled clinical trials of renal, cardiac, and hepatic transplant patients.  In pediatric patients, no other malignancies 
besides lymphoproliferative disorder (2/148 patients) have been observed. 
Combination with Other Immunosuppressive Agents  
MMF has been administered in combination with the following agents in clinical trials: antithymocyte globulin 
(ATGAM®), OKT3 (Orthoclone OKT® 3), cyclosporine (Sandimmune®, Neoral®) and corticosteroids. The efficacy and 
safety of the use of MMF in combination with other immunosuppressive agents have not been determined. 
Serious Infections 
Patients receiving immunosuppressants, including MMF, are at increased risk of developing bacterial, fungal, protozoal 
and new or reactivated viral infections, including opportunistic infections.  These infections may lead to serious, 
including fatal outcomes.  Because of the danger of over suppression of the immune system which can increase 
susceptibility to infection, combination immunosuppressant therapy should be used with caution. 
New or Reactivated Viral Infections 
Polyomavirus associated nephropathy (PVAN), JC virus associated progressive multifocal leukoencephalopathy (PML), 
cytomegalovirus (CMV) infections, reactivation of hepatitis B (HBV) or hepatitis C (HCV) have been reported in patients 
treated with immunosuppressants, including MMF.  Reduction in immunosuppression should be considered for patients 
who develop evidence of new or reactivated viral infections.  Physicians should also consider the risk that reduced 
immunosuppression represents to the functioning allograft. 
PML, which is sometimes fatal, commonly presents with hemiparesis, apathy, confusion, cognitive deficiencies, and 
ataxia.  Risk factors for PML include treatment with immunosuppressant therapies and impairment of immune function. 
In immunosuppressed patients, physicians should consider PML in the differential diagnosis in patients reporting 
neurological symptoms and consultation with a neurologist should be considered as clinically indicated.  The risk of CMV 
viremia and CMV disease is highest among transplant recipients seronegative for CMV at time of transplant who receive 
a graft from a CMV seropositive donor.  Therapeutic approaches to limiting CMV disease exist and should be routinely 


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provided.  Patient monitoring may help detect patients at risk for CMV disease.  Viral reactivation has been reported in 
patients infected with HBV or HCV.  Monitoring infected patients for clinical and laboratory signs of active HBV or HCV 
infection is recommended. 
Neutropenia 
Severe neutropenia [absolute neutrophil count (ANC) <0.5 x 103/uL] developed in up to 2.0% of renal, up to 2.8% of 
cardiac, and up to 3.6% of hepatic transplant patients receiving MMF 3g daily.  Patients receiving MMF should be 
monitored for neutropenia. The development of neutropenia may be related to CellCept itself, concomitant 
medications, viral infections, or some combination of these causes.  If neutropenia develops (ANC <1.3 x 103/uL), dosing 
with MMF should be interrupted or the dose reduced, appropriate diagnostic tests performed, and the patient managed 
appropriately.  Neutropenia has been observed most frequently in the period from 31 to 180 days post-transplant in 
patients treated for prevention of renal, cardiac, and hepatic rejection. 
Patients receiving MMF should be instructed to report immediately any evidence of infection, unexpected bruising, 
bleeding, or any other manifestation of bone marrow depression. 
Pure Red Cell Aplasia (PRCA)  
Cases of pure red cell aplasia (PRCA) have been reported in patients treated with MMF in combination with other 
immunosuppressive agents.  The mechanism for MMF induced PRCA is unknown; the relative contribution of other 
immunosuppressants and their combinations in an immunosuppression regimen are also unknown.  In some cases, 
PRCA was found to be reversible with dose reduction or cessation of MMF therapy.  In transplant patients, however, 
reduced immunosuppression may place the graft at risk. 
Gastrointestinal Disorders 
Gastrointestinal bleeding (requiring hospitalization) has been observed in approximately 3% of renal, in 1.7% of cardiac, 
and in 5.4% of hepatic transplant patients treated with MMF 3 g daily.  In pediatric renal transplant patients, 5/148 cases 
of gastrointestinal bleeding (requiring hospitalization) were observed.  Gastrointestinal perforations have rarely been 
observed. Most patients receiving MMF were also receiving other drugs known to be associated with these 
complications.  Patients with active peptic ulcer disease were excluded from enrollment in studies with MMF.  Because 
MMF has been associated with an increased incidence of digestive system adverse events, including infrequent cases of 
gastrointestinal tract ulceration, hemorrhage, and perforation, MMF should be administered with caution in patients 
with active serious digestive system disease. 
Patients with Renal Impairment  
Subjects with severe chronic renal impairment (GFR <25 mL/min/1.73 m2) who have received single doses of MMF 
showed higher plasma MPA and MPAG AUCs relative to subjects with lesser degrees of renal impairment or normal 
healthy volunteers.  No data are available on the safety of long-term exposure to these levels of MPAG.  Doses of MMF 
greater than 1 g administered twice a day to renal transplant patients should be avoided and they should be carefully 
observed. 
Patients with HGPRT Deficiency  
MMF is an IMPDH (inosine monophosphate dehydrogenase) inhibitor; therefore, it should be avoided in patients with 
rare hereditary deficiency of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) such as Lesch-Nyhan and 
Kelley-Seegmiller syndrome. 
Phenylketonurics 


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 MMF Oral Suspension contains aspartame, a source of phenylalanine (0.56 mg phenylalanine/mL suspension). 
Therefore, care should be taken if MMF Oral Suspension is administered to patients with phenylketonuria. 
 Risks of Study Procedures  
 
Risks of Blood Draws 
Risks of blood draw or venipuncture are typically minimal with temporary local discomfort. More serious risks would 
include ecchymosis and, rarely, localized infection. The amount of blood that may be drawn from adult subjects for 
research purposes will not be more than 600 mL over an eight-week period. The additional amount of blood could 
contribute to the development of anemia. The subject’s clinical condition will be taken into consideration to determine 
if research blood tests can be performed. 
 
Risks of Leukapheresis 
The common risks of leukapheresis include bruising and discomfort at the site of needle placement, typically in the 
antecubital fossae.  Calcium level in blood may fall due the citrate anticoagulant used to prevent clotting in the 
leukapheresis instrument.  Hypocalcemia can lead to perioral or digital numbness and tingling.  Calcium replacement 
may be used during the procedure and is routinely used at the conclusion of the procedure.  Platelet count may fall due 
to platelet loss during processing.  Hemorrhagic complications due to thrombocytopenia have not been reported in 
normal donors.  Thrombosis and bleeding could theoretically occur, although they are rarely if ever observed.  
 
Risks of Kidney Biopsy 
There is a risk of bleeding associated with transplant kidney biopsies. Transient hematuria occurs in 3 to 10% of patients 
and may prolong hospitalization, require bladder catheterization for clot drainage, or in approximately 1% of patients, 
require blood transfusion. Ureteral obstruction from blood clot may require percutaneous nephrostomy in <1% of 
patients. Massive hemorrhage requiring surgical exploration, transplant nephrectomy, or arterial embolization occurs in 
~0.1 % of patients. Death from massive hemorrhage is rare. 
 Potential Benefits 
This study might not provide direct or immediate benefit to the participants.   
 
Decrease in Graft Inflammation 
It is possible that infusions of Tregs may modify the infiltrate in the renal allograft to the benefit of the kidney (Taflin C, 
2010). 
 
CNI Reduction 
Subjects who have a significant decrease in graft inflammation will be eligible for reduction and potentially elimination 
of tacrolimus therapy. This may help to reduce/ prevent adverse effects associated with CNI therapy, including 
nephrotoxicity (Mjörnstedt L, 2012).   


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 Study Visits 
 
Enrollment 
The research study will be explained in lay terms to each potential research participant. The potential participant will 
sign an informed consent form before undergoing any study procedures. A subject is considered enrolled in the trial 
once the consent form has been signed.   
 
Study Eligibility and Study Group Allocation 
Subjects will have a renal allograft biopsy performed 5 months (±8 weeks) after transplantation.  Biopsy results will be 
an integral part of screening for study eligibility and will be confirmed by the central pathologist prior to enrollment.  
During the screening period the study personnel will review the subject’s medical record for previous and current 
medical history, demographic information (age, gender, and race), medications, and laboratory test results.  Additional 
laboratory tests, such as a pregnancy test (if applicable), will be performed at the transplant center for screening 
purposes.  Blood, urine and tissue will be collected for central laboratory analysis. 
Eligible patients will be assigned according to the allocation scheme (Section 3.7) to 2 groups: group 1 CNI maintenance; 
group 2 infusion of 550 ± 450 x 106 polyTregs.   The day of study group assignment is designated day 0 on the Schedule 
of Events (Appendix 2 and 3).   
 
Specific Visits for Group 2 
 
Whole Blood Collection or Leukapheresis for Treg Manufacture (PBMC Collection Visit)  
Participants in Group 2 will provide 450-500 ml of whole blood via phlebotomy or have leukapheresis; either will be 
shipped to UCSF for manufacturing.  The actual visit dates of recipient collections will be based on the selected infusion 
date. 
 
Treg Infusion Visit 
Eligibility for Treg infusion will be reviewed at the time of the study visit.  When applicable, a urine pregnancy test must 
be confirmed negative prior to infusion.  Blood will be drawn for local laboratory tests.  Subjects will receive the 
polyTreg infusion no later than 62 days after central biopsy confirmation.   The Treg infusion visit requires an overnight 
stay in the hospital or research unit for observation. 
 
Follow Up Visits  
Group 2 participants will have study visits on day 1, 7, 14, 28, 84, 182, and 364 after Treg infusion.  Participants in Group 
1 will have the same visits in parallel with Group 2 patients, starting 48 days after study group assignment.  Please refer 
to Appendix 2 for Group 1 schedule of events and Appendix 3 for Group 2 schedule of events. 
10.4.1 Provisions for COVID-19 
Participating centers should follow site-specific guidance related to COVID-19 in regard to telemedicine visits in leiu of 
visits at the study site.  For CTOT-21, Visit 2/ Day 14 and Visit 5/ Day 182 are considered essential or ciritical for 
assessment study endpoints.  Efforts should be made to have subjects return to the study site for these two visits in 
order to obtain required assessments, including blood, biopsy, and urine specimens.  In addition, study assessments 
should be obtained if a subject has a clinically indicated biopsy. 
 
 


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Unscheduled Visits/ Clinically Indicated Biopsies  
If creatinine increases or other concerns arise between regularly scheduled visits, participants will be instructed to 
return to the study site for an “unscheduled” visit.   Study assessments will be performed, and research specimens 
collected if a subject has a clinically indicated biopsy.  Please see Appendix 2 for details. 
 
Visit Windows 
Study visits should take place within the time limits specified below (Table 2).  The designated visit windows for each 
scheduled visit are also indicated on the Schedules of Events. 
Table 2. Visit Windows  
Study Visit 
Visit Window 
Study Eligibility Biopsy  
5 months ± 8 weeks  
Study Eligibility/ Study Group Allocation 
≤ 2 weeks after qualifying biopsy 
Group 1 Only 
Day 41/ Visit T0-G1 
± 3 days 
Day 48/ Visit 1 
Day 55/ Visit 2 
Day 69/ Visit 3 
Day 125/ Visit 4 
± 5 days 
Day 223/ Visit 5 
± 14 days 
 
Day 405/ Visit 6 
Group 2 
PBMC Collection  
Visit T-16 
Dictated by infusion date and manufacturing 
timeline. 
Treg Infusion Day / Visit T0  
Infusion can occur any time up to and 
including day 62 after central pathology 
confirmation. 
Visit T1 / Day 1 
± 1 hour (Group 2) 
Visit 1 / Day 7 
± 1 day 
Visit 2 / Day 14 
Visit 3 / Day 28 
 Visit 4 / Day 84 
± 3 days 
Visit 5 / Day 182 
± 14 days 
 Visit 6 / Day 364 
± 14 days 
 


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 Mechanistic Assays 
The goal of the mechanistic studies in this trial is to evaluate the impact of polyTregs on the immunological profiles of 
kidney transplant recipients.  We will accomplish this by determining: 1) impact of Treg infusion on the numbers of 
circulating and graft-infiltrating Tregs; 2) impact of Treg infusion on the inflammation in the graft; 3) impact of Treg 
infusion on the peripheral blood biomarkers of rejection and graft injury. 
 
Impact of Treg Infusion on Numbers of Circulating and Graft-Infiltrating Tregs 
 
Detection of Infused Tregs Using Deuterium Labeling (Deu-bl) 
Infused polyTregs will be indistinguishable from endogenous Tregs by standard surface markers, prohibiting the tracking 
of infused cells using conventional means. The Hellerstein group and others have developed stable-isotope-labeling 
technology to measure the survival, replication, and trafficking of murine and human cells. Importantly, deuterium, a 
naturally occurring stable isotope of hydrogen, is nonradioactive and non-toxic, and has been safely used as a cellular, 
molecular, and metabolic marker in patients and healthy controls for more than 6 decades (Busch R, 2007) (Macallan 
DC, 1998). The use of deuterium-labeled glucose in polyTreg cultures led to ~60% labeling of DNA that was detectable 
using gas chromatography-mass spectrometry (GC-MS). Deuterium labeling is permanent such that the percentage of 
deuterated DNA on a per cell basis only decreases with DNA replication. After intravenous administration of 3.2 x 108 
deuterium-labeled polyTregs in the UCSF type I diabetes trial, infused Tregs were readily detected up to 180 days post 
infusion in all 3 patients, demonstrating the feasibility of this approach (Figure 4).  In addition, early experience with a 
kidney transplant recipient shows that Tregs from immunosuppressed patients can be labeled with similar efficiency 
during in vitro expansion and the in vivo pharmacokinetics of Tregs is similar to that seen type 1 diabetes patients who 
were not on immunosuppression (Figure 4). 
 
Figure 4.  Deuterium labeling for Treg tracking 


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Following infusion of stable isotope-labeled Tregs, 15 ml of whole blood will be collected at visits specified in the 
schedule of events (Appendix 2 and 3). The cells will be processed into PBMC and Tregs will be sorted based on 
CD4+CD127lo/-CD25+ markers. In addition, CD4+ T cells that are outside the Treg gate will also be collected and analyzed 
to determine if infused Tregs give rise to non-Tregs in vivo after infusion.  Genomic DNA will be isolated from the 
purified cells and hydrolyzed before assessing the 2H isotopic enrichment of the purine deoxyribonucleosides using GC-
MS.  The level of 2H enrichment among the purified Treg and non-Treg cells will be determined. 
 
Detection of Infused Tregs in Graft Using Deuterium Labeling (Deu-biopsy) 
Tregs have a propensity to traffic to and accumulate in sites of inflammation.  Since patients in this trial are selected to 
have inflammation in the kidney allograft, the infused Tregs may infiltrate the graft.  To determine if this occurs, fresh 
biopsy samples (1/2 to a full 16G core) will be collected 2 weeks after Treg infusion.  The tissue will be digested to dissociate 
the cells into single cell suspension.  Infiltrating CD45+immune cells and its various subsets will be isolated using FACS and 
the level of 2H enrichment among the purified cells will be determined as described above for blood samples. 
 
Peripheral Blood Treg Number (MFC-Treg) 
Blood (1 ml) will be collected at visits specified in the schedule of events (Appendix 2 and 3), processed into PBMC, and 
cryopreserved for batched analysis.  Percentage of Tregs among CD4 T cells will be determined using multiparameter 
flow cytometry (MFC) as described in section 11.3.1 below.  The absolute number of peripheral blood Tregs will be 
calculated using the number of CD4 counts obtained from clinical lab test. 
 
Treg TruCount Analysis 
Samples collected for this assay will be used directly for analysis without cryopreservation.  Blood will be aliquoted into a 
TruCount tube, stained with fluorochrome-conjugated antibodies to CD4, CD45, CD25 and CD127, and analyzed on a 
flow cytometer to enumerate the numbers of Tregs. 
This assay would allow us to obtain the absolute Treg counts in one microliter of blood.   
 
Impact of Treg Infusion on Graft Inflammation  
 
Detection of Intragraft Inflammation by Multiplexed Immunofluorescence and in situ 
Hybridization (mIF/ISH) 
Histopathology combined with immunohistochemistry (IHC) or immunofluorescence (IF) provides information on the 
location, nature, and magnitude of the inflammatory response, but traditionally is not a reliable approach for 
quantifying changes in inflammatory mediators. To quantitatively assess cytokine expression in the graft tissue in 
response to intervention, the Laszik lab has adapted the RNAScope® in situ hybridization (ISH) platform to analyze 
formalin-fixed paraffin-embedded biopsies.  Furthermore, a novel technology that combines ISH with IF was developed 
and optimized for various targets; this highly sensitive and specific ISH/IF methodology combined with computer-
assisted image analysis allows precise quantitative phenotypical analysis of immune infiltrates and parenchymal cells 
along with their gene expression profiles in the biopsy tissue. We plan to use this technology to assess change in 
inflammatory and tolerance markers in the grafts after Treg therapy in biopsy samples obtained at 2 weeks and 6 
months after Treg infusion and compared to the baseline in the eligibility biopsy samples. The following structural and 
inflammatory markers will be evaluated using IHC/IF technology: collagen 3 for interstitial fibrosis, and CD4, CD8, Treg 
(FoxP3), CD20, and CD68 for inflammation. ISH IL-6 mRNA will be detected via ISH.  Any for-cause biopsies performed 
during the study will also be evaluated using the same parameters.    
 


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Graft Common Response Module (CRM) Gene Expression 
Increased transcriptional profiling of transplant biopsies has provided useful insights into allograft injury mechanisms 
such as acute and chronic rejection (Samanta A, 2008) (Morgun A, 2006) (Snyder TM, 2011). Identifying a common 
rejection mechanism could facilitate novel diagnostics without requiring details about tissue-specific injury. Recently, 
the Sarwal Lab and collaborators analyzed whole genome expression profiles in 13 independent transplant cohorts 
consisting of 1164 graft biopsy samples from four different organs (kidney, heart, lung, and liver) and thus representing 
the largest study of its kind in transplantation. This study was able to define a CRM for the prediction of cross-organ 
acute rejection and identification of novel drug targets in transplantation (Khatri P, 2013). The CRM consists of 12 genes 
that were significantly up regulated during rejection independent of the organ type (Figure 5 and Table 3). Additional 
pathway analyses revealed the relevant association of these genes with T cell receptors, interleukins, chemokines and 
transcription factors. In order to avoid (1) the influence of a single large experiment on the meta-analysis results and (2) 
organ-specific bias due to unequal number of data sets (and samples) used in the metaanalysis, a “leave-one-organ out” 
meta-analysis was performed leading to the definition of 12 ubiquitously overexpressed rejection associated genes 
BASP1, CD6, CD7, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, and TAP1. 
Figure 5. 12-Gene CRM across heterogeneous microarray datasets on different platforms. 
 


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Gene ID 
Protein name 
Function 
BASP1 
Brain acid soluble protein 1 
Transmembrane protein involved in signaling, found in renal epithelial 
cells and other cells with high turnover 
CD6 
T-cell differentiation antigen 
CD6 
Surface protein expressed on lymphocytes, involved in cell adhesion 
CXCL9 
C-X-C motif chemokine 9 
Cytokine that affects growth, movement, or activation of immune cells, 
recruits T-cells. 
CXCL10 
C-X-C motif chemokine 10 
Cytokine that recruits monocytes and T-cells. 
INPP5D 
Phosphatidylinositol 3,4,5-
triphosphatase 1 
Negative regulator of B-cell antigen receptor signaling 
ISG20 
Interferon-stimulated gene 
20kDa 
Cleaves single-stranded RNA and DNA; induced by interferon gamma 
(IFNγ) has antiviral effects against RNA viruses 
LCK 
Tyrosine-protein kinase Lck 
Protein kinase involved in signaling to promote T cell response and 
proliferation 
NKG7 
Natural killer cell protein 7 
Surface membrane protein expressed on activated T cells, kidney, liver 
and pancreas 
PSMB9 
Proteasome subunit beta 
type-9 
Cleaves proteins to create MHC class I binding peptides 
RUNX3 
Runt-related transcription 
factor 3 
Regulates transcription by binding enhancers and promoters of 
polyomavirus, T-cell receptor, lck, IL-2 and GM-CSF 
TAP1 
Antigen peptide transporter 1 
Transports antigens into endoplasmic reticulum for presentation by MHC 
class I 
Table 3. CRM: Upregulated genes during rejection  
The significance of the CRM genes in AR was validated in an independent cohort of publicly available microarray data 
from 282 renal allograft biopsies (GSE 36059;  (Halloran PF, 2010)) and classified AR (irrespective of cellular, antibody or 
mixed rejection calls) and stable samples with high accuracy (Figure 7A). Most importantly, the CRM expression 
correlated with molecular inflammation and the extent of graft injury when formulated into a statistically derived CRM-
Score using 11/12 defined genes, calculated by using the geometric mean of the expression of each of the CRM genes in 
each sample. In AR biopsies, the CRM quantitative score correlated with the Banff t-score (p = 6.04e-12) and the Banff i-
score (p=2.72e-12) supporting the value of this molecular measurement in the assessment of alloimmune inflammation 
in the graft. The CRM score in histologically normal 6-month protocol biopsies also correlated strongly with the Banff ct 
score (p-value = 1.995e-5) and Banff ci score (p-value = 6.195e-7) in transplants that went on to develop progressive 
chronic allograft injury, versus those that had stable histology and graft function (p=4.9e-6, Figure 7B). Given that 
histological analysis of the graft can miss subtle changes in inflammation, the inclusion of the CRM score as a surrogate 
for SCI is a strength of this study in the interpretation of the inflammation response in the kidney and retrospective 
interrogation for its interplay with other peripheral markers of graft inflammation in the blood and the urine.  The 
finding that the CRM score associates with the extent of graft injury (Figure 7B) as assessed by Banff criteria is of special 
importance for the current study, as it provides a measure of integrating gene expression profiles with the findings of 
the immune histopathology core. Moreover, as the CRM score also correlates with an increased risk of subsequent graft 
fibrosis, it may inform investigators of those at risk for late graft failure and provide mechanistic insight into the success 
of the proposed interventions.  


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Total RNA will be extracted from 3x10¥ìm-thick sections of the FFPE tissue using the PureLink FFPE Total RNA Isolation 
Kit. RNA quantity and integrity will be determined with the Thermo Scientific NanoDrop ND-2000 UV-Vis 
Spectrophotometer and Agilent Bioanalyzer, respectively.  Gene expression will be analyzed using a set of 800 immune 
genes that includes the CRM genes. The RNA samples will be added to a Barcoded Codeset designed by NanoString 
Technologies consisting of Reporter and Capture probes that hybridize to the target sequences of interest, forming a 
tripartite complex.  The raw counts for each assay will be collected using the NanoString data analysis software, 
nSolver¢ç (NanoString Technologies, Seattle, WA). Normalization of the data will be performed using nSolver¢ç for the 
following two methods. (1) Positive control normalization: gene expression data is normalized to the mean of the POS 
control probes for each assay. (2) RNA content normalization: gene expression data is normalized to the geometric mean 
of housekeeping genes in the CodeSet. 
The CRM score is defined as geometric mean of the CRM genes expression.  Distribution of CRM scores in AR and STA 
groups and receiver operating characteristic (ROC) curve are shown for GSE21374 (Figure 8A-B), GSE36059 (Figure 8C-D). 
The x axes represent false positive rate, and the y axes represents true positive rate when using the CRM scores for 
predicting AR. Error bars indicate the standard error of the mean (Fig.8). In each independent dataset, the CRM score 
was significantly higher in the AR group (P < 1.5 ¡¿ 10£¬7; Fig. 8). Each unit increment in the CRM score increased the 
odds of AR by 4.17 and 5.45 in GSE21374 and GSE36059. It was also able to distinguish AR and STA samples with high 
specificity and sensitivity in GSE21374 area under the curve (AUC = 0.83; Fig. 8B), GSE36059 (AUC = 0.8; Fig. 8D). The 
quantitative CRM score will be used to quantify SCI at a molecular level in the 2 week and 7-month protocol biopsies as 
well as any clinically indicated biopsies in the TASK study. The rest of the ~780 immune genes will also be analyzed along 
with the CRM genes. The gene expression data will be correlated with the Banff scores and the inflammatory cell load in 
the biopsies as measured by quantitative image analysis of CD45-stained sections of the FFPE tissue.    


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Figure 7.   The intragraft CRM gene expression score can distinguish AR and STA samples with high sensitivity and specificity. 
 
Urinary Biomarkers of Graft Inflammation and Injury 
Urine protein biomarkers correlate highly with the graft milieu and may directly reflect alloreactive inflammation and 
injury  (Lei J, 2010) (Ling XB, 2010) (Schaub S, 2004) (Schaub S W. J., 2006) (Sigdel TK K. A., 2010). The Sarwal lab has 
utilized a cohort of over 2000 highly clinically annotated urine samples from ~800 prospectively monitored renal 
transplant recipients, collected in multiple clinical trials (CCTPT SNSO1 study, U01 AI55795-02; CTOTC IMPACT trial 1U01 
AI077821; AARA UO1AI063594-06). By following an unbiased high-throughput 2D-LCMS/MS based proteomic approach, 
the Sarwal Lab selected 11 urine proteins (fibrinogen β (FBB), fibrinogen γ (FBG), Class II histocompatibility protein, HLA-
DRB1 and SUMO2) to develop a composite biomarker panel for transplant injury, distinguishing acute rejection, chronic 
rejection and BKVN (p<0.01; fold increase >1.5). This panel has been validated in 154 independent urine samples, each 
with biopsy matched phenotypic diagnoses. Interestingly, when histological scores of inflammation from the biopsies 
matched with the urine sample collections were analyzed, there was a significant correlation (r=0.73; p<0.0003) 
between these urinary proteins and mononuclear cell interstitial inflammation score (i-score) and the tubulitis score (t-
score), suggesting that these urine proteins in addition to being valuable clinical biomarkers for rejection monitoring, 
may also segregate biopsies with higher grades of T cell mediated rejection.  In addition, mRNA analysis for the CRM 
genes in urine may also provide a means to monitor for SCI in a non-invasive, serial manner. 
Total RNA will be extracted from the urine pellet (Qiagen). Total RNA will be measured for RNA integrity using the RNA 
6000 Nano LabChip Kit on a 2100 Bioanalyzer (both from Agilent Technologies, Santa Clara, CA), with suitable RNA 
defined by an RNA integrity number exceeding 5. Using a urine sample processing method that we developed, urinary 
proteins will be isolated by filtering the supernatant through Amicon Ultra centrifugal filtration tubes (10K molecular 
weight cutoff, Millipore, Bedford, MA) to separate small MW peptides and other pigments (<10 kDa) from the larger 
proteins. Commercially available ELISA assays for the urine protein panels will be purchased from Antibodies-online 
(Atlanta, GA), and the urine protein levels will be normalized to urine creatinine levels. The relative amount of mRNA 
expression in each sample will be calculated for each of the CRM genes using the comparative threshold cycle (CT) 
method. Expression values for all genes will be normalized to ribosomal18S RNA (18S). The levels of specific proteins and 


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genes will be measured in samples with and without SCI and their abundance also will be correlated with extent of graft 
fibrosis.  
 
Impact of Treg Infusion on Peripheral Blood Biomarkers of Rejection and Graft Injury 
 
Peripheral Blood Leukocyte Population and Phenotype by Multiparameter Flow Cytometry 
(MFC) 
Blood (1 ml) will be collected at visits specified in the schedule of events (Appendix 2 and 3), processed into PBMC, and 
cryopreserved for batched analysis to assess impact of Treg infusion on the frequencies and phenotypes of peripheral 
blood leukocytes.  The MFC analysis is focused on defining leukocyte subsets, T cell activation/exhaustion status, and 
CD4+ and CD8+ Treg frequencies.  Extensively validated MFC panels will be used to quantify changes in leukocyte 
populations in blood (Table 4) collected prior to and after Treg infusion.  The absolute number of each cell types in the 
peripheral blood will be calculated by referencing to the number of CD4 counts obtained from clinical lab test. 
Table 4. TASK MFC Panels 
Panel 
Names 
Markers 
Rationale 
Leuko 
CD3, CD4, CD8, CD14, CD16, 
CD19, CD56, HLA-DR 
To determine the numbers and percentages of T cells, B cells, 
subsets of monocytes, subsets of NKs and dendritic cells 
Treg 
CD3, CD4, CD8, CD25, CD127, 
FXOP3, HELIOS 
To determine the numbers and percentages of Tregs 
Tact 
CD3, CD4, CD8, CD27, CD28, 
CD45RA, CCR7 
To determine the activation status of  T cells 
Texh 
CD3, CD4, CD8, CD57, PD1, 
Tim3 
To determine the percentage of T cells that express 
exhausted or inhibited phenotype 
 
Peripheral Blood Biomarkers of Acute Rejection (kSORT) 
Building on a recent publication of the identification of 10 genes in peripheral blood for the diagnosis of pediatric renal 
allograft rejection in low-risk patients (Rose-John, 2012), an additional 7 genes for the diagnosis and prediction of acute 
rejection in both pediatric and adult kidney transplant recipients (Roedder S S. T., 2014) was identified, independent of 
the cause of end stage renal disease, co-morbidities, transplant center or immunosuppression usage. This signature 
comprises the following genes (CFLAR, DUSP1, ITGAX, RNF130, PSEN1, NKTR, RYBP, NAMPT, MAPK9, IFNGR1, CEACAM4, 
RHEB, GXMK, RARA, SLC25A37, EPOR, RXRA) which combines the previous 10-gene set in addition to 7 genes obtained 
through additional inclusion of adult renal transplant data, into the final developed assay that will be used in this study 
called kSORT (kidney solid organ response test) (Table 5). 
The kSORT genes are highly specific and sensitive for the prediction of biopsy confirmed acute rejection at the time of 
AR, and additionally 3-4 months prior to graft dysfunction and histologically confirmed AR. The 17 genes can be easily 
used for the prediction of acute rejection using a novel plug-in reference-correlation classification approach called 
Lineage Profiler; this program has been customized in the Sarwal Lab and calculates an AR risk score where a score >0 
indicates AR and a score <0 indicates non-AR (Figure 8). The SORT genes will be analyzed in our trials to define their 
association with inflammation and borderline rejection and to examine whether they are relevant in the response to 
therapy with Tregs. 


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Gene ID 
Protein name 
Function 
DUSP1 
Dual specificity protein 
phosphatase-1 
Dephosphorylates MAP kinase to regulate cell cycle 
CFLAR 
CASP8 and FADD-like apoptosis 
inhibitor 
Cytosolic protein that inhibits tumor necrosis factor related 
apoptosis 
ITGAX 
Integrin alpha–X 
Membrane receptor for fibrinogen that mediates cell-cell 
interaction during inflammatory responses; important in 
monocyte adhesion and chemotaxis 
NAMPT 
Nicotinamide 
phosphoribosyltransferase 
Catalyzes formation of NAD; also exists as nonsecreted form that 
acts as cytokine and adipokine; plays a role in regulating circadian 
clock 
MAPK9 
Mitogen-activated protein kinase 9 
Activated by cytokines and/or stress response to promote cell 
proliferation, differentiation, migration, transformation and 
programmed cell death 
RNF130 
E3 ubiquitin-protein ligase RNF130 
Cytoplasmic zinc-binding protein 
IFNGR1 
Interferon-receptor gamma-1 
Membrane protein that binds interferon gamma; mutation causes 
Immunodeficiency 27A 
PSEN1 
Presenilin-1 
Stimulates cell-cell adhesion; may play role in signaling and 
apoptosis; mutations associated with familial early-onset 
Alzheimer’s 
RYBP 
RING1 and YY1-binding protein 
Nucleic and cytosolic protein that inhibits transcription, promotes 
apoptosis 
NKTR 
NK-tumor recognition protein 
Membrane protein that is a component of a putative tumor 
recognition complex on the surface of NK cells; binds cyclosporin A 
SLC25A37 
Mitoferrin-1 
Mitochondrial iron transporter that takes up iron for developing 
erythroid cells 
CEACAM4 
Carcinoembryonic antigen-related 
cell adhesion molecule 4 (CD66a) 
Surface protein found on lymphocytes for cell to cell adhesion 
RARA 
Retinoic acid receptor alpha 
Suppresses transcription when unbound, loss of suppression when 
bound, associated with spermatocyte survival when bound 
RXRA 
Rxra protein 
Nuclear protein that regulates transcription 
EPOR 
Erythropoietin receptor 
Promotes erythroblast proliferation and differentiation when 
bound by EPO 
GZMK 
Granzyme K 
Serine protease released by NK or T cells for killing of cancerous 
cells, viruses, or bacteria 
RHEB 
GTP-binding protein Rheb 
Promotes proliferation and inflammation through activation of 
mTOR complex 
Table 5.  kSORT genes 


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Figure 8.  Performance of the kSORT assay. (A) kSORT score and classification category were calculated for each sample: the kSORT 
assay correctly classified 36 out of 39 AR samples as high risk for AR (red bars, 92.3%; risk score ≥9) and 43 out of 46 No-AR samples 
as low risk for AR (green bars, 93.5%, risk score ≤9) across four different sample collection sites (UPMC, Mexico, Barcelona and UCSF), 
and adult  versus pediatric recipient age groups, with samples collected in clinical trial and non-clinical-trial settings; remaining 15 
samples classified as indeterminate risk for AR (grey bars; risk score (scores <9 and >-9) (B) Mean aggregated kSORT scores (error 
bars give standard error of mean) were significantly higher in all true AR samples than in all true No-AR samples by two-sided 
Student’s t test. (C) ROC analysis demonstrated high sensitivity and specificity for the 100 samples evaluated in this dataset. 
Total RNA will be extracted using the column-based method kits of PreAnalytiX (Qiagen). Total RNA will be measured for 
RNA integrity using the RNA 6000 Nano LabChip Kit on a 2100 Bioanalyzer (both from Agilent Technologies, Santa Clara, 
CA), with suitable RNA defined by an RNA integrity number exceeding 7. cDNA synthesis will be performed using 250 ng 
of extracted quality mRNA from the PB samples using the SuperScript II first strand cDNA synthesis kit. Standard 
protocols developed in the Sarwal Lab will be used for QPCR reactions on the ABI Quant-Studio 6 sing the same TaqMan 
gene expression assays. The relative amount of mRNA expression in each sample will be calculated for each of the 17 
kSORT genes using the comparative threshold cycle (CT) method. Expression values for all genes will be normalized to 


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ribosomal18S RNA (18S). All samples will be evaluated against our customized database (kSAS) of centroids for AR and 
No-AR, with the greatest correlation to the evaluated sample. Classification of a sample as AR, No-AR, or indeterminate 
will be derived by computing the Pearson correlation coefficients for all sample–centroid comparisons, assigning a score 
of 1 to samples with a greater correlation to an AR centroid and a score of -1 to samples with a greater correlation to a 
No-AR centroid. A kSAS aggregated AR risk score (-13 to 13) will then be determined by summing the scores for all 
evaluated gene models. Indeterminate samples will be defined as those with a score less than 9 and greater than -9, 
based on evaluation of sample classification. The blood sample kSORT scores will be correlated with SCI and clinical AR 
(expected kSORT scores of ≥9) as well as stable, quiescent samples (expected kSORT scores of ≥ -9) in the TASK study.  
 
Alloimune Quiescence and Operational Tolerance (kSPOT) 
Sarwal Lab has identified an initial tolerance footprint of 49 genes by microarray and qPCR in peripheral blood samples 
from 75 renal allograft recipients and 16 healthy individuals from the U.S. and in Europe (Brouard S, 2007). 21/49 genes 
correctly segregated tolerance and chronic rejection phenotypes with 99% and 86% specificity, and the signature was 
shared with 8% and 50% respectively of stable patients on triple immunosuppression and low-dose steroid monotherapy 
respectively. The gene signature suggested a pattern of reduced co-stimulatory signaling, immune quiescence, 
apoptosis, and memory T cell responses, and additionally suggested that TGFβ might contribute to this process. This 
panel has been reduced to a minimal 3-gene signature (BNC2, CYP1B1, KLF6) called SPOT (Spontaneous Tolerance Test) 
using penalized logistic regression modeling. The inclusion of SPOT in the proposed trials will therefore provide an 
assessment of immune quiescence that may then be deployed as a measure for the success of Treg therapy. 
Additionally, these results provide a proof of concept for successfully linking the gene expression profiles in peripheral 
blood with the peripheral blood phenotype measured by flow cytometry. 
Sample collection and analysis plan for kSPOT. Please refer to kSORT for all sample collection and processing. The 21 
gene logistic regression score for a tolerance like signature (Roedder S, 2014) will determine immune quiescence profile 
for all samples in the TASK study and will evaluate if this signature is more likely to be found in patients who have 
received Treg therapy. 
 
Non-HLA Antibody Assays for Prediction of Chronic Allograft Injury (nHLA-Ab) 
The Sarwal Lab has identified and validated a de novo panel of 6 nHLA- Ab (MIG, ITAC, IFN-γ, GABPA, IL8, CCL21, GDNF) 
at 6 months post-transplant as important and previously unrecognized triggers of subsequent chronic graft injury (Sigdel 
TK, 2012). This panel positively correlated (r=0.78; p<0.01) with the chronic allograft damage index (Yilmaz S, 2003) 
scores at 2 years with 80% sensitivity and 100% specificity. This discovery involved the use of high-throughput screening 
of serum IgG interactions in post-transplant recipient sera to 9000 unique full-length human proteins using the 
Protoarray (Invitrogen) protein array technology and data analysis by customized algorithms (Li L, 2009). The Sarwal Lab 
has generated customized reverse ELISA assays for this panel of nHLA-Ab to be easily multiplexed on the Meso Scale 
Discovery® (MSD) platform. These specific nHLA-Ab panels will be correlated with histological chronicity and change in 
eGFR in the different treatment arms of the study. 
Sample processing analysis plan for nHLA MSD ELISA will be carried out following the protocol developed in the Sarwal 
lab. Briefly 15 ng antigen will be coated on to the wells using 50 µL coating at 0.3 µg/ml. The plates will be stored at 4 °C 
overnight. The following day the coating solution is removed and the plate is blocked by 150 µl Blocker A (at RT, 450 
rmp) for 1 hr.  After 1 hr the plate will be washed 3 times with 300 µl wash buffer. Next, 25 µl of diluted samples will be 
added and incubated at RT with 450 rpm for 2 hr. This will be followed by washing step followed by addition of 25 µl 
detection antibody SULFO-TAG Goat anti Human (Meso Scale Discovery, Gaithersburg MD, Cat# R32AJ-5) (1:1000 
dilution) for 1 hr. After this step the plates will be washed and 150ul/well of 1X read buffer (MSD Gaithersburg MD, Cat# 


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R92TC-2) will be added to each well. The plates will be read immediately using MESO QuickPlex SQ 120 scanner. Relative 
signal intensity will be used to measure relative abundance of the nHLA antibodies in the samples. The data will be 
analyzed by Mann-Whitney test using GraphPad Prism software. Spearman correlation will be done to correlate nHLA Ab 
signal with histological chronicity and change in eGFR in the different treatment arms of the study. These specific nHLA-
Ab panels will be correlated with histological chronicity and change in eGFR in the different treatment arms of the study.  
 
Markers of Tubular Epithelial Injury and Renal Fibrosis 
Urine and serum markers of renal injury have been associated with subsequent graft fibrosis and failure. Preliminary 
studies in Dr. Mannon’s laboratory have identified urinary TGFβ, connective tissue growth factor (CTGF) and the integrin 
ligand vitronectin as associated with allograft injury and fibrosis (Cheng O, 2008) (Mannon RB, 2010). In addition, urinary 
ANXA11, α3 and β3 integrins and TNF-α have been found to be closely associated with acute alloimmune mediated 
injury in kidney transplant recipients (Srivastava M, 2011) but have not yet been analyzed prospectively.  A novel link 
under study is between innate immune activation and allograft fibrosis, and measures of HMGB1 and HSP 27 in serum 
and urine will be of value (Harris HE, 2012) (Lotze M, 2005).  The recent association of resistin with brain death and 
delayed graft function (Oltean S, 2013), as well as the association of acute inflammation, endothelial activation and 
fibrosis (Jackson AM, 2014) suggest this as a protein that can be measured in this prospective cohort.  Having such 
targets is critical while investigating therapies which may mitigate inflammatory injury that leads to subsequent graft 
failure in order to monitor the success or failure of treatment. Other relevant targets for monitoring include expression 
of matrix molecules such as collagens I and III (Mannon R, 1999)and fibroblast infiltration such as α-SMA as indicators of 
developing fibrosis (Cheng O, 2008) as well as recently described markers of myeloid fibroblast infiltration including 
MCP-1, CXCL16 and adiponectin (Yang J, 2013). Establishing a link between these markers and graft histology/ renal 
function is a unique feature of this proposal in which protocol biopsies provide a “hard endpoint” in the subjects’ 
management.  
 
Stored Plasma and Serum 
Plasma and serum will be collected on the day of planned infusion, prior to polyTreg administration for subjects in Group 
2 .  If a need arises during the course of the study, testing may be performed on archived specimens (e.g. CMV, BK, DSA 
etc.). 


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 Biospecimen Storage 
Biological specimens (i.e., whole blood, plasma, serum, urine supernatant, urine pellet) obtained under this protocol 
may be used in future assays to reevaluate biological responses as additional research tests are developed over time. 
Residual specimens collected at time points planned for the core mechanistic studies will be maintained to allow 
specimens to be stored for possible use in new assays that have yet to be optimized or conceived, or assays performed 
by other CTOT members for cross-validation studies.  Appropriate informed consent will be obtained for both the 
collection and storing of samples.  During the funding period, samples will be identifiable, which means samples will be 
coded with a subject ID number that could be directly linked to the subject and the subject’s medical record.  The 
specimens from these evaluations may be stored beyond the funding period. Samples will be identifiable, which means 
samples will be coded with a subject ID number that could be directly linked to the subject and the subject’s medical 
record.  When the funding period is over, data from the clinical database will be de-identified, meaning patient 
identifiers will be removed, and all associated date information will be transformed to study days.  Core laboratories 
and/or repositories maintaining specimens will not have the ability to identify a subject or access data associated with a 
specimen without specific approval from the study team.    
   


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 Criteria for Participant Completion and Premature Study Termination 
 
Participant Stopping Rules and Withdrawal Criteria 
Participants may be prematurely terminated from the study for the following reasons: 
1. The participant elects to withdraw consent from all future study activities, including follow-up. 
2. The participant is “lost to follow-up” 3 months after the date of a missed study visit (i.e., no further follow-up is 
possible because attempts to reestablish contact with the participant have failed).  
3. The participant dies.  
4. The Investigator no longer believes participation is in the best interest of the participant. 
5. CTCAE Grade 3 or higher infusion reaction. 
6. Failure to manufacture and supply the cellular product two times for the same subject.   
 
Participant Replacement 
Participants who are prematurely terminated from this study following infusion of at least 100 x 106 Tregs will not be 
replaced.  Subjects who are prematurely terminated prior to polyTreg infusion will be replaced. 
 
Follow-up after Early Study Withdrawal 
If a participant in Group 2 is withdrawn from the study for any reason, the participant will be asked to complete a final 
visit for safety assessment at 52 weeks after polyTregs (Day 364/Visit 6 on Appendix 3). 
 
Study Stopping Rules 
The study may be prematurely terminated for the following reasons: 
1. Any CTCAE grade 4 or higher infusion reaction  
2. Any study defined grade 4 or higher infection  
3. Any diagnosis of malignancy and post-transplant lymphoproliferative disease (except non-melanoma skin cancer) 
4. Any graft loss  
5. Acute Rejection (Banff grade 2A or higher) or acute antibody mediated rejection within 6 weeks after Treg infusion 
in 3 of first 5 subjects 
6. Any death  
7. Inability to manufacture and supply polyTregs in 3 of 5 consecutive subjects assigned to Group 2 
8. If there is a total of 8 failed lots of polyTregs at any time during the study. 
Any of these events will stop trial enrollment and Treg infusions and require DSMB review (See Section 14.8).  Subjects 
who have already received Treg infusion are eligible for mTOR therapy will remain on current therapy during the data 
review.     


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 Safety Monitoring and Reporting 
 
Overview 
This section defines the types of safety data that will be collected under this protocol and outlines the procedures for 
appropriately collecting, grading, recording, and reporting those data. Adverse events that are classified as serious 
according to the definition of health authorities must be reported promptly to the sponsor [DAIT/NIAID].  Appropriate 
notifications will also be made to site principal investigators, Institutional Review Boards (IRBs), and health authorities.  
Information in this section complies with ICH Guideline E2A: Clinical Safety Data Management: Definitions and Standards 
for Expedited Reporting, ICH Guideline E-6: Guideline for Good Clinical Practice, 21CFR Parts 312 and 320, and applies 
the standards set forth in the National Cancer Institute (NCI), Common Terminology Criteria for Adverse Events (CTCAE), 
Version 4.0: http://ctep.cancer.gov/reporting/ctc.html. 
 
Definitions 
 
Adverse Event (AE) 
Any untoward or unfavorable medical occurrence associated with the subject’s participation in the research, whether or 
not considered related to the subject’s participation in the research (modified from the definition of adverse events in 
the 1996 International Conference on Harmonization E-6 Guidelines for Good Clinical Practice) (from OHRP "Guidance 
on Reviewing and Reporting Unanticipated Problems Involving Risks to Subjects or Others and Adverse Events 
(1/15/07)" http://www.hhs.gov/ohrp/policy/advevntguid.html#Q2 )  
 
Suspected Adverse Reaction (SAR)  
Any adverse event for which there is a reasonable possibility that the investigational drug [or investigational study 
therapy regimen] caused the adverse event. For the purposes of safety reporting, ‘reasonable possibility’ means there is 
evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies 
a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug (21 
CFR 312.32(a)). 
 
Unexpected Adverse Event  
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the Investigator Brochure 
or package insert or is not listed at the specificity, severity or rate of occurrence that has been observed. 
“Unexpected” also refers to adverse events or suspected adverse reactions that are mentioned in the Investigator 
Brochure or package insert as occurring with a class of drugs or as anticipated from the pharmacological properties of 
the drug, but are not specifically mentioned as occurring with the particular drug under investigation (21 CFR 312.32(a)] 
 
Serious Adverse Event (SAE) 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either the investigator or 
Sponsor, it results in any of the following outcomes (21 CFR 312.32(a)): 
1. Death. 
2. A life-threatening event: An AE or SAR is considered “life-threatening” if, in the view of either the 
investigator or Sponsor [add DAIT/NIAID or other Sponsor, if applicable], its occurrence places the 
subject at immediate risk of death. It does not include an AE or SAR that, had it occurred in a more 
severe form, might have caused death.  
3. Inpatient hospitalization or prolongation of existing hospitalization. 


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4. Persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
5. Congenital anomaly or birth defect.  
6. Important medical events that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may jeopardize 
the subject and may require medical or surgical intervention to prevent one of the outcomes listed 
above. 
Elective hospitalizations or hospital admissions for the purpose of conduct of protocol mandated procedures are not to 
be reported as an SAE unless hospitalization is prolonged due to complications. 
 Other Significant Adverse Events 
The events below should be reported (entered on the AE/SAE form) within 24 hours of awareness even if the event does 
not meet serious criteria: 
• 
Biopsy proven or clinical (Treated) AR 
• 
Chronic rejection 
• 
Infusion Reactions (CTCAE grade 2 or higher) 
• 
Malignancy, PTLD 
• 
Infections (study defined grade 3 or higher) 
• 
COVID-19 infections of any grade  
If no other serious criteria are met for COVID-19 infection, the event should be considered of medical importance. 
 
Grading and Attribution of Adverse Events 
 
Grading Criteria 
The study site will grade the severity of adverse events experienced by the study subjects according to the criteria set 
forth in the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.    
This document (referred to herein as the NCI-CTCAE manual) provides a common language to describe levels of severity, 
to analyze and interpret data, and to articulate the clinical significance of all adverse events. The NCI-CTCAE has been 
reviewed by the principal investigator and protocol chair and has been deemed appropriate for the subject population 
to be studied in this protocol.  
Infections will be graded using the study-specific scale as described below: 
Grade 1 = asymptomatic; clinical or diagnostic observation only; intervention with oral antibiotic, antifungal, or 
antiviral agent only; no invasive intervention required 
Grade 2 = symptomatic; intervention with intravenous antibiotic, antifungal, or antiviral agent; invasive 
intervention may be required  
Grade 3 = any infection associated with hemodynamic compromise requiring pressors; any infection necessitating 
ICU level of care; any infection necessitating operative intervention; any infection involving the central nervous 
system; any infection with a positive fungal blood culture; any proven or probable aspergillus infection; any tissue 
invasive fungal infection; any pneumocystis jiroveci infection 
Grade 4 = life-threatening infection 
Grade 5 = death resulting from infection 


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All other adverse events will be graded on a scale from 1 to 5 according to the following standards in the NCI-CTCAE 
manual: 
Grade 1 = mild adverse event. 
Grade 2 = moderate adverse event. 
Grade 3 = severe and undesirable adverse event. 
Grade 4 = life-threatening or disabling adverse event. 
Grade 5 = death. 
Infection events grade 2 or higher; and all other events CTCAE grade 2 or higher will be recorded on the appropriate AE 
eCRF for this study. 
For grading an abnormal value or result of a clinical or laboratory evaluation (including, but not limited to, a radiograph, 
an ultrasound, an electrocardiogram etc.), a treatment-emergent adverse event is defined as an increase in grade from 
baseline or from the last post-baseline value that doesn’t meet grading criteria. Changes in grade from screening to 
baseline will also be recorded as adverse events but are not treatment-emergent. If a specific event or result from a 
given clinical or laboratory evaluation is not included in the NCI-CTCAE manual, then an abnormal result would be 
considered an adverse event if changes in therapy or monitoring are implemented as a result of the event/result. 
 
Attribution Definitions 
The relationship, or attribution, of an adverse event to the study therapy regimen or study procedure(s) will initially be 
determined by the site investigator and recorded on the appropriate AE/SAE eCRF.  Final determination of attribution 
for safety reporting will be determined by DAIT/NIAID.  The relationship of an adverse event to study therapy regimen or 
procedures will be determined using the descriptors and definitions provided in Table 6.  
For additional information and a printable version of the NCI-CTCAE manual, consult the NCI-CTCAE web site: 
http://ctep.cancer.gov/reporting/ctc.html.        
Table 6.  Attribution of Adverse Events 
Code 
Descriptor 
Relationship (to primary investigational product and/or other 
concurrent mandated study therapy or study procedure) 
Unrelated Category 
1 
Unrelated 
The adverse event is clearly not related:  there is insufficient 
evidence to suggest a causal relationship. 
 Related Categories 
2 
Possible 
The adverse event has a reasonable possibility to be related; 
there is evidence to suggest a causal relationship. 
3 
Related 
The adverse event is clearly related. 
Attribution of adverse event to tacrolimus, MMF/MPA, and prednisone will not be assessed in this study because these 
medications are used as standard of care for kidney transplant recipients. 
Attribution assessment for the following study interventions and procedures will be made when a SAE is reported: 
 


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Study therapy regimen:  
1. polyTregs 
Study mandated procedures:  
1. Leukapheresis 
2. Kidney biopsy  
3. Blood Draw (Donor or Recipient) 
4. IS regimen conversion / Everolimus 
 
Collection and Recording of Adverse Events 
 
Collection Period 
Adverse events will be collected from the time of first study mandated procedure until a subject completes study 
participation or until 30 days after he/she prematurely withdraws (without withdrawing consent) or is withdrawn from 
the study. 
Note that the study eligibility is performed as a standard of care surveillance biopsy with no collection of research 
specimens.  The eligibility biopsy is not considered a study mandated procedure for these reasons.  
 
Collecting Adverse Events 
Adverse events (including SAEs) may be discovered through any of these methods: 
• Observing the subject. 
• Interviewing the subject [e.g., using a checklist, structured questioning, diary, etc.]  . 
• Receiving an unsolicited complaint from the subject. 
• In addition, an abnormal value or result from a clinical or laboratory evaluation can also indicate an 
adverse event, as defined in Section 14.2.4, Grading and Attribution of Adverse Events. 
 
Recording Adverse Events 
Throughout the study, the investigator will record adverse events and serious adverse events as described previously 
(Section 14.2, Definitions) on the appropriate AE/SAE eCRF regardless of the relationship to study therapy regimen or 
study procedure.   
Once recorded, an AE/SAE will be followed until it resolves with or without sequelae, or until the end of study 
participation, or until 30 days after the subject prematurely withdraws (without withdrawing consent)/or is withdrawn 
from the study, whichever occurs first. 
 
Reporting of Serious Adverse Events and Adverse Events 
 
Reporting of Serious Adverse Events to Sponsor  
This section describes the responsibilities of the site investigator to report serious adverse events to the sponsor via the 
AE/SAE eCRF.  Timely reporting of adverse events is required by 21 CFR and ICH E6 guidelines.  
Site investigators will report all serious adverse events (see Section 14.2.3, Serious Adverse Event), regardless of 
relationship or expectedness within 24 hours of discovering the event. 


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For serious adverse events, all requested information on the AE/SAE eCRF will be provided.  However, unavailable 
details of the event will not delay submission of the known information.  As additional details become available, the 
AE/SAE eCRF will be updated and submitted. 
14.5.2. Reporting to Health Authority 
After an adverse event requiring 24-hour reporting (per Section 14.5.1, Reporting of Serious Adverse Events to Sponsor) 
is submitted by the site investigator and assessed by DAIT/NIAID, there are two options to report the adverse event to 
the appropriate health authorities: 
14.5.2.1    Annual Reporting 
DAIT/NIAID will include in the annual study report to health authorities all adverse events classified as: 
• 
Serious, expected, suspected adverse reactions (see Section 14.2.1.1, Suspected Adverse Reaction, and Section 
14.2.2, Unexpected Adverse Event). 
• 
Serious and not a suspected adverse reaction (see Section 14.2.1.1, Suspected Adverse Reaction). 
• 
Pregnancies. 
Note that all adverse events (not just those requiring 24-hour reporting) will be reported in the Annual IND Report. 
14.5.2.2 
Expedited Safety Reporting  
DAIT/NIAID shall notify the FDA and all participating investigators of expedited Safety Reports within 15 calendar days; 
unexpected fatal or immediately life-threatening suspected adverse reaction(s) shall be reported as soon as possible or 
within 7 calendar days.  
This option, with 2 possible categories, applies if the adverse event is classified as one of the following: 
Category 1:  Serious and unexpected suspected adverse reaction [SUSAR] (see Section 14.2.1.1, Suspected Adverse 
Reaction and Section 14.2.2, Unexpected Adverse Event and 21 CFR 312.32(c)(1)i).  
The sponsor shall report any suspected adverse reaction that is both serious and unexpected.  The sponsor shall report 
an adverse event as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the 
study drug and the adverse event, such as: 
1. A single occurrence of an event that is uncommon and known to be strongly associated with drug 
exposure (e.g., angioedema, hepatic injury, or Stevens-Johnson Syndrome); 
2. One or more occurrences of an event that is not commonly associated with drug exposure, but is 
otherwise uncommon in the population exposed to the drug (e.g., tendon rupture); 
3. An aggregate analysis of specific events observed in a clinical trial (such as known consequences of the 
underlying disease or condition under investigation or other events that commonly occur in the study 
population independent of drug therapy) that indicates those events occur more frequently in the 
drug treatment group than in a concurrent or historical control group. 
 
Category 2: Any findings from studies that suggests a significant human risk  
The sponsor shall report any findings from other epidemiological studies, analyses of adverse events within the current 
study or pooled analysis across clinical studies or animal or in vitro testing (e.g., mutagenicity, teratogenicity, 
carcinogenicity) that suggest a significant risk in humans exposed to the drug that would result in a safety-related 
change in the protocol, informed consent, investigator brochure or package insert or other aspects of the overall 
conduct of the study.   


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Reporting of Adverse Events to IRBs/IECs 
All investigators shall report adverse events, including expedited reports, in a timely fashion to their respective IRBs/IECs 
in accordance with applicable regulations and guidelines. All Safety Reports to the FDA shall be distributed by 
DAIT/NIAID or designee to all participating institutions for site IRB/IEC submission. 
 
Pregnancy Reporting 
The investigator shall be informed immediately of any pregnancy in a study subject or a partner of a study subject.  A 
pregnant subject shall not receive Tregs, and will be instructed to stop taking everolimus, if applicable.  The investigator 
shall counsel the subject and discuss the risks of continuing with the pregnancy and the possible effects on the fetus.  
Monitoring of the pregnant subject shall continue until the conclusion of the pregnancy.    
The investigator shall report to the Statistical and Clinical Coordinating Center (SACCC) all pregnancies within 1 business 
day of becoming aware of the event using the Pregnancy eCRF.  All pregnancies identified during the study shall be 
followed to conclusion and the outcome of each must be reported.  The Pregnancy eCRF shall be updated and submitted 
to the DAIT/NIAID via the SACCC when details about the outcome are available.  When possible, similar information shall 
be obtained for a pregnancy occurring in a partner of a study subject. 
Information requested about the delivery shall include: 
1. Gestational age at delivery 
2. Birth weight, length, and head circumference 
3. Gender 
4. Appearance, pulse, grimace, activity, and respiration (APGAR) score at 1 minute, 5 minutes, and 24 
hours after birth, if available 
5. Any abnormalities. 
All pregnancy complications that result in a congenital abnormality, birth defect, miscarriage, and medically indicated 
abortion - an SAE shall be submitted to the DAIT/NIAID via the SACCC using the SAE reporting procedures described 
above.  Pregnancies reported as SAE’s will be reported to the FDA as described above. 
 
Reporting of Other Safety Information 
An investigator shall promptly notify the site IRB as well as the SACCC using the AE/SAE eCRF when an “unanticipated 
problem involving risks to subjects or others” is identified, which is not otherwise reportable as an adverse event. 
 
Review of Safety Information 
 
Medical Monitor Review 
The PI, Protocol Chair, and NIAID Medical Monitor will review safety data on an ongoing basis.  Enrollment and initiation 
of study treatment may be suspended at any time if these reviews conclude that there are significant safety concerns.  
In addition, the Medical Monitor shall review and make decisions on the disposition of the SAE and pregnancy reports 
received by the SACCC in a timely manner.  
 
Planned DSMB Reviews  


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The Data and Safety Monitoring Board (DSMB) shall review safety data at least yearly during planned DSMB Data Review 
Meetings. Data for the planned safety reviews will include, at a minimum, a listing of all reported AEs and SAEs.   
The DSMB will be informed of an Expedited Safety Report in a timely manner.   
 
Ad hoc DSMB Reviews  
In addition to the pre-scheduled data reviews and planned safety monitoring, the DSMB may be called upon for ad hoc 
reviews. The DSMB will  review any event that potentially impacts safety at the request of the PI, protocol chair or 
DAIT/NIAID.  After review of the data, the DSMB will make recommendations regarding study conduct and/or 
continuation. 


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 Statistical Considerations and Analytical Plan 
 
Overview  
CTOT-21 is a Phase 2 open label study, in which 14 adult kidney transplant recipients will be allocated to one of 2 
treatment arms: CNI-only, or polyTregs.  Subjects allocated to the Treg treatment arm will receive a single infusion of 
polyTregs approximately 7 months after transplant.  The primary safety objective is to evaluate the safety of polyTregs in 
adult kidney transplant recipients.  The primary efficacy objective of the study is to evaluate whether polyTregs reduce 
graft inflammation when compared to CNI-based maintenance therapy.   
 
Measures to Minimize Bias  
The study groups will be assigned in an un-blinded fashion using a variation of the Pocock and Simon adaptive 
randomization algorithm designed to maintain an overall 1:1 ratio.  Centralized laboratories will be used to minimize 
bias and mechanistic core laboratory personnel, including the core pathologist, will be blinded to study treatment 
assignments.  Clinical sites will not be blinded to treatment assignments and reports for site use and monitoring 
purposes will not be blinded. 
 
Analysis Plan 
Statistical analyses of the safety and clinical outcomes will be performed for the analysis samples defined below in 
section 15.3.1, employing standard methods for the estimation of incidence rates and their exact two-sided 95% 
confidence intervals. Statistical analyses of most mechanistic outcomes will be exploratory in nature. The subject 
enrolled and treated in the darTreg arm, prior to the removal of that treatment arm in protocol version 9.0, will be 
individually summarized, including but not limited to adverse events, infusion details, and inflammation scores and 
changes.  The plans for statistical analyses of study data will be described in more detail in a Statistical Analysis Plan 
(SAP). 
 
Analysis Samples 
The statistical analyses will be performed on the following subject samples: 
1. The Modified Intent-to-Treat Sample 1 (mITT1) consists of all subjects allocated to the CNI-based 
treatment regimen or received polyTregs while on study. 
2. The Per Protocol Sample (PP) consists of all subjects allocated to the CNI-based treatment regimen or 
received at least 300 x 106 of polyTregs while on study that did not have major protocol deviations of 
concern. 
3. The modified intent-to-Treat Sample 2 (mITT2) consists of all subjects who received polyTregs (Group 2). 
 
Endpoint Assessments 
15.3.2.1 Primary and Secondary Safety Endpoints 
Table 7.  Analyses of Safety Endpoints describes the safety endpoints and their corresponding parameters to be 
estimated in the study.  Safety endpoints will be listed or summarized, as appropriate, using standard descriptive 
statistics for continuous and categorical data. 
 
 


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Table 7.  Analyses of Safety Endpoints 
ENDPOINT 
DESCRIPTIVE PARAMETER 
ANALYSIS 
POPULATION 
Primary Endpoint  
 
Incidence of severe acute rejection or antibody mediated 
rejection in subjects receiving polyTregs in comparison with CNI-
based maintenance IS therapy. 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Incidence of severe infection in subjects receiving polyTregs in 
comparison with CNI-based maintenance IS therapy. 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Secondary Endpoints – Treg safety 
 
Incidence of polyTregs infusion reactions (episodes of fever, 
bronchospasm, hypoxia, cytokine release syndrome, or infusion 
site reaction) 
Proportion with exact 
binomial 95% confidence 
limits  
mITT2 
Severity of polyTregs infusion reactions 
Descriptive CTCAE categories 
mITT2 
Incidence of culture-proven and clinically diagnosed infections  
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Severity of culture-proven and clinically diagnosed infections  
Descriptive CTCAE categories  
mITT2 
Incidence of severe infection in subjects receiving polyTregs in 
comparison with CNI-based maintenance IS therapy. 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Timing of acute rejection 
Mean time to rejection and 
standard deviation  
mITT1 
Incidence of acute rejection 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Severity of acute rejection 
Descriptive Banff criteria 
mITT1 
Incidence of CMV reactivation 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Incidence of BK viremia  
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Incidence of >10% decrease in eGFR from baseline 
Proportion with exact 
binomial 95% confidence 
limits 
mITT1 
Secondary Endpoints – mTOR therapy 
Incidence of acute rejection 
Proportion with exact 
binomial 95% confidence 
limits 
mITT2 (Treg 
mTOR vs Treg 
no mTOR) 
15.3.2.2 Supportive Analyses of the Primary Safety Endpoints  
Although the study is not powered to detect clinically significant differences in safety event incidence between 
treatment groups, in a supportive analysis, the incidence of the composite endpoint will be compared between the two 
treatment groups by an overall Fisher’s exact test using the mITT1 analysis sample to compare the incidence between 
the two groups.  


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15.3.2.3 Primary Efficacy Endpoint 
The primary efficacy endpoint is the mean change from baseline in percent inflammation on biopsy at 7 months and it 
will be compared between the two treatment groups by ANOVA using the mITT1 analysis sample.  The primary endpoint 
data will be summarized in the 2 groups using boxplots, along with means and 95% confidence intervals. 
15.3.2.4 Secondary Efficacy Endpoints 
Table 8 describes secondary efficacy endpoints and their corresponding parameters to be estimated in the study.  
Efficacy endpoints will be listed or summarized, as appropriate, using standard descriptive statistics for continuous and 
categorical data, including confidence intervals.  All secondary endpoints will be descriptive in nature due to the small 
sample size. 
Table 8.  Analyses of Secondary Efficacy Endpoints 
SECONDARY ENDPOINT 
DESCRIPTIVE PARAMETER 
ANALYSIS SAMPLE* 
Number of subjects who exhibit a relative decrease of 25% 
or more inflammation on kidney biopsy 7 months after 
study group allocation compared to baseline 
Proportion with exact binomial 
95% confidence limits 
mITT1  
Number of subjects who exhibit a relative decrease of 25% 
or more inflammation on kidney biopsy 2 weeks after Treg 
infusion compared to baseline 
Proportion with exact binomial 
95% confidence limits 
mITT2 
Number of subjects who exhibit a relative decrease of 50% 
or more inflammation on kidney biopsy 2 weeks after Treg 
infusion compared to baseline 
Proportion with exact binomial 
95% confidence limits 
mITT2 
15.3.2.5 Supportive Analyses of the Primary Efficacy Endpoints 
In addition to the primary efficacy endpoint analysis described above, several supportive analyses will be performed: 
1. Since the occurrence of treated rejection prior to or at the time of the 7 month biopsy may confound 
the measurement of inflammation, the ANOVA described above will be repeated deleting any subjects 
from the analysis who had treated rejection prior to the 7 month biopsy. 
2. Since clinical site, donor type, and race may confound the interpretation of the primary analysis, 
ANCOVA will be used to separately test for the effect of site, donor type, and race on the primary 
endpoint analysis. 
3. If the PP sample differs from the mITT1 sample, the primary endpoint analysis will be repeated on the 
PP analysis sample. 
 
 


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15.3.2.6 Mechanistic Endpoints 
Three of the mechanistic assays, CRM, SORT, and DEU, which have high level of sensitivity and specificity, will be used to 
evaluate mechanistic efficacy endpoints (Table 9).   
Table 9.  Analysis of mechanistic endpoints 
Endpoints 
Assays 
Measurements 
Pharmacokinetics of infused 
Tregs in blood and graft 
DEU¶ 
% deuterium enrichment in blood and graft 
Rejection gene in blood 
SORT¶  
Pearson’s Correlation Coefficient based Z score; z = χ - µglobal / σglobal 
Inflammatory gene in graft 
CRM¶ 
Geometric mean score of the expression of 11 of the 12 CRM genes in each 
sample 
¶ DEU, SORT and CRM assays are for evaluation of efficacy endpoints 
15.3.2.7 Analyses of Exploratory Endpoints 
Planned mechanistic assays other than CRM, SORT, and DEU are for exploratory assessment of the impact of specific 
therapies on inflammation and borderline rejection. Any statistically significant results from these tests will be reported 
as requiring independent verification and will provide a basis for further studies.   
Treatment groups and comparators: We will analyze the readouts on assays longitudinally. Patient measurements will 
be obtained before and after treatment. There will be at least 2 time points recorded for the TASK trial (before and after 
Treg infusion). The primary interest of analysis is to compare longitudinal changes in assays between different groups: 
SCI with SOC, and SCI with polyTregs. Two groups of patients, those with normal histology and those with acute 
rejection on the 6-month surveillance biopsy, will serve as negative and positive controls respectively for the 
mechanistic studies. We expect the changes over time in these assays to be approximately linear. If after examination of 
the data appears to not be the case then we will either transform the assay measures or model the time axis time points 
as an unordered factor (similar to repeated measures ANOVA, but without the requirement for fully balanced data).  
Table 10.  Analysis of exploratory mechanistic endpoints 
Endpoints 
Assays 
Measurements 
Total Tregs in blood and graft 
MFC 
% and absolute numbers of Tregs  
mIHC 
% FOXP3+ among CD3+ cells  
Infused Tregs in blood and 
graft 
TCR* 
# and frequency of clones from Treg product 
Cellular alloimmune profile 
MFC  
% and absolute numbers of various cell subsets 
 
 
SUPP 
% suppression at 1 Treg:4 PBMC ratio 
Cellular viral immune profile 
VIR 
Frequencies of CMV and EBV reactive CD8 T cells; % polyfunctional anti-viral T 
cells 
Antibody responses 
HLA  
DSA+ or DSA-; MFI if DSA+ 
nHLA 
Concentration of antibodies to 6 nHLA antigens 
Immune tolerance gene in 
blood 
SPOT 
Penalized Logistic Regression score for 3 genes 
Inflammation in graft 
HIS 
% inflammatory cell load by area; % area with interstitial fibrosis 
IL6 signaling in the graft 
mIF 
ISH§ 
+cells /high power field; number of ISH dots/high power field 
Graft fibrosis 
FIB  
Concentration of protein associated with tubular injury; concentration of pro-
fibrotic proteins 
uFIB 
Concentration of protein associated with fibrosis 
Inflammation markers in urine 
uPRO 
Concentration of proteins associated with kidney injury and inflammation in urine 
uRNA 
Absolute copy number of mRNA for genes associated with acute rejection 
 
Descriptive Analyses  


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We will use descriptive analyses to summarize subject characteristics of our study populations across treatment groups. 
Dichotomous variables will be summarized as proportions with 95% confidence intervals. Continuous variables will be 
summarized using means, standard deviations, and 95% confidence intervals if they are symmetric and unimodal. 
Otherwise, they will be summarized using the median and the interquartile range. Simple t-, chi-squared, or Fisher's 
exact test, as appropriate, will be used to compare quantitative measures across treatment groups.  The following 
variables will be summarized:  
1. baseline and demographic characteristics 
2. use of concomitant medications 
3. reasons for early termination 
4. all reported AEs  
 
Interim Analyses  
No formal interim analyses of this study are planned. 
 
Sample Size Considerations  
No formal sample size calculations will be performed on the primary safety endpoint as it is descriptive in nature.  
However, sample sizes calculations were done based on the primary efficacy endpoint.   There is no previous data to 
guide the estimation of treatment effect size and variance for the assessment of clinical efficacy. For the primary efficacy 
endpoint analysis, which is the mean reduction in inflammation from baseline at 7 months after study group allocation 
in the mITT1 analysis sample compared between the 2 groups, we have used a two sample 2-sided t-test to determine 
the estimated power for differences in mean reduction between the treatment groups (ranging from 10-50% reduction 
in inflammation) at different standard deviations (SD) of the mean change in inflammation (ranging from10-30%).  These 
power estimates are shown in the following Figure 9 and 
Table 10.  Analysis of exploratory mechanistic endpoints for sample size of 7 in each group and an alpha level of 0.05 and 
2-tailed tests.   
Figure 9.  Revised Sample Size Calculations 


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Table 11. Power estimates by mean difference in percent reduction in inflammation 
Mean difference in 
percent reduction in 
inflammation 
Power estimates for different standard deviations 
SD 10 
SD 15 
SD 20 
SD 25 
SD 30 
10% 
40.6% 
21.0% 
X 
X 
X 
20% 
92.9% 
63.0% 
40.6% 
28.1% 
21.0% 
30% 
100% 
92.9% 
73.1% 
54.1% 
40.6% 
40% 
100% 
99.5% 
92.9% 
78.5% 
63.0% 
50% 
100% 
100% 
99.0% 
92.9% 
81.6% 
 
With group sizes of 7, we would achieve at least 90% power in the primary efficacy endpoint analysis to detect a 30% 
difference between the groups in the percent reduction in inflammation if the standard deviation is 15 or less.  Due to 
the relatively small sample size, all secondary endpoint analyses will be performed as descriptive analyses rather than 
statistical comparisons between groups. 
 
 
Power vs M2 by S1
M1=0.0 S2=30.0 Alpha=0.05 N1=7 N2=N1 2-Sided T test
M2
Power
10
20
30
40
50
0.0
0.2
0.4
0.6
0.8
1.0
S1
10.0
15.0
20.0
25.0
30.0


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 Identification and Access to Source Data 
 
Source Data 
Source documents and source data are considered to be the original documentation where subject information, visits 
consultations, examinations and other information are recorded.   Documentation of source data is necessary for the 
reconstruction, evaluation and validation of clinical findings, observations and other activities during a clinical trial.  
 
Access to Source Data 
The site investigators and site staff will make all source data available to the DAIT/NIAID, as well as to relevant health 
authorities.  Authorized representatives as noted above are bound to maintain the strict confidentiality of medical and 
research information that may be linked to identified individuals. 


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 Protocol Deviations 
 
Protocol Deviation Definitions 
Protocol Deviation – The investigators and site staff will conduct the study in accordance to the protocol; no deviations 
from the protocol are permitted.  Any change, divergence, or departure from the study design or procedures constitutes 
a protocol deviation.  As a result of any deviation, corrective actions will be developed by the site and implemented 
promptly. 
Major Protocol Deviation - A Protocol Deviation is a variance from the IRB approved protocol that may affect the 
subject's rights, safety, or well-being and/or the completeness, accuracy and reliability of the study data.  In addition, 
protocol deviations include willful or knowing breaches of human subject protection regulations, or policies, any action 
that is inconsistent with the NIH Human Research Protection Program’s research, medical, and ethical principles, and a 
serious or continuing noncompliance with federal, state, local or institutional human subject protection regulations, 
policies, or procedures.  
Non-Major Protocol Deviation - A non-major protocol deviation is any change, divergence, or departure from the study 
design or procedures of a research protocol that does not have a major impact on the subject's rights, safety or well-
being, or the completeness, accuracy and reliability of the study data. 
 
Reporting and Managing Protocol Deviations 
The study site principal investigator has the responsibility to identify, document and report protocol deviations as 
directed by the study Sponsor.  However, protocol deviations may also be identified during site monitoring visits or 
during other forms of study conduct review.  
Upon determination that a protocol deviation has occurred, the study staff will complete a Protocol Deviation form. 
Protocol deviation reports will be compiled and reviewed by the NIAID/DAIT and the DSMB.  Sites will be responsible for 
reporting deviations to local IRB’s, as per local requirements.   


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 Ethical Considerations and Compliance with Good Clinical Practice 
 
Statement of Compliance 
This clinical study will be conducted using good clinical practice (GCP), as delineated in Guidance for Industry: E6 Good 
Clinical Practice Consolidated Guidance, and according to the criteria specified in this study protocol.  Before study 
initiation, the protocol and the informed consent documents will be reviewed and approved by each site IRB.  Any 
amendments to the protocol or to the consent materials will also be approved by the respective IRB before they are 
implemented. 
 
Informed Consent Process 
The consent process will provide information about the study to a prospective participant and will allow adequate time 
for review and discussion prior to his/her decision.  The principal investigator or designee listed on the FDA 1572 will 
review the consent and answer questions.  The prospective participant will be told that being in the trial is voluntary and 
that he or she may withdraw from the study at any time, for any reason.  All participants (or their legally acceptable 
representative) will read, sign, and date a consent form before undergoing any study procedures.  Consent materials will 
be presented in participants’ primary language. A copy of the signed consent form will be given to the participant. 
The consent process will be ongoing.  The consent form will be revised when important new safety information is 
available, the protocol is amended, and/or new information becomes available that may affect participation in the 
study.  
 
Privacy and Confidentiality 
A participant’s privacy and confidentiality will be respected throughout the study.  Each participant will be assigned a 
unique identification number and these numbers rather than names will be used to collect, store, and report participant 
information.  Site personnel will not transmit documents containing personal health identifiers (PHI) to the study 
sponsor or their representatives. 


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 Publication Policy 
The CTOT policy on the publication of study results will apply to this trial. 


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Appendix 1.  Donor Assessments  
 
  
Screening 
  
Visit Number 
D1 
Study Assessments (All Donors) 
Recipient Eligibility and Consent 
x 
Demographics (Age at Donation, Gender, Ethnicity) 
x 
Donor HLA Typing (retrospective chart review) 
x 
EBV and CMV IgG1 
x 
HBV cAb, HBV sAg, HBV sAb quantitative1 
x 
Limited Medical History  
x 
1 HLA Typing and viral serologies performed as part of evaluation for donation can be collected retrospectively.   
 


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Appendix 2.  Group 1 Recipient Schedule of Events 
 
Study 
Eligibility
Randomization
Day 
41
Day 
48 
Day 
55
Day 
69
Day 
125
Day 
223
Day 
405
Clinically 
Indicated 
Biopsy
Visit Number
Screen 1
Day 0
T0-G1
1
2
3
4
5
6
CIB
Visit Window
± 2 w eeks of 
6 month 
biopsy Ɨ
≤ 2 w eeks after 
Qualifying Biopsy
± 3 
days
- 14 days prior 
to last study visit
Study Eligibility Ɨ
x
Informed Consent
x
Randomization
x
Demographics, Transplant History
x
Medical History
→
→
→
→
→
→
→
→
→
Physical Examination/ Vital Signs
x
x
x
x
x
x
x
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
Recipient HLA Typing1 
x
Donor Specific Anti-HLA Antibodies Class 1 and 21
x
x
x
x
CMV, EBV, HIV1, HIV2, HBV (HBsAg, HBcAb), HCV (HCVAb) serology1
x
CMV, EBV by PCR
x
x
x
x
x
x
x
BKV by PCR in serum 
x
x
x
x
HCV PCR (for subjects with positive HCVAb)
x
SARS-COV2 RT-PCR
x4
CBC (with differential and platelets) 
x
x
x
x
x
x
x
x
x
CD4 Count
x
x
x
x
x
x
x
PT/INR
x
x2
x
Basic Chemistry (Na, K, Cl, CO2, BUN, Glucose, Creatinine)
x
x
x
x
x
x
x
x
x
Liver Tests (ALT, AST, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
Tacrolimus Level
x
x
x
x
x
x
x
x
x3
Fasting Lipid Panel
x
x
x
x
Urine Protein and Creatinine Ratio
x
x
x
x
x
x
x
x
Urine Pregnancy Test
x
Local Pathology Results - Graft Histology (Standard of Care Biopsy)
x
x
x
TruCount3 (UCSFa)
x
MFC Panels (Percentage Tregs/ Leukocyte phenotypes in Blood) - PBMC (UCSFb)
x
x
x
x
x
x
kSORT and kSPOT (Gene Expression in Blood) - Paxgene Tube (UCSFb)
x
x
x
x
x
x
non- HLA Alloantibodies in Blood - 5 ml Red Top Tube (UCSFb)
Fibrosis Biomarkers in Serum - Serum from Red Top Tube (UAB)
mIF/ISH (Intragraft Cytokine Expression) - 1/2 16g Core Formalin Fixed Paraffin-Embedded (UCSFc)
x
x
x
CRM (Transcriptional Profiling of Graft) - performed with FFPE tissue (UCSFc)
x2
x
x
Pathology Slides (biopsy case) for Central Pathology Reading (UCSFc)
x
x
x
Protein Biomarkers and RNA in Urine (Shared 100ml Urine Collection (Pellet & Supernatant to UCSFb)
Fibrosis Biomarkers in Urine (Shared 100ml Urine Collection (Supernatant to UAB)
2 PT/INR should be collected as part of standard of care assessment prior to biopsy.  
x
x
x
± 5 days
± 14 days
x
Central Laboratory Assessments
General Study Assessments
Blood Specimens
Renal Biopsy Specimens
Urine Specimens
x
Local Laboratory Assessments
x
1 HLA Typing, DSA, and viral serologies performed as part of evaluation for transplantation can be collected retrospectively.  If not available, serologic testing should be done within 2 weeks of screening biopsy.  
EBV, should be repeated within screening window if previously negative.
4 SARS-COV2 testing does not have to be repeated if negative at time of biopsy. 
3 TruCount will only be performed at the screening visit.
Ɨ Screening biopsy date must be 5 months after transplant ± 8 weeks.  Except as marked, all screening labs should be dated ± 2 weeks of biopsy date, keeping in mind lab results must be available within 
window for randomization.
x
x
x
x
x
x
x
x


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Appendix 3.  Group 2 Recipient Schedule of Events  
 
Visit Label/ Days after Treg Infusion
Visit Number
Screen 1
T0
T1
1
2
3
4
5
6
CIB
Visit Window
± 2 w eeks of 6 
month biopsy Ɨ
≤ 2 w eeks after 
Qualifying Biopsy
± 1 
hr
± 1 
day
± 3 
days
- 14 days prior 
to last study visit
Study Eligibility Ɨ, Treg Eligibility, mTOR Eligibility
x
x
x
Informed Consent
x
Randomization
x
Demographics, Transplant History
x
Medical History
→
→
→
→
→
→
→
→
→
→
→
Physical Examination/ Vital Signs
x
x
x
x
x
x
x
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
Recipient HLA Typing1 
x
Donor Specific Anti-HLA Antibodies Class 1 and 21
x
x
x
x
CMV, EBV, HIV1, HIV2, HBV (HBsAg, HBcAb), HCV (HCVAb) serology1
x
CMV, EBV by PCR
x
x
x
x
x
x
x
BKV by PCR in serum 
x
x
x
x
HCV PCR (for subjects with positive HCVAb)
x
SARS-COV2 RT-PCR
x6
x6
CBC (with differential and platelets) 
x5
x
x
x
x
x
x
x
x
x
CD4 Count
x
x
x
x
x
x
x
x
x
PT/INR
x
x
x
x
Basic Chemistry (Na, K, Cl, CO2, BUN, Glucose, Creatinine)
x
x
x
x
x
x
x
x
x
x
Liver Tests (ALT, AST, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
Tacrolimus Level
x
x
x
x
x
x2
x2
x2
x2
x2
Everolimus Level (Group 2 subjects who convert only)
x2
x2
x2
x2
x2
Fasting Lipid Panel
x
x
x
x
Urine Protein and Creatinine Ratio
x
x
x
x
x
x
x
x
x
Urine Pregnancy Test
x
x
Local Pathology Results - Graft Histology (Standard of Care Biopsy)
x
x
x
Blood Collection (450-500ml) or Leukapheresis for PBMC Isolation/ Treg Manufacturing (UCSFa)
Deu-bl (Detection of Deuterated Cells in Blood) - PBMC (UCSFa)
TruCount4 (UCSFa)
MFC Panels (Percentage Tregs/ Leukocyte phenotypes in Blood) - PBMC (UCSFb)
kSORT and kSPOT (Gene Expression in Blood) - Paxgene Tube (UCSFb)
x
x
x
x
x
x
non- HLA Alloantibodies in Blood - 5 ml Red Top Tube (UCSFb)
Fibrosis Biomarkers in Serum - Serum from Red Top Tube (UAB)
Plasma for Banking (6 ml Lavender EDTA Tube (UCSFb)
x
Serum for Banking (4 ml Red Top Tube (UCSFb)
x
Deu-biopsy (Detection of Deuterated Cells in Graft) - 1/2 to 1 16g core in PBS (UCSFa)
x
Central Pathology Reading - shared specimen with mIF/ISH FFPE (UCSFa)
x
mIF/ISH (Intragraft Cytokine Expression) - 1/2 16g Core Formalin Fixed Paraffin-Embedded (UCSFc)
x
x
x3
x3
CRM (Transcriptional Profiling of Graft) - performed with FFPE tissue (UCSFc)
x
x
x
x
Pathology Slides (biopsy case) for Central Pathology Reading (UCSFc)
x
x
x
Protein Biomarkers and RNA in Urine (Shared 100ml Urine Collection (Pellet & Supernatant to UCSFb)
Fibrosis Biomarkers in Urine (Shared 100ml Urine Collection (Supernatant to UAB)
3 If inflammation is detected in 6 month biopsies, deuterium detection may be performed on histological sections.    
4 TruCount will only be performed at the screening visit.
Blood Specimens
Manufacturing 
x4
x
x
x
x
x
x
x
x
x
x
x
x
2 Collect relevant trough level only, not necessarily both.  EVR trough levels should be obtained at least weekly until target level is reached/maintained.  
5 CBC at screening should be utilized for eligibility and determination of PBMC collection method.  CBC should be repeated prior to PBMC collection.    
Renal Biopsy Specimens
Ɨ Entry into the study is based on results of a post-transplant standard of care surveillance biopsy.  This biopsy must be obtained at 5 months (+/- 8 w eeks) from the day of transplantation.  Screen 1 labs must be draw n ± 2 w eeks from the date of the biopsy, keeping 
in mind lab results must be available within window for randomization.  Study group allocation must take place no more than 2 weeks after the central pathologist confirms eligibility.
1 HLA Typing, DSA, and viral serologies performed as part of evaluation for transplantation can be collected retrospectively.  If not available, serologic testing should be done within 2 weeks of screening biopsy.  EBV must be repeated 
within screening window if previously negative.
x
x
x
x
x
x
x
Urine Specimens
x
x
x
Day 
1
Day 
7
Day 
14
Day 
28
Group 2
T -16
Day 
84
Day 
182
x
x5
x
6 SARS-COV2 testing does not have to be repeated if negative at time of biopsy.  SARS-COV2 testing must be done w ithin 1 w eek of polyTreg infusion.
 Infusion can occur any time up to 
and including day 62 after central 
pathology confirmation.
Day 
364
PBMC 
Collection
± 3 days
± 14 days
General Study Assessments
Local Laboratory Assessments
Central Laboratory Assessments
→
x
→
→
Study 
Eligibility
Clinically 
Indicated 
Biopsy
Randomization
Treg 
Infusion


CTOT-21 
Confidential 
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TASK 
                                               Version 11.0 / January 11, 2022 
Appendix 4.  Manufacturing Timeline  
 
Randomization Day ## ## ## ## ## -9 -8 -7 -6 -5 -4 -3 -2 -1 0
1
2
3
4
5
6
7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Post Biopsy Day
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
*Group 2 recipient draw must occur 16 days (14-16 days for UCSF) prior to infusion date.
Central Biopsy 
Reading
Randomization to occur as soon as eligibility is 
confirmed, no later than 14 days after central 
biopsy confirmation.
Group 2 
Recipient 
Draw*
Group 2 
PolyTreg 
Infusion
Infusion can occur any time up 
to and including day 62 after 
central biopsy reading.  There is 
no low er limit other than those 
imposed by site and 
manufacturing scheduling.
Amendment 8: Group 1 SOE will maintain Day 0 as day of study group assignment.  Day 0 label was removed from the Group 2 and 3 SOE to avoid confusion.  For Group 2 and 3, the day of infusion will no longer be linked to study group 
assignment but rather to central biopsy reading confirmation.

SAP_003.pdf: 
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STATISTICAL ANALYSIS PLAN 
TREG ADOPTIVE THERAPY IN SUBCLINICAL INFLAMMATION IN 
KIDNEY TRANSPLANTATION 
PROTOCOL NUMBER: CTOT-21 
SHORT TITLE: 
Tregs in Subclinical Inflammation 
NCT #: 
NCT02711826 
CLIENT: 
NIH/NIAD/DAIT 
IND #: 
16626 
PREPARED BY: 
Rho 
2635 E NC Hwy 54 
Durham, NC 27713 
Telephone: (919) 408-8000 
Fax: (919) 408-0999 
This document is confidential and proprietary to NIH/NIAID/DAIT. This study is being conducted in compliance 
with good clinical practice, including the archiving of essential documents. Acceptance of this document 
constitutes agreement by the recipient that no unpublished information contained herein will be reproduced, 
published, or otherwise disclosed without the prior written approval of NIH/NIAID/DAIT, except that this 
document may be disclosed to appropriate Institutional Review Boards under the condition that they keep the 
information confidential. 


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ACKNOWLEDGEMENT AND SIGNATURE SHEET 
Approved: 
Approved: 


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VERSION HISTORY 
 
SAP Version 
Version Date 
Change(s) 
Rationale 
1.0 
17May2024 
Initial Version 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
1. 
LIST OF ABBREVIATIONS.......................................................................................7 
2. 
PURPOSE OF THE ANALYSES ................................................................................9 
3. 
PROTOCOL SUMMARY ..........................................................................................10 
4. 
ANALYSIS SAMPLES..............................................................................................15 
5. 
STUDY SUBJECTS ...................................................................................................16 
5.1. 
Disposition of Participants ..........................................................................................16 
5.2. 
Demographic and Other Baseline Characteristics ......................................................16 
5.3. 
Prior and Concomitant Medications ...........................................................................16 
5.4. 
Medical History ..........................................................................................................16 
6. 
STUDY OPERATIONS .............................................................................................17 
6.1. 
Protocol Deviations ....................................................................................................17 
6.2. 
Randomization ............................................................................................................17 
6.3. 
Measurement of Treatment Compliance ....................................................................17 
7. 
GENERAL ANALYSIS AND REPORTING CONSIDERATIONS ........................18 
7.1.1. 
Multicenter Studies .....................................................................................................18 
7.1.2. 
Assessment Time Windows ........................................................................................18 
7.1.3. 
Timing of Analyses .....................................................................................................18 
7.1.4. 
Multiple Comparisons/Multiplicity ............................................................................18 
8. 
ENDPOINT EVALUATION .....................................................................................19 
8.1. 
Primary Endpoint ........................................................................................................19 
8.1.1. 
Computation of the Primary Endpoint ........................................................................19 
8.1.2. 
Primary Analysis of the Primary Endpoint .................................................................20 
8.1.3. 
Sensitivity Analyses of the Primary Analysis ............................................................20 
8.1.4. 
Secondary Analyses of the Primary Endpoint ............................................................20 
8.2. 
Secondary Endpoints ..................................................................................................20 
8.2.1. 
Endpoints ....................................................................................................................20 
8.3. 
Other Endpoints ..........................................................................................................23 
9. 
SAFETY EVALUATION ..........................................................................................24 
9.1. 
Overview of Safety Analysis Methods .......................................................................24 
9.2. 
Extent of Exposure .....................................................................................................24 
9.3. 
Adverse Events ...........................................................................................................24 


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9.4. 
Deaths, Serious Adverse Events, and Other Significant Adverse Events ..................24 
9.5. 
Clinical Laboratory Evaluation ...................................................................................24 
9.6. 
Vital Signs, Physical Findings, and Other Observations Related to Safety ...............25 
9.6.1. 
Vital Signs ..................................................................................................................25 
9.6.2. 
Physical Examinations ................................................................................................25 
9.6.3. 
Other Safety Measures ................................................................................................25 
10. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL .....................26 
11. 
REFERENCES ...........................................................................................................27 
12. 
APPENDICES ............................................................................................................28 
13. 
ATTACHMENTS .......................................................................................................29 
 
 


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LIST OF TABLES 
Table 1: 
List of Abbreviations ....................................................................................................7 
 


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1. 
LIST OF ABBREVIATIONS 
Table 1: 
List of Abbreviations 
Abbreviation 
Term 
AB 
Antibody 
AE 
Adverse Event 
ANC 
Absolute Neutrophil Count 
ANOVA 
Analysis of Variance 
BK 
Human Polyomavirus 1 
CDC 
Centers for Disease Control and Prevention 
CMV 
Cytomegalovirus 
CNI 
Calcineurin Inhibitor 
CRM 
Common Response Module 
CSR 
Clinical Study Report 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTOT 
Clinical Trials in Organ Transplantation 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DSA 
Donor Specific Antibodies 
DSMB 
Data and Safety Monitoring Board 
EBNA 
Epstein-Barr Nuclear Antigen 
EBV 
Epstein-Barr Virus 
eCRF 
Electronic Case Report Form 
eGFR 
Estimated Glomerular Filtration Rate 
ELISPOT 
Enzyme-linked Immunosorbent Spot 
FDA 
Food and Drug Administration 
HB 
Hepatitis B 
HBcAB 
Hepatitis B Core Antibody Test 
HBSAg 
Hepatitis B Surface Antigen 
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
ICU 
Intensive Care Unit 
IgM 
Immunoglobulin M 


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IND 
Investigational New Drug 
IRB 
Institutional Review Board 
IS 
Immunosuppression 
ITT 
Intent-to-Treat Sample 
IVIg 
Intravenous Immunoglobulin 
kSORT 
Kidney Solid Organ Response Test 
LCA 
Leukocyte Common Antigen 
MedDRA 
Medical Dictionary for Regulatory Activities 
MFI 
Mean Fluorescence Intensity 
mITT1 
Modified Intent-to-Treat Sample 1 
mITT2 
Modified Intent-to-Treat Sample 2 
MMF/MPA 
Mycophenolate Mofetil/Mycophenolic Acid 
mRNA 
Messenger Ribonucleic Acid 
mTOR 
Mammalian Target of Rapamycin 
NCI 
National Cancer Institute 
NIAID 
National Institute of Allergy and Infectious Disease 
NIH 
National Institutes of Health 
PCR 
Polymerase Chain Reaction 
PPD 
Purified Protein Derivative 
REMS 
Risk Evaluation and Mitigation Strategy 
RT-PCR 
Reverse Transcription Polymerase Chain Reaction 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SARS-CoV2 
Severe Acute Respiratory Syndrome Coronavirus 2 
SOC 
System Organ Class 
TB 
Tuberculosis 
TdAP 
Tetanus, Diphtheria, and Pertussis 
VCA 
Viral Capsid Antigen 
 


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2. 
PURPOSE OF THE ANALYSES 
The purpose of this statistical analysis plan (SAP) is to describe the planned analyses and 
associated data displays to be included in the end of study analyses for Protocol CTOT-21 being 
conducted under IND 16626. This document provides details on the analysis population, 
derivation of variables, and statistical methods to be used in these analyses. 
We will conduct one or possibly more analyses to evaluate whether inflammation is suppressed 
in the graft and markers of inflammation in graft and urine are reduced via an infusion of 
polyTregs in comparison to participants treated with CNI-based maintenance 
immunosuppression therapy. 
 
 


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3. 
PROTOCOL SUMMARY 
Title 
Treg Adoptive Therapy in Subclinical Inflammation in Kidney Transplantation 
Short Title 
Tregs Infusion for Inflammation Suppression 
Clinical Phase 
I/II 
Number of Sites 
7 
IND Sponsor/Number 
NIH/NIAID/DAIT/ #16626 
Study Objectives 
1. To assess the safety of polyTregs infusions and the safety of converting 
from CNI-based maintenance therapy to mTOR inhibitors after Treg 
therapy in adult kidney transplant recipients (see Study Definitions page). 
2. To assess the efficacy of polyTregs infusions in reducing inflammation in 
adult kidney transplant recipients (see Study Definitions Page). 
Study Design 
This is a multi-center, open-label, randomized, controlled safety and efficacy 
trial with 2 cohorts, maintenance CNI-based immunosuppression therapy and 
polyTregs infusion, to achieve inflammation suppression in adult kidney 
transplant recipients. Eligible participants will either continue maintenance 
CNI-based immunosuppression therapy or will receive a single infusion of 
polyTregs (550 ± 450x106). All participants will undergo biopsies to determine 
study eligibility and at 7 months after study group allocation. Participants who 
received a polyTregs infusion will undergo an additional biopsy 14 days after 
polyTregs infusion.  
The original study design included a third treatment arm of donor alloantigen 
reactive Tregs (darTregs). One participant was treated with darTregs prior to 
protocol version 9.0 in which this treatment arm was eliminated due to 
excessive manufacturing failures. 
Primary Endpoint(s) 
Primary Safety Endpoint 
Participants will be followed for safety for the full duration of study. The safety 
of polyTregs (Group 2) will be described in comparison with CNI-based 
maintenance IS therapy (Group 1) by: 
1. The timing and incidence of Banff 2A or higher acute cell-
mediated rejection and/or acute antibody mediated rejection, 
2. The timing and incidence of study defined Grade 3 or higher 
infection 
Primary Efficacy Endpoint 
The change in inflammation as measured by the percentage area of cortex 
occupied by inflammatory cells using computer-assisted quantitative image 
analysis on the biopsy 7 months after group allocation, expressed as the percent 
change relative to the baseline biopsy.  
Primary Mechanistic Endpoint 
The immunologic profiles of kidney transplant recipients using graft common 
response module (CRM) gene expression of rejection and/or evidence of 


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inflammation in biopsies at 2 weeks after infusion (Group 2) and 7 months 
after group allocation (Groups 1 and 2). 
Secondary Endpoint(s)  
Secondary Endpoints for polyTregs – Clinical 
1. Proportion of participants exhibiting a relative decrease of 25% or more 
inflammation between the baseline kidney biopsy and the biopsy 2 weeks 
after polyTregs, measured as the percentage area of cortex occupied by 
inflammatory cells using computer-assisted quantitative image analysis. 
2.  Number of participants who exhibit a relative decrease of 50% or more 
inflammation on kidney biopsy at 2 weeks after polyTregs 
3. Timing, incidence, and severity of polyTreg infusion reactions 
4. Timing, incidence, and severity of culture-proven and clinically diagnosed 
infections after polyTreg infusion 
5. Timing, incidence, and severity of acute rejection using Banff grading 
6. Timing and incidence of BK viremia and cytomegalovirus (CMV) 
reactivation 
7. Timing and incidence of >10% decrease in eGFR compared to baseline 
Secondary Endpoints for mTOR Therapy – Clinical 
1. Incidence and timing of acute rejection, comparing participants who 
receive Tregs and convert to mTOR therapy to subjects who do not convert 
to mTOR therapy and participants in Group 1. 
Secondary Endpoints – Mechanistic 
1. Persistence of infused Tregs in blood and biopsies using deuterium labeling 
and T-cell repertoire analysis (Group 2) 
2.  Cytokine and CRM mRNA and protein profiles in the urine as correlates 
of acute rejection and/or histologic evidence of inflammation and graft 
fibrosis (Groups 1 and 2) 
3. Peripheral blood kSORT (kidney solid organ response test) mRNA 
expression of rejection/increased immune response (Groups 1 and 2) 
Accrual Objective 
14 participants total: 
7 participants on CNI maintenance therapy 
7 participants to receive polyTregs 
Study Duration 
6.5 Year Accrual, 12 Months Follow Up 
Treatment Description  
Subjects will be on maintenance immunosuppresiont herapy using CNI + 
MMF/MPA with or without steroids at the time of study entry. 
Eligible participants will be randomized to receive: 
1. Standard CNI maintenance IS (no Tregs) 
2. 
550 ± 450x106 polyTregs 
After receiving at least 300x106 polyTregs infusion, eligible participants will 
start mTOR inhibitor. 


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Inclusion Criteria 
Individuals who meet all of the following criteria are eligible for enrollment as 
study participants:  
1. Participant must be able to understand and provide informed consent 
2. Age ≥18 years of age at the time of study entry 
3. Recipients of non-HLA identical living or deceased donor renal transplants 
4. Protocol renal allograft biopsy at 5 months (± 8 weeks) after 
transplantation with Banff i1 and/or ti1 with concomitant t scores t0, t1, t2 
or t3; Banff i2 and/or ti2 with concomitant t scores t0 or t1; and without v 
>0, [ptc + g] ≥2, C4d >1 (by IF), or C4d > 0 (by IHC) confirmed by central 
pathologist. Participants must not be treated for pathologic criteria (e.g., 
steroids). 
5. eGFR ≥30 ml/min at the time of study entry 
6. Maintenance immunosuppression consisting of tacrolimus, MMF/MPA ± 
prednisone (≥10 mg/day) 
7. Current immunizations including TdAP, pneumococcal and seasonal 
influenza vaccines prior to study treatment, completed prior to 
randomization and no less than 14 days prior to planned manufacturing 
collection 
8. Hepatitis B serologies must be:  
(1) Positive HB surface antibody, negative HB core antibody and 
negative HB surface antigen for recipients immune to Hepatitis B 
(2) Negative HB surface antibody, negative HB core antibody and 
negative HB surface for non-immune/HBV naïve recipients provided 
donor had negative HB core antibody and negative HB surface 
antigen at the time of donation 
9. Negative TB test (PPD, interferon-gamma release assay, ELISPOT testing) 
within 1 year prior to enrollment. Participants with a history of TB 
(positive TB test without active infection) must have completed one of the 
latent TB infection treatment regimens endorsed by the CDC (Division of 
TB Elimination, 2016). Alternative regimens for latent TB infection 
eradication will be adjudicated by the site’s infectious disease specialist. 
10. Female participants with childbearing potential must have reviewed 
Mycophenolate REMS and have a negative pregnancy test upon study 
entry  
11. Female participants of childbearing potential must agree to use FDA 
approved methods of birth control for the duration of the study; 
participants must consult with their physician and determine the most 
suitable method(s) that are greater than 80% effective 
(http://www.fda.gov/birthcontrol) 
Exclusion Criteria 
Individuals who meet any of these criteria are not eligible for enrollment as 
study participants:  
1. Inability or unwillingness of a participant to give written informed consent 
or comply with study protocol 
2. History of malignancy; except adequately treated basal cell carcinoma 


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3. History of graft loss from acute rejection within 1 year after any previous 
transplant 
4. History of transplant renal artery stenosis  
5. History of cellular rejection prior to enrollment that did not respond to 
steroids and/or subsequent creatinine after treatment for rejection greater 
than 15% above baseline  
6. Known hypersensitivity to mTOR inhibitors or contraindication to 
everolimus (such as history of wound healing complications)  
7. Any chronic illness requiring uninterrupted anti-coagulation after kidney 
transplantation  
8. Post-transplant DSA >5000 MFI or post-transplant treatment with IVIg for 
DSA. Enrolled participants with post-transplant DSA >2000 MFI will not 
be eligible for mTOR conversion.  
9. Positive HIV 1 or HIV 2 serology prior to transplantation  
10. Known positive HBSAg, or HBcAb serology  
11. Proteinuria with urine pr/cr > 1.0 g/g  
12. Any condition requiring chronic use of corticosteroids >10mg/day at the 
time of study entry  
13. Subjects requiring treatment for pathologic findings on study eligibility 
biopsy (see inclusion 4).  
14. Active infection at the time of study entry  
15. History of active TB or latent TB without adequate treatment (see 
inclusion 10).  
16. Serum BK virus >1,000 copies/ml by PCR at the time of study entry  
17. Hematocrit <27%; ANC < 1,000/μL; lymphocytes <500/μL; at the time of 
study entry  
18. Participation in any other studies with investigational drugs or regimens in 
the preceding year  
19. Any condition or prior treatment which, in the opinion of the investigator, 
precludes study participation  
20. Unable to provide adequate biopsy specimen (paraffin embedded formalin 
fixed) from eligibility biopsy (3-7 months post-transplant) for quantitative 
analysis.  
21. EBV naïve recipient of a kidney from an EBV positive donor, historically 
EBV naïve recipient with primary EBV infection at the time of screening 
(primary anti-VCA IgM, without antibody to EBNA), positive EBV PCR  
22. Hepatitis C Virus AB positive participants with negative HCV PCR are 
eligible if they have spontaneously cleared infection or are in sustained 
virologic remission for at least 12 weeks after treatment.  
23. Positive SARS-CoV2 testing by RT-PCR  
Treg Infusion Inclusion Criteria 
1. Individuals randomized to Group 2 who continue to meet all of the 
enrollment criteria are eligible for Treg infusion 
2. Negative SARS-COV2 RTPCR testing within 1 week of Treg infusion 


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Treg Infusion Exclusion 
Criteria 
1. Received any vaccination within 14 days prior to blood collection for Treg 
manufacture 
2. Unacceptable Treg product 
3. Positive pregnancy test for women of childbearing potential 
mTOR Conversion Inclusion 
Criteria 
1. Received at least 300 x 106 polyTreg infusion 
2. Resolution of inflammation on the 2-week post-infusion biopsy as 
compared to the baseline biopsy, confirmed by central pathologist 
mTOR Conversion Exclusion 
Criteria 
1. Post-transplant DSA >2000 MFI 
2. Any condition or clinical variable, which in the opinion of the site 
investigator, precludes conversion to mTOR 
Study Stopping Rules 
Any of the following after subjects have initiated their study regimen: 
1. Any CTCAE grade 4 or higher infusion reaction 
2. Any diagnosis of malignancy and post-transplant lymphoproliferative 
disease (except non-melanoma skin cancer) 
3. Any graft loss 
4. Acute Rejection (Banff grade 2A or higher) or acute antibody mediated 
rejection within 6 weeks after Treg infusion in 3 of first 5 participants 
5. Any death 
6. Inability to manufacture and supply polyTregs in 3 of 5 consecutive 
subjects assigned to Group 2 
7. If there is a total of 8 failed lots of polyTregs at any time during the study 
 
 
 


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4. 
ANALYSIS SAMPLES 
Modified Intent-to-Treat Sample 1 (mITT1) – The mITT1 sample consists of all subjects 
allocated to the CNI-based treatment regimen or received polyTregs while on study. 
Modified intent-to-Treat Sample 2 (mITT2) – The mITT2 sample consists of all subjects who 
received polyTregs (Group 2). 


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5. 
STUDY SUBJECTS 
5.1. 
Disposition of Participants 
The disposition of all enrolled (i.e., consented) participants will be summarized in tables and 
listed, as necessary. 
 
The numbers and percentages of various participant statuses will be presented. Reasons for 
screen failure will be presented. For participants discontinuing study treatment early, the reasons 
for discontinuing study treatment early will also be presented. 
5.2. 
Demographic and Other Baseline Characteristics 
Summary descriptive statistics for baseline and demographic characteristics will be reported for 
all participants in the analysis sample. Characteristics to be summarized include age, sex, race, 
and ethnicity. Additional characteristics may be included.  
5.3. 
Prior and Concomitant Medications 
If needed for the CSR, a prior and concomitant medication listing will be prepared. 
5.4. 
Medical History 
If needed for the CSR, a medical history listing will be prepared. 


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6. 
STUDY OPERATIONS 
6.1. 
Protocol Deviations 
The study site principal investigator has the responsibility to identify, document and report 
protocol deviations as directed by the study Sponsor. However, protocol deviations may also be 
identified during site monitoring visits or during other forms of study conduct review.  
Upon determination that a protocol deviation has occurred, the study staff will complete a 
Protocol Deviation form. Protocol deviation reports will be compiled and reviewed by 
NIAID/DAIT and the DSMB. Sites will be responsible for reporting deviations to local IRBs, as 
per local requirements. 
6.2. 
Randomization 
Participants will be assigned to the treatment groups in an unblinded fashion using a variation of 
the Pocock and Simon adaptive randomization strategy. A web-based randomization system will 
be used in order to minimize the possibility of bias entering into those assignments and to 
maintain approximate equality in the group sizes. The initial treatment allocation will weight 
each treatment equally. In subsequent allocations, the less-populated arm will be weighted more 
in order to restore the balance between the groups. If a recipient is assigned to polyTreg and the 
recipient does not receive the intended polyTreg infusion, that recipient will be re-assigned to 
Group 1. The allocation schedule algorithm/scheme will not incorporate stratification variables. 
6.3. 
Measurement of Treatment Compliance 
Study subjects will have monitoring of trough levels at 1 week, 2 weeks, 1 month, 3 months, 6 
months, 12 months. 


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7. 
GENERAL ANALYSIS AND REPORTING CONSIDERATIONS 
The following is a list of general analysis and reportion conventions to be applied for this study. 
1. Categorical variables will be summarized using counts (n) and percents (%) and will 
be presented in the form n (%). 
2. Continuous variables will be summarized using appropriate statistics (e.g., mean, 
median, standard deviation, min, max, interquartile range) using an appropriate level 
of precision. 
3. Following SAS default rules, the median will be reported as the average of the two 
middle numbers if the dataset contains even numbers. 
4. P-values will be reported to 3 decimal places if greater than 0.001. If less than 0.001 
then report ‘<0.001’. Report p-values and significant levels as 0.05 rather than .05. 
5. No preliminary rounding should be performed; rounding should only occur after 
analysis. To round, consider digit to right of last significant digit: if <5 then round 
down, if ≥5 then round up. 
6. All listings will be sorted in order of subject, and time of assessment (e.g., visit, time, 
and/or event). 
7. All analysis will be performed using the SAS System version 9.4 or higher. 
7.1.1. 
Multicenter Studies 
Study participants will be recruited from 7 study sites. 
Following database lock, if potentially impactful site-to-site variation is noted, basic descriptive 
analyses of baseline demographics, medical history, or other key points will be repeated for each 
site individually in order to allow qualitative exploration of site-to-site variability. 
 
7.1.2. 
Assessment Time Windows 
Allowable visit windows are detailed in Section 10.6 of the protocol. 
Unscheduled visits may also occur throughout the study if creatinine increases or other concerns 
arise between regularly scheduled visits. 
Generally, all data will be included in analyses, regardless of time of assessment. 
7.1.3. 
Timing of Analyses 
All analyses will be conducted following database lock. 
 
7.1.4. 
Multiple Comparisons/Multiplicity 
The analysis is primarily descriptive and will not be adjusted for multiple comparisons. 


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8. 
ENDPOINT EVALUATION 
 
8.1. 
Primary Endpoint 
The primary efficacy endpoint will be the change in inflammation as measured by the percentage 
area of cortex occupied by inflammatory cells using computer-assisted quantitative image 
analysis on the biopsy 7 months after study group allocation, expressed as the percent change 
relative to the baseline biopsy. The subject in the darTregs group will be excluded from efficacy 
analyses. 
The safety of polyTregs (Group 2) and darTregs (Group 3) will be described in comparison with 
CNI-based maintenance IS therapy (Group 1) by:  
1. The timing and incidence of Banff 2A or higher acute cell-mediated rejection and/or 
acute antibody mediated rejection.  
2. The timing and incidence of study defined Grade 3 or higher infection. 
 
8.1.1. 
Computation of the Primary Endpoint 
• Primary Efficacy Endpoint 
o Percent change in inflammation at 7 months after group allocation relative to 
baseline biopsy. 
• Primary Safety Endpoint 
o The timing and incidence of Banff IIA or higher acute cell-mediated rejection 
and/or acute antibody mediated rejection.  
 Severe Acute Cell-Mediated Rejection- Acute cell-mediated rejection 
was defined using the Banff 2007 criteria. Participants with a Banff grade 
of greater than or equal to IIA were determined to have met the endpoint. 
Severity is graded as IA, IB, IIA, IIB, or III, with IA being the mildest 
form of cellular rejection and III being the most severe form of cellular 
rejection. 
 Acute Antibody Mediated Rejection- Antibody mediated rejection was 
defined as diffusely positive staining for C4d, presence of circulating anti-
donor antibodies, and morphologic evidence of acute tissue injury. 
o The timing and incidence of study defined Grade 3 or higher infection. 
 Severe infection was defined in the study as a Grade 3 or higher infection 
that was culture-proven and clinically diagnosed. Severity is graded as 1 
through 5, with 1 being least severe to 5 being most severe. Grade 3 is any 
infection associated with hemodynamic compromise requiring pressors; 
any infection necessitating ICU level of care; any infection necessitating 
operative intervention; any infection involving the central nervous system; 
any infection with a positive fungal blood culture; any proven or probable 
aspergillus infection; any tissue invasive fungal infection; any 


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pneumocystis jiroveci infection. Grade 4 is any life-threatening infection. 
Grade 5 is any infection resulting in death. 
8.1.2. 
Primary Analysis of the Primary Endpoint 
The ANOVA method or Kruskal-Wallis test (or t-test for comparison of Groups 1 and 2) will be 
used to compare treatment groups on the mean or median percent change in inflammation at 7 
months post-group allocation, relative to baseline biopsy. This method will be run on the mITT1 
population, subset to participants that have available LCA data from the 7 month visit post-group 
allocation. 
The Fisher’s Exact or Cochran-Mantel-Haenszel tests will be used to compare the treatment 
groups on the incidence of severe acute cell-mediated rejection and/or acute antibody mediated 
rejection. This method will be run on the mITT1 population, subset to participants that have 
available Banff data. 
The ANOVA method or Kruskal-Wallis test (or t-test for comparison of Groups 1 and 2) will be 
used to compare treatment groups on the mean or median time of event of severe acute cell-
mediated rejection and/or acute antibody mediated rejection. This method will be run on the 
mITT1 population, subset to participants that have available Banff data. 
The Fisher’s Exact or Cochran-Mantel-Haenszel tests method will be used to compare the 
treatment groups on the incidence of severe infection. This method will be run on the mITT1 
population. 
The ANOVA method or Kruskal-Wallis test (or t-test for comparison of Groups 1 and 2) will be 
used to compare treatment groups on the mean or median time of event of severe infection. This 
method will be run on the mITT1 population. 
8.1.3. 
Sensitivity Analyses of the Primary Analysis 
No sensitivity analysis is planned. 
8.1.4. 
Secondary Analyses of the Primary Endpoint 
No secondary analysis of the primary endpoint is planned. 
8.2. 
Secondary Endpoints 
All secondary endpoint analyses will use the mITT1 population, unless otherwise specified. 
8.2.1. 
Endpoints 
Variable: Secondary incidence endpoints including: 
Endpoint 
Time Frame 
polyTreg infusion reactions 
405 days post-group allocation 
Culture-proven and clinically diagnosed 
infections 
405 days post-group allocation 


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Acute rejection using Banff grading 
405 days post-group allocation 
BK viremia after polyTregs infusion 
405 days post-group allocation 
CMV reactivation after polyTregs infusion 
405 days post-group allocation 
>10% decrease in eGFR compared to baseline 405 days post-group allocation 
Acute rejection after converting to mTOR 
therapy after polyTregs infusion 
69 days post-group allocation to 405 days 
post-group allocation  
Proportion of participants exhibiting >=25% 
decrease of inflammation between baseline 
kidney biopsy and the week 2 kidney biopsy 
Baseline biopsy to Week 2 kidney biopsy 
Proportion of participants exhibiting >=50% 
decrease of inflammation between baseline 
kidney biopsy and the week 2 kidney biopsy 
Baseline biopsy to Week 2 kidney biopsy 
Proportion of participants exhibiting >=25% 
decrease of inflammation between baseline 
kidney biopsy and the month 6 kidney biopsy 
Baseline biopsy to 6 month kidney biopsy 
(for polyTregs infusion group). 
Baseline biopsy to 7 months post-group 
allocation (for maintenance therapy group). 
Analysis: For each of the above incidence endpoints, the proportion of participants with events 
will be reported by treatment group and compared using Fisher’s Exact test or a Cochran-
Mantel-Haenszel test. 
Population-level summary:  
• polyTreg infusion reactions - mITT 2 
• Acute rejection using Banff grading – mITT1, subset to participants that have available 
Banff lab data 
• Acute rejection after converting to mTOR therapy after polyTregs infusion- mITT2 
• Proportion of participants exhibiting >=25% decrease of inflammation between 
baseline kidney biopsy and the week 2 kidney biopsy – mITT1, subset to participants 
with available LCA data from week 2 visit 
• Proportion of participants exhibiting >=50% decrease of inflammation between 
baseline kidney biopsy and the week 2 kidney biopsy – mITT1, subset to participants 
with available LCA data from week 2 visit 
• Proportion of participants exhibiting >=25% decrease of inflammation between 
baseline kidney biopsy and the month 6 kidney biopsy – mITT1, subset to participants 
with available LCA data from 6 month visit 
• All other endpoints will use the mITT1 sample 


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Variable: Secondary timing endpoints including: 
Endpoint 
Time Frame 
polyTreg infusion reactions 
405 days post-group allocation 
Culture-proven and clinically diagnosed 
infections 
405 days post-group allocation 
Acute rejection using Banff grading 
405 days post-group allocation 
BK viremia after polyTregs infusion 
405 days post-group allocation 
CMV reactivation after polyTregs infusion 
405 days post-group allocation 
>10% decrease in eGFR compared to baseline 405 days post-group allocation 
Acute rejection after converting to mTOR 
therapy after polyTregs infusion 
69 days post-group allocation to 405 days 
post-group allocation  
Analysis: For each of the above timing endpoints, the mean or median time of event (study days 
since start of treatment) will be reported by treatment group and compared using ANOVA or the 
Kruskal-Wallis test (or t-test for comparison of Groups 1 and 2). 
Population-level summary:  
• polyTreg infusion reactions – mITT2 
• Acute rejection using Banff grading – mITT1, subset to participants that have available 
Banff lab data 
• Acute rejection after converting to mTOR therapy after polyTregs infusion- mITT2 
• All other endpoints will use the mITT1 sample 
Variable: Secondary severity endpoints including: 
Endpoint 
Time Frame 
polyTreg infusion reactions 
405 days post-group allocation 
Culture-proven and clinically diagnosed 
infections 
405 days post-group allocation 
Acute rejection using Banff grading 
405 days post-group allocation 
Analysis: For each of the above severity endpoints, the count of participants will be reported by 
treatment group and compared using Fisher’s Exact test or a Cochran-Mantel-Haenszel test. 
Population-level summary:  
• polyTreg infusion reactions – mITT2 


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• Culture-proven and clinically diagnosed infections – mITT1, subset to those with 
culture-proven and clinically diagnosed infections 
• Acute rejection using Banff grading – mITT1, subset to participants that have available 
Banff lab data 
 
8.3. 
Other Endpoints 
Any mechanistic data received, aside from data that’s been noted above in sections 8.1 and 8.2, 
will be summarized using appropriate statistics, provided that the data is sufficient and complete 
to do so. 
 
 


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9. 
SAFETY EVALUATION 
 
9.1. 
Overview of Safety Analysis Methods 
General safety analyses will be carried out using the mITT1 population defined in Section 4 
(with the addition of the one subject who received darTregs), unless otherwise noted. Missing 
safety information will not be imputed. These analyses will not be stratified by site. 
Safety will be analyzed in each treatment group through the reporting of AEs, vital signs, and 
changes in routine laboratory measures. 
Listings will be prepared, if needed to fulfill regulatory reporting obligations, for all safety 
measurements. All listings will be sorted in order of subject identifier and time point of 
assessment (e.g., visit and/or time). 
9.2. 
Extent of Exposure 
If needed to fulfill regulatory reporting obligations, a listing summarizing polyTreg and darTreg 
infusion status will be created. 
9.3. 
Adverse Events 
All AEs will be classified by system organ class (SOC) and preferred term, according to a 
standardized thesaurus (Medical Dictionary for Regulatory Activities [MedDRA] version 18.1). 
The severity of AEs will be classified using the National Cancer Institute’s (NCI’s) Common 
Toxicity Criteria for Adverse Events (CTCAE) version 4.0. Each AE is entered on the electronic 
case report form (eCRF) once at the highest severity. As such, no additional data manipulation is 
needed to identify events. 
A summary table will present the total number of events as well as the number and percentage of 
subjects experiencing the events organized by relatedness and severity for all AEs and SAEs 
separately. If a subject experiences the same AE on multiple occasions, the event will be counted 
once for each occurrence when reporting the number of AEs. When reporting the number of 
subjects experiencing the events, a subject will only be counted once if they experience an event 
within the particular SOC or preferred term. A data listing will be provided for all AEs. Separate 
listings will be provided for the CSR as needed. 
9.4. 
Deaths, Serious Adverse Events, and Other Significant Adverse 
Events 
Serious adverse events (SAEs) will be listed. If needed to fulfill regulatory reporting obligations, 
a separate display listing summarizing death, including time of death relative to treatment start 
date and cause of death, will also be created. 
9.5. 
Clinical Laboratory Evaluation 
If needed to fulfill regulatory reporting obligations, a separate listing summarizing clinical 
laboratory measurement data will also be created.  


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9.6. 
Vital Signs, Physical Findings, and Other Observations Related to 
Safety 
9.6.1. 
Vital Signs 
If needed to fulfill regulatory reportion obligations, a separate listing summarizing vital signs 
related to safety will be created. 
9.6.2. 
Physical Examinations 
If needed to fulfill regulatory reporting obligations, listings of physical examination data will be 
produced. 
9.6.3. 
Other Safety Measures 
No other safety measures have been identified. 


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10. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
The analysis populations for the endpoints 1) incidence of severe acute cell-mediated rejection 
and/or acute antibody mediated rejection, 2) incidence of acute rejection, 3) severity of acute 
rejection, and 4) timing of acute rejection were changed from the modified intent-to-treat 
population to the modified intent-to-treat population subset to participants that have available 
Banff data. 
The analysis population for the severity of culture-proven and clinically diagnosed infections 
endpoint was changed from the modified intent-to-treat population with polyTregs infusion to 
the modified intent-to-treat population subset to those with culture-proven and clinically 
diagnosed infections. 
The analysis populations for the endpoints 1) percent change in inflammation at 7 months after 
group allocation relative to baseline biopsy and 2) proportion of participants exhibiting >=25% 
relative decrease of inflammation between baseline kidney biopsy and the 6 month kidney 
biopsy were changed from the modified intent-to-treat population with polyTregs infusion to the 
modified intent-to-treat population subset to participants that have available LCA data from 6 
month visit. 
The analysis populations for the endpoints 1) proportion of participants exhibiting >=25% 
relative decrease of inflammation between baseline kidney biopsy and the week 2 kidney biopsy 
and 2) proportion of participants exhibiting >=50% relative decrease of inflammation between 
baseline kidney biopsy and the week 2 kidney biopsy were changed from the modified intent-to-
treat population with polyTregs infusion to the modified intent-to-treat population subset to 
participants that have available LCA data from Week 2 visit. 


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11. 
REFERENCES 
None 


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12. 
APPENDICES 
None 
 


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13. 
ATTACHMENTS 
None