A Prospective Cohort Study of Operationally Tolerant Allograft Recipients (ITN063ST)
Conditions: Kidney Transplant, Liver Transplant
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

 
ALLTOL 
A Prospective Cohort Study of Operationally Tolerant Allograft 
Recipients 
Protocol ITN063ST 
Version 5.0 (September 4, 2019) 
Protocol Chair 
 
 
 
 
 
 
ITN Clinical Trial Physician 
 
 
 
 
NIAID Medical Monitor 
 
 
 
 
 
  
 
NIAID Regulatory Affairs 
 
 
 
  
  
ITN Lead Biologist 
 
 
 
 
 
 
 
   
 
 
ITN Clinical Operations Manager 
 
 
 
 
  
NIAID Project Manager 
 
 
 
 
 
  
 

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Rho Scientist 
 
 
 
  
  
    
 
Rho Biostatistician 
 
 
 
  
  
   
  
This clinical study is supported and conducted by the Immune Tolerance Network, which is 
sponsored by the National Institute of Allergy and Infectious Diseases. 
This document is confidential. It is provided for review only to investigators, potential 
investigators, consultants, study staff, and applicable independent ethics committees or institutional 
review boards. It is understood that the contents of this document will not be disclosed to others without 
written authorization from ITN and NIAID unless it is necessary to obtain informed consent from 
potential study participants. 
 
 

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Protocol Approval 
Trial ID:  ITN063ST 
Protocol Version: 5.0 
 
Dated: September 4, 2019 
IND # N/A 
Protocol Chair: 
 
 
Title: ALLTOL: A Prospective Cohort Study of Operationally Tolerant Allograft Recipients 
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 US 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 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 written permission of the 
NIAID. 
 
 
 
__________________________________________                                      
Principal Investigator                        (Print)                     
 
 
__________________________________________         _________________ 
Principal Investigator                        (Sign)                      Date 
 
 
 

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Table of Contents 
1. 
BACKGROUND AND RATIONALE ................................................................................ 14 
1.1 
BACKGROUND ............................................................................................................ 14 
1.2 
SCIENTIFIC RATIONALE ............................................................................................. 14 
1.3 
SUMMARY OF KNOWN AND POTENTIAL RISKS AND BENEFITS FOR HUMAN 
PARTICIPANTS ........................................................................................................... 15 
1.3.1 
Risks .......................................................................................................................................... 15 
1.3.2 
Benefits ..................................................................................................................................... 15 
2. 
OBJECTIVES ................................................................................................................... 15 
2.1 
PRIMARY OBJECTIVE ................................................................................................. 15 
2.2 
SECONDARY OBJECTIVES.......................................................................................... 15 
2.3 
EXPLORATORY OBJECTIVE ....................................................................................... 15 
3. 
STUDY DESIGN ............................................................................................................... 16 
3.1 
DESCRIPTION .............................................................................................................. 16 
3.1.1 
Recruitment Plan ....................................................................................................................... 17 
3.1.2 
Enrollment and Accrual ............................................................................................................ 18 
3.1.3 
Screen Failures .......................................................................................................................... 18 
3.2 
STUDY DURATION ...................................................................................................... 18 
3.3 
STUDY ENDPOINTS ..................................................................................................... 18 
3.3.1 
Timing of endpoints and subsets for analysis of endpoints ....................................................... 18 
3.3.2 
Primary Endpoint ...................................................................................................................... 18 
3.3.3 
Secondary Endpoints ................................................................................................................. 19 
3.3.4 
Exploratory Endpoints .............................................................................................................. 19 
3.4 
PREMATURE TERMINATION OR SUSPENSION OF THE TRIAL / STOPPING RULES 19 
4. 
ELIGIBILITY ................................................................................................................... 19 
4.1 
INCLUSION CRITERIA ................................................................................................. 19 
4.2 
EXCLUSION CRITERIA ................................................................................................ 20 
4.3 
PREMATURE TERMINATION OF A PARTICIPANT FROM THE STUDY .................... 20 
5. 
STUDY MEDICATIONS .................................................................................................. 20 
5.1 
INVESTIGATIONAL MEDICATION ............................................................................. 20 
5.2 
CONCOMITANT MEDICATIONS ................................................................................. 21 
6. 
STUDY PROCEDURES .................................................................................................... 21 

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6.1 
VISIT WINDOWS .......................................................................................................... 21 
6.1.1 
Scheduled Visits ........................................................................................................................ 21 
6.2 
GENERAL ASSESSMENTS ........................................................................................... 21 
6.3 
CLINICAL LABORATORY ASSESSMENTS ................................................................. 22 
6.4 
MECHANISTIC ASSESSMENTS ................................................................................... 22 
7. 
TOLERANCE ASSAYS .................................................................................................... 23 
7.1 
MECHANISTIC STUDIES ............................................................................................. 23 
7.2 
FUTURE / UNPLANNED STUDIES ............................................................................... 23 
7.3 
SPECIMEN LOGISTICS ................................................................................................. 23 
7.3.1 
Specimen Tracking Procedures ................................................................................................. 24 
7.3.2 
Sample Storage.......................................................................................................................... 24 
8. 
SAFETY MONITORING .................................................................................................. 24 
8.1 
OVERVIEW ................................................................................................................... 24 
8.2 
DEFINITIONS ............................................................................................................... 25 
8.2.1 
Adverse Event ........................................................................................................................... 25 
8.2.2 
Suspected Adverse Reaction (SAR) .......................................................................................... 25 
8.2.3 
Serious Adverse Event .............................................................................................................. 26 
8.3 
GRADING AND ATTRIBUTION OF SERIOUS ADVERSE EVENTS ............................. 26 
8.3.1 
Grading Criteria ........................................................................................................................ 26 
8.3.2 
Attribution Definitions .............................................................................................................. 27 
8.4 
COLLECTION AND RECORDING OF SERIOUS ADVERSE EVENTS .......................... 27 
8.4.1 
Collection Period ....................................................................................................................... 27 
8.4.2 
Collecting Serious Adverse Events ........................................................................................... 27 
8.4.3 
Exceptions to Collection ........................................................................................................... 28 
8.4.4 
Recording Adverse Events ........................................................................................................ 28 
8.5 
REPORTING OF SERIOUS ADVERSE EVENTS ............................................................ 28 
8.5.1 
Reporting of Serious Adverse Events to Sponsor (DAIT/NIAID) ............................................ 28 
8.5.2 
Reporting of Adverse Events to IRBs/IECs .............................................................................. 29 
8.6 
PREGNANCY REPORTING........................................................................................... 29 
8.7 
REPORTING OF OTHER SAFETY INFORMATION ...................................................... 29 
8.8 
REVIEW OF SAFETY INFORMATION ......................................................................... 29 
8.8.1 
Medical Monitor Review .......................................................................................................... 29 
8.8.2 
DSMB Review .......................................................................................................................... 29 
9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ................................... 30 

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9.1 
ANALYSIS SAMPLE ..................................................................................................... 30 
9.2 
ANALYSIS PLAN .......................................................................................................... 30 
9.2.1 
Primary Endpoint ...................................................................................................................... 30 
9.2.2 
Secondary Endpoints ................................................................................................................. 30 
9.2.3 
Description of Baseline Characteristics and Demographics ...................................................... 31 
9.2.4 
Medical History ......................................................................................................................... 31 
9.2.5 
Use of Medications ................................................................................................................... 31 
9.2.6 
Study Completion...................................................................................................................... 31 
9.2.7 
Adverse Events.......................................................................................................................... 31 
9.3 
SAMPLE SIZE ............................................................................................................... 31 
9.4 
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL PLAN ................... 31 
10. ACCESS TO SOURCE DATA/DOCUMENTS .................................................................. 32 
11. QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 32 
12. ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE
 ..................................................................................................................................... 33 
12.1 
STATEMENT OF COMPLIANCE .................................................................................. 33 
12.2 
INFORMED CONSENT ................................................................................................. 33 
12.3 
PRIVACY AND CONFIDENTIALITY ............................................................................ 33 
13. PUBLICATION POLICY ................................................................................................. 33 
14. REFERENCES ................................................................................................................. 35 
APPENDIX 1. SCHEDULE OF EVENTS ................................................................................. 36 
APPENDIX 2. HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, 18TH EDITION ...... 37 
APPENDIX 3. HARRIET LANE HANDBOOK, 19TH EDITION ............................................... 38 
 
LIST OF TABLES 
Table 1. Attribution of Serious Adverse Events ...................................................................................... 27 
 
LIST OF FIGURES 
Figure 1. Schematic of Study Conduct .................................................................................................... 17 

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Synopsis 
Title 
 
ALLTOL: A Prospective Cohort Study of Operationally Tolerant Allograft 
Recipients 
IND Sponsor 
NIAID 
Conducted by 
Immune Tolerance Network 
Protocol Chair(s)  
 
Accrual Objective 
There is no upper limit on the number of study participants, but we estimate 
that approximately 70 participants may be enrolled from former ITN studies 
and from de novo referrals. 
Study Treatment 
N/A 
Study Design 
This trial is a multi-center, prospective, observational study in which 
operationally tolerant recipients of liver or kidney allografts will be 
followed longitudinally with annual collections of clinical data and 
biological samples. All participants will be followed for the duration of the 
trial regardless of changes in their tolerance status. 
 
Participants will be recruited along three main pathways: 
 
1. Tolerant participants from current and past ITN trials; including those 
who have already completed trial participation and those who are 
anticipated to complete trial participation; 
 
2. Tolerant participants referred by ITN affiliated investigators, academic 
and community transplant physicians and directly through outreach to 
transplant affinity groups such as the National Kidney Foundation 
(NKF); 
 
3. Tolerant participants from the general transplant community who are 
reachable through general media channels such as clinicaltrials.gov, the 
ITN website, word-of-mouth referrals from existing participants and 
social media. 
 
Participants will be encouraged to complete their study visits on site. 
However, participants who are unable or unwilling to travel to a study site 
will have the option of completing their study visits remotely (Figure 1).  
 
Study Duration 
The trial will continue through January 31, 2021. 

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Primary Objective 
The primary objective of this study is to identify and longitudinally follow 
individuals who have received a kidney or liver transplant and who have 
achieved a state of operational tolerance to their allograft. 
Operational tolerance is defined as meeting all of the criteria listed below: 
1. Absence of biopsy-proven or clinical rejection, as determined by 
medical history 
2. Maintenance off immunosuppressive medication, with the exception of 
short courses of steroids for non-allograft related conditions; e.g., 
asthma 
3. Normal and stable allograft function (see Study Definitions). Allograft 
function tests used in primary endpoint calculations must be obtained in 
the absence of confounding factors. A Primary Endpoint Adjudication 
Committee (see Study Definitions) will review cases where 
confounding factors are suspected of having impact on the participant’s 
test results and make recommendations toward resolution of the 
participant’s status. 
Primary Endpoint 
The primary endpoint of this study is the time to loss of operational 
tolerance.  
Secondary 
Endpoints 
Safety 
The following endpoints will be assessed for all participants: 
1. Time to development of de novo anti-HLA antibody or DSA  
2. Time to the first episode of biopsy-proven or clinical acute rejection, 
steroid resistant rejection and chronic rejection 
3. Time to graft loss, not including death with functioning graft 
 
Mechanistic 
Mechanistic endpoints will longitudinally examine the following parameters 
with regard to operational tolerance: 
4. Time course of changes in previously identified tolerance signatures in 
individual participants. 
5. Expression levels for a wide variety of genes measured by large-scale 
microarray or by PCR assessments. 
6. Flow-cytometric measurements of cell populations distinguished by 
cell-surface phenotype. 
7. miRNA levels in peripheral blood 
8. Gut microbiome profile 
Inclusion Criteria 
Participants must meet all of the following criteria to be eligible for this 
study: 
1. Recipient of single organ liver or kidney allograft from a living or 
deceased donor.  

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2. At screening, operationally tolerant defined as meeting both of the 
following criteria: 
a. Absence of any immunosuppressive therapy for ≥ 52 weeks prior 
to the screening visit. 
b. No evidence of allograft rejection in the 52 weeks prior to the 
screening visit, based on the medical history. 
3. Normal allograft function (see Study Definitions) 
If the site investigator judges that a participant exhibits stable allograft 
function despite values outside these criteria, then the participant is 
eligible if the ITN clinical trial physician/protocol chair, NIAID 
medical monitor and a subject matter expert concur.  
4. Receiving regular follow up for a kidney or liver transplant by a local 
physician. Participants must be willing to allow the study team to 
contact and share medical information with this local physician. 
5. Ability to sign informed consent.  
Exclusion Criteria 
Participants who meet any of the following criteria will not be eligible for 
this study: 
1. Current malignancy requiring recent surgery, ongoing chemotherapy, or 
radiation. 
2. Transplant of another organ. 
3. Current drug or alcohol dependency. 
4. Any medical condition that in the opinion of the principal investigator 
would interfere with safe completion of the trial. 
5. Inability to comply with the study visit schedule and required 
assessments. 
 
 

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Abbreviations  
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
AUC 
area under the curve 
CBC 
complete blood count 
CFR 
Code of Federal Regulations 
CKD 
chronic kidney disease 
CRF 
case report form 
CRO 
contract research organization 
DAIT 
Division of Allergy, Immunology and Transplantation 
DSMB 
Data and Safety Monitoring Board 
EDC 
electronic data capture 
FDA 
US Food and Drug Administration  
GCP 
good clinical practice 
GFR 
glomerular filtration rate 
GGT 
gamma-glutamyl transferase 
HLA 
human leukocyte antigen 
ICH 
International Conference on Harmonisation 
IRB 
institutional review board 
ITN 
Immune Tolerance Network 
LFT 
Liver function test 
MSAP 
mechanistic statistical analysis plan 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
manual of operations 
NCI 
National Cancer Institute 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for 
Adverse Events (version 4.3, June 10, 2010) 

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NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NKF 
National Kidney Foundation 
PBL 
peripheral blood lymphocytes 
PBMC 
peripheral blood mononuclear cells 
PCR 
polymerase chain reaction 
PD 
pharmacodynamics 
PTLD 
Post-transplant lymphoproliferative disorder 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SAR 
suspected adverse event 
SACCC 
Statistical and Clinical Coordinating Center 
SOE 
schedule of events 
STS 
Specimen Tracking System 
TCR 
T-Cell Receptor 
ULN 
upper limit of normal 
WHO 
World Health Organization 
 
 
 
 
 
 
 
 

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Study Definitions Page 
 
Adverse Event 
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 (Section 8.2.1). 
Biliary 
Complications 
Incidence of biliary complications including any of the following: 
• 
Biliary stricture requiring stenting 
• 
Other evidence of biliary obstruction 
• 
Cholangitis 
Enrollment 
The signing of informed consent (Section 3.1.1). 
Liver Graft Loss 
The incidence of any of the following: 
• 
Re-transplantation 
• 
Re-listing for transplantation  
• 
Participant death with a non-functioning graft 
Normal and stable 
graft function 
Baseline (Values collected at screening):   
Liver transplant recipients: Liver function tests (ALT, GGT) both less than or 
equal to the upper limit of normal (ULN).   
Kidney transplant recipients: Serum creatinine value correspond to an 
estimated GFR > 45 ml/min/1.73 m2.  
Post-Baseline  
Liver function tests (ALT, GGT) (for liver transplant recipients) that have not 
increased more than 50% above the participant’s baseline values or serum 
creatinine (for kidney transplant recipients) that have not increased more than 
25% above the participant’s baseline values, and these tests were collected in 
the absence of confounding factors. 
Operational 
Tolerance  
Participants must meet all of these criteria for at least 52 weeks after the last 
documented dose of immunosuppressive medication: 
1. Absence of rejection (biopsy-proven or clinical rejection) 
2. Maintenance off immunosuppressive medication 
3. Normal and/or stable allograft function  
Primary Endpoint 
Adjudication 
Committee 
The Primary Endpoint Adjudication Committee is comprised of the Study 
Protocol Chair/ITN Clinical Trial Physician, NIH Medical Monitor, a subject 
matter expert, and Statistician. The Committee will review the participant’s 
clinical history to determine whether the participant is operationally tolerant. 
Renal Function 
Estimated GFR calculated for adults by CKD-EPI and for pediatric 
participants (<18 years) by both CKD-EPI and modified Schwartz formulae1. 
Renal Graft Loss 
The incidence of any of the following: 
• 
Institution of chronic dialysis (at least 6 consecutive weeks) 
• 
Transplant nephrectomy 
• 
Re-transplantation 
• 
Re-listing for transplantation 
Serious Adverse 
Event  
Any adverse event that meets one or more serious criterion and occurs within 
24 hours of the protocol mandated blood draw for research specimens. Events 
that occur outside the 24 hour period that meet serious criteria should be 
reported if the site investigator deems a possible or definite association with a 
protocol mandated blood draw (Section 8.2.3). 

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Time Zero 
Defined as 52 weeks from the participant’s last documented dose of 
immunosuppression and in the absence of rejection or evidence for loss of 
normal and stable graft function since that last dose. 
Upper Limit of 
Normal (ULN) 
ULN values will be defined by age appropriate ranges from Harrison’s 
Principles of Internal Medicine, 18th edition (Appendix 2) or Harriet Lane 
Handbook, 19th edition (Appendix 3).  

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1. 
BACKGROUND AND RATIONALE 
1.1 
BACKGROUND 
Life-long immunosuppression is typically regarded as obligatory for solid-organ recipients to 
avoid the risk of graft loss from alloimmune attack. Evidence that not all patients require 
perpetual immunosuppression to maintain their graft is found in a small subset of patients who 
successfully discontinue immunosuppression through non-compliance or out of medical necessity 
(i.e. PTLD), and yet sustain normal graft function apparently indicating a state of operational 
tolerance. For kidney, this fraction is estimated at ~5-10%2. For liver it has traditionally been 
estimated at 20%3,4. However recent studies demonstrate that the prevalence of operational 
tolerance may be as high as 40% in adult liver recipients and as high as 60% in a highly selected 
cohort of pediatric liver recipients5,6.  
 
Longitudinal study of an operationally tolerant cohort will allow us to assess the durability of the 
tolerance phenotype, the effect of allograft subtype on the maintenance of tolerance and potential 
triggers associated with breaking of tolerance. Study of these patients might provide clues to 
tolerance mechanisms, thereby facilitating its prospective replication in non-spontaneously 
tolerant recipients. Finally the study of these patients may allow us to identify and validate 
biomarkers predictive of the tolerant phenotype. 
1.2 
SCIENTIFIC RATIONALE 
Recent studies suggest that gene expression analysis in peripheral blood and/or urine lymphocytes 
may identify the immune status of the graft. Gene signatures predicting rejection in heart and 
kidney recipients7-11 and a state of functional tolerance in liver and kidney patients12-14 have been 
identified. In the kidney setting, Newell et al made the unexpected observation in a cross-
sectional analysis of operationally tolerant renal transplant recipients that tolerance was 
associated with an expression signature dominated by B cell transcripts12. A three gene set 
validated by PCR was found to have high positive and negative predictive value. Interestingly, 
each of the three genes was involved in B cell maturation and development. These molecular 
signals were confirmed in PBL flow analysis which revealed parallel B cell subset differences 
with expansion of immature stage B cells that correlated with tolerance. Of note, the kidney 
signature identified by Newell has not yet been validated by a prospective weaning trial.  
 
In the liver transplant setting, Martinez-Llordella et al studied 16 operationally tolerant liver 
recipients by PBL transcriptional profiling and detailed flow subset characterization and 
compared them to 16 patients on immunosuppression and 10 normal controls. A gene expression 
signature indicative of tolerance was identified that included genes encoding γδT cells and NK 
cells. By flow there was an increase in the Treg subset13. An attempt was recently undertaken to 
validate these findings prospectively in a multicenter trial by weaning from immunosuppression. 
The trial’s molecular signature analysis published in JCI in 2012 revealed a robust tissue 
signature unexpectedly dominated by transcripts associated with iron metabolism15. 
 Establishment of a prospective cohort study of tolerant patients will allow cross-validation of 
these and other signatures as well as discovery of de novo signatures associated with tolerance. 

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1.3 
SUMMARY OF KNOWN AND POTENTIAL RISKS AND BENEFITS FOR 
HUMAN PARTICIPANTS  
1.3.1 Risks 
1.3.1.1 Risks associated with Blood Draws 
Risks to participants will be limited to collection of biological samples every year. During these 
collections, participants may experience some discomfort, minor bruising, or fainting during 
blood collection. There is also a very small chance (less than 1%) of infection at the needle 
puncture site.  
1.3.2 Benefits 
Participation in this trial will not result in any direct benefit, however, the information gathered 
during this study may someday be of benefit to transplant recipients.  
2. 
OBJECTIVES 
2.1 
PRIMARY OBJECTIVE 
The primary objective of this study is to identify and longitudinally follow individuals who have 
received a kidney or liver transplant and who have achieved a state of operational tolerance to 
their allograft. Operational tolerance is defined as meeting all three and loss of operational 
tolerance is defined as failing to meet any one of the following criteria: 
 
1. Absence of rejection (biopsy-proven or clinical rejection), as determined by medical history 
2. Maintenance off immunosuppressive medication, with the exception of short courses of 
steroids for non-allograft related conditions; i.e. asthma 
3. Normal and stable allograft function (see Study Definitions). Allograft function tests used in 
primary endpoint calculations must be obtained in the absence of confounding factors. A 
Primary Endpoint Adjudication Committee (see Study Definitions) will review cases where 
confounding factors are suspected of having impact on the participant’s test results and make 
recommendations toward resolution of the participant’s status. 
2.2 
SECONDARY OBJECTIVES 
1. To create an electronic database of operationally tolerant allograft recipients.  
2. To create a repository for samples obtained from operationally tolerant allograft recipients. 
3. To assess the durability of operational tolerance in allograft recipients over time. 
4. To analyze changes over time in previously identified biomarkers predictive of or associated 
with operational tolerance in allograft recipients. 
5. To follow markers of immunologic activity over time for possible use in exploratory 
biomarker analysis.  
2.3 
EXPLORATORY OBJECTIVE 
To assess the medical outcomes of operational tolerance over time, as determined by medical 
history, in allograft recipients.  

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3. 
STUDY DESIGN 
3.1 
DESCRIPTION 
This trial is a multi-center, prospective, observational study in which operationally tolerant 
recipients of liver or kidney allografts will be followed longitudinally with annual collections of 
clinical data and biological samples. All participants will be followed for the duration of the trial 
regardless of changes in their tolerance status. 
 
Participants will be recruited along three main pathways: 
 
1. Tolerant participants from current and past ITN trials; including those who have already 
completed trial participation and those who are anticipated to complete trial participation; 
 
2. Tolerant participants referred by ITN affiliated investigators, academic and community 
transplant physicians and directly through outreach to transplant affinity groups such as the 
National Kidney Foundation (NKF); 
 
3. Tolerant participants from the general transplant community who are reachable through 
general media channels such as clinicaltrials.gov, the ITN website, word-of-mouth referrals 
from existing participants and social media. 
 
Participants will be encouraged to complete their study visits on site. However, participants who 
are unable or unwilling to travel to a study site will have the option of completing their study 
visits remotely (Figure 1).  
 
 

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Figure 1. Schematic of Study Conduct 
 
3.1.1 Recruitment Plan 
This initiative will be publicized at national and international meetings that focus on liver and 
kidney transplantation using announcements included in the scientific program. Materials will 
also be distributed at the national and regional meetings of organizations such as the National 
Kidney Foundation (NKF) and Juvenile Diabetes Foundation (JDF) that have a major 
involvement with liver and kidney transplantation. Announcements will also be included in the 
newsletters of the aforementioned organizations. This project will also be advertised in major 
transplant journals (i.e., American Journal of Transplantation, Transplantation, Clinical 
Transplantation, etc.) and on the web pages of the ITN, National Institutes of Health (NIH), JDF, 
NKF, American Society of Transplantation (AST), American Society of Transplant Surgeons 
(ASTS), and the Transplantation Society. All advertisement materials will receive appropriate 
IRB/EC approval. 
The medical and surgical directors of all major liver and kidney transplant programs will also be 
contacted by mail, telephone and e-mail in an attempt to locate tolerant participants. These 
contacts will also include investigators from previous ITN liver and kidney transplant trials who 
have enrolled and followed tolerant participants. A potential candidate’s primary care physician 
may inform him/her of the study and if the potential candidate is interested in learning more about 
the study he/she may contact the study investigator. These primary care physicians will also be 

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provided with an IRB/EC approved “Dear Patient” letter describing the study and providing key 
contact information, which includes the internet address of a web page that outlines the trial and 
allows entry of basic information that will be used as a preliminary screening tool. Furthermore, 
traditional contact information (telephone, fax numbers, and email address) will also be provided. 
Study candidates who have participated in previous ITN studies will be first contacted by a 
physician at their prior ITN study site notifying them of the ALLTOL study. Interested candidates 
will be instructed to contact ALLTOL study sites. Study candidates who have not previously 
participated in an ITN study will be identified through referral by their physician or the ALLTOL 
study recruitment website. 
3.1.2 Enrollment and Accrual 
Enrollment will occur on an ongoing basis through November 1, 2020. There is no upper limit on 
the number of study participants, but we estimate that approximately 70 participants may be 
enrolled from former ITN studies and from de novo referrals. Enrollment is defined as the signing 
of informed consent and will be obtained prior to the initiation of any screening or study 
mandated procedures. Enrolled participants who do not fulfill eligibility criteria or who do not 
complete any mechanistic sample collection for any reason will be considered screen failures. 
Screening values will be considered the participant’s baseline for those who meet all eligibility 
criteria. If a repeat assessment is needed to confirm eligibility, the repeat value will be considered 
the participant’s baseline value. There is no upper limit on screen failures. 
3.1.3 Screen Failures 
Screening data will be collected on all participants who have signed informed consent and 
undergone screening but who fail to meet eligibility criteria or who do not complete any 
mechanistic sample collection for any reason. The reasons for ineligibility will be tabulated.  
3.2 
STUDY DURATION 
The trial will continue through January 31, 2021. 
3.3 
STUDY ENDPOINTS 
3.3.1 Timing of endpoints and subsets for analysis of endpoints 
Study endpoints for all participants will be evaluated relative to Time Zero. Time Zero will be 
considered the time that operational tolerance was obtained prior to study entry (see Study 
Definitions).  If the exact date of the last dose is not known, the date will be imputed to the 15th of 
the month when the day is missing, and July 1st when both day and month are missing. The year 
must be known.  
The following subset analyses will be performed on all study endpoints: 
a. allograft type (i.e.- liver or kidney)  
b. previous ITN study participation or non-ITN study-referral 
3.3.2 Primary Endpoint 
The primary endpoint of this study is the time to loss of operational tolerance. 

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3.3.3 Secondary Endpoints 
 
Safety 
The following endpoints will be assessed for all participants: 
1. Time to development of de novo anti-HLA antibody or DSA 
2. Time to the first episode of biopsy-proven or clinical acute rejection, steroid resistant 
rejection and chronic rejection 
3. Time to graft loss, not including death with a functioning graft 
 
 
Mechanistic 
Mechanistic endpoints will longitudinally examine the following parameters with regard 
to operational tolerance: 
4. Time course of changes in previously identified tolerance signatures in individual 
participants 
5. Expression levels for a wide variety of genes measured by large-scale microarray or by 
PCR assessments 
6. Flow-cytometric measurements of cell populations distinguished by cell-surface 
phenotype 
7. MiRNA levels in peripheral blood 
8. Gut microbiome profile 
 
3.3.4 Exploratory Endpoints 
1. Time to all-cause mortality 
2.  Changes in renal function (defined as estimated GFR calculated for adults  by CKD-EPI 
and for pediatric participants (< 18 years) by both CKD-EPI and modified Schwartz 
formulae) over time in renal transplant recipients1 
3. Changes in liver tests (ALT, GGT) over time in liver transplant recipients 
4. Time to incidence of biliary complications in liver transplant recipients  
5. Incidence of diabetes or use of concomitant medications for hypertension and/or  
hyperlipidemia  
3.4 
PREMATURE TERMINATION OR SUSPENSION OF THE TRIAL / 
STOPPING RULES 
There will be no stopping rules for this trial. 
4. 
ELIGIBILITY 
4.1 
INCLUSION CRITERIA 
Participants must meet all of the following criteria to be eligible for this study: 

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1. 
Recipient of single organ liver or kidney allograft from a living or deceased donor.  
2. 
At screening, operationally tolerant defined as meeting both of the following 
criteria: 
a. Absence of any immunosuppressive therapy for ≥ 52 weeks prior to the 
screening visit. 
b. No evidence of allograft rejection in the 52 weeks prior to the screening visit, 
based on the medical history. 
3. 
Normal allograft function (see Study Definitions) 
If the site investigator judges that a participant exhibits stable allograft function 
despite values outside these criteria, then the participant is eligible if the ITN 
clinical trial physician/protocol chair, the NIAID medical monitor and a subject 
matter expert concur.  
4. 
Receiving regular follow-up for a kidney or liver transplant by a local physician. 
Participants must be willing to allow the study team to contact and share medical 
information with this local physician. 
5. 
Ability to sign informed consent.  
4.2 
EXCLUSION CRITERIA 
Participants who meet any of the following criteria will not be eligible for this study: 
1. 
Current malignancy requiring recent surgery, ongoing chemotherapy, or radiation.  
2. 
Transplant of another organ. 
3. 
Current drug or alcohol dependency. 
4. 
Any medical condition that in the opinion of the principal investigator would 
interfere with safe completion of the trial. 
5. 
Inability to comply with the study visit schedule and required assessments. 
4.3 
PREMATURE TERMINATION OF A PARTICIPANT FROM THE STUDY 
Withdrawal of consent. Participants who withdraw consent for further follow-up.  
Investigator decision. The principal investigator may choose to withdraw a participant from the 
study for any reason.  
Failure to return. Participants who do not return for visits and who do not respond to repeated 
attempts by the site staff to have them return will be considered lost to follow-up.  
Participants who prematurely terminate from the study will not be replaced.  
5. 
STUDY MEDICATIONS 
5.1 
INVESTIGATIONAL MEDICATION 
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5.2 
CONCOMITANT MEDICATIONS 
All immunosuppressive medications and any concomitant medications taken within 30 days prior 
to enrollment and to each annual visit will be documented at each visit. There are no prohibited 
medications. 
6. 
STUDY PROCEDURES 
Study procedures will be limited to yearly venipuncture and urine for clinical labs and 
mechanistic sample collection. A stool sample will also be acquired yearly for mechanistic 
purposes. 
6.1 
VISIT WINDOWS 
6.1.1 Scheduled Visits 
Appendix 1 presents the schedule of events for this trial. Visit 0 must occur within 60 days of 
enrollment. All other scheduled study visits must occur within ±60 days of the scheduled time 
point listed in Appendix 1. 
6.2 
GENERAL ASSESSMENTS 
Recipient  
• 
Informed consent 
• 
Medical and demographic history including solid organ transplant specifics, any 
induction regimens, history of rejection and immunosuppression medication use 
since transplant: 
o For participants who previously participated in an ITN study, the participant ID 
and site ID (if separate) will be collected on the ALLTOL eCRF. The participant 
ID will be obtained by asking the ALLTOL participant for their previous ITN 
study participant ID and clinical study site. 
• 
Contact information for local physician providing regular follow-up care for the 
kidney or liver transplant. Local care providers will be notified of any critical clinical 
information that may indicate allograft dysfunction. This process is further outlined 
in the MOP. 
• 
Inclusion/Exclusion Criteria 
• 
HLA and blood typing (if available) 
• 
Change in adverse events and concomitant medications  
• 
Development of clinical outcomes of interest, which include incidence of diabetes 
and/or any biliary complications  

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• 
If there is a 25% increase in the value(s) collected during the scheduled annual visit 
compared to the participant’s baseline value, collect all liver function tests (for liver 
transplant recipients) or all serum creatinine levels (for kidney transplant recipients) 
since the last visit  
• 
All biopsies 
• 
All rejection episodes, including acute rejection, chronic rejection, antibody mediated 
rejection, and interstitial fibrosis and tubular atrophy rejection episodes 
Donor 
• 
Donor type (living-related donor, living-unrelated donor, deceased-donor) 
• 
Demographics 
• 
HLA and blood typing (if available) 
6.3 
CLINICAL LABORATORY ASSESSMENTS 
These laboratory assessments may be performed at study sites, at local laboratories, or 
though the central laboratory: 
• 
Hematology – CBC with differential and platelets 
• 
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, BUN, creatinine, calcium, 
glucose, albumin, total bilirubin, direct bilirubin, GGT, AST, ALT, and alkaline 
phosphatase) 
o Screening allograft function tests must be obtained in the absence of confounding 
factors such as dehydration, infection etc. Abnormal test results should be 
repeated to confirm eligibility after any confounding conditions have resolved. 
• 
Fasting lipid panel 
• 
Urine albumin-creatinine ratio  
• 
Urinalysis (protein, glucose, WBC, and hematuria) 
6.4 
MECHANISTIC ASSESSMENTS 
• 
Frozen PBMC – flow cytometry panel staining 
• 
Frozen PBMC – cellular assays 
• 
Serum – HLA alloantibodies, includes DSA, flow PRA 
• 
Whole Blood – RNA gene expression profiling 
• 
Urine pellet – gene expression profiling (not indicated for remote visits) 

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• 
Fecal – microbiome profiling 
See section 7 for detailed discussion of additional mechanistic assays.  
7. 
TOLERANCE ASSAYS 
7.1 
MECHANISTIC STUDIES 
The aim of this trial is to create a repository for samples obtained from operationally tolerant 
transplant recipients that have either completed ITN studies or have otherwise successfully 
withdrawn from immunosuppressive therapy. These samples will allow the ITN to longitudinally 
assess the phenotype, maintenance, generation, and stability of immune transplant tolerance in both 
kidney and liver transplant recipients. In addition, mechanistic data and samples collected from 
ALLTOL participants during previous ITN studies may be used to supplement the ALLTOL 
specimens and data, as needed.  
 
We propose to monitor related parameters over time and collect blood, cells, serum, urine, urine 
pellet, and fecal microbiome samples from tolerant participants. As the field of tolerance is 
evolving, markers of interest will be determined based on the literature and state-of-the-art at the 
time of experimentation.  
7.2 
FUTURE / UNPLANNED STUDIES 
Exploratory ancillary studies may be performed at a future date. Appropriate comparator arms 
will be determined at the time of experimentation and may include comparison with previously 
banked specimens or recruitment of control participants. Participants will be consented for 
sample storage and future use. Specimens stored during the trial may also be used in future assays 
to reevaluate biological responses as research tests are developed over time. Additionally, 
samples may be used for assays/ experiments outside the scope of this proposal, such 
investigation of miRNA expression, differences in the TCR repertoire as evaluated by 
sequencing, proteomics or other explorations that may emerge and be compelling during the trial 
period. Reevaluations or new assays will only be performed on samples of participants who have 
consented for future research. Blood samples will be collected and saved to allow for the possible 
use in later gene association studies. Specific consent will be obtained for the storage and use of 
participant DNA. The ITN sample sharing policy will apply for the provision of samples to study 
or outside investigators.  
7.3 
SPECIMEN LOGISTICS  
Sites will be trained in the collection, processing, shipment, and tracking of mechanistic research 
specimens. The ITN will monitor specimen quality, shipping compliance, etc., and retrain any 
clinical site that is not producing optimum quality mechanistic samples. Sites will process all 
mechanistic samples according to the ITN standard procedures and use the ITN Specimen 
Tracking System (STS) software to identify and track all mechanistic specimens. The sites will be 
required to have certain laboratory equipment for use in following ITN procedures, such as a 
centrifuge for spinning primary blood tubes, a micropipettor to aliquot specimens, and freezer to 
store frozen specimens until they can be shipped. Sites will use appropriate courier service for 
shipping specimens to repositories/ core labs, per ITN standard procedures. All shipping will 

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conform to Department of Transportation regulations (49 CFR 173.199) for Diagnostic 
Specimens.  
 
If samples cannot be collected at the study site, the site study coordinator will arrange for a 
representative from an ITN approved vendor to come to the participant’s location to complete the 
study assessments.  The ITN approved vendor will be trained in the ITN standard procedures for 
collection, processing and shipment of mechanistic research specimens. The site study 
coordinator(s) will use the ITN Specimen Tracking System (STS) software to identify and track 
all mechanistic specimens.  
7.3.1  Specimen Tracking Procedures  
The ITN will track all mechanistic specimens until the final disposition of all material is known. 
Samples will remain in the ITN repository until used for assays or destroyed.  
7.3.2 Sample Storage  
Samples sent to the ITN repository will be stored under specific conditions to maintain long-term 
sample integrity, as well as specimen tracking from receipt to shipment to alternate locations. A 
21 CFR Part 11 validated database system can be used to track shipment date, location shipped to, 
carrier, items shipped, amount shipped, barcode numbers, protocol number, and associated 
comments about each individual specimen. Storage temperature, location, processing and 
aliquoting, and freeze/thaw events may also be recorded. 
 
If the study participant allows storage, the participant’s specimens will be stored indefinitely. The 
participant can change their mind at any time and have their stored specimens destroyed by 
notifying the study physician in writing. In such cases, the site coordinator would send all 
requests for sample destruction to the ITN. The site will receive confirmation that the specimen 
was destroyed as requested. If the participant’s samples have already been analyzed, then the data 
will be used as part of the overall analysis. The participant can only request to have samples 
destroyed if they still exist, i.e. have not already been used in an experiment.  
 
Specimens at the ITN core or repository can only be transferred to another destination with 
appropriate authorization per ITN standard procedures. Purpose for accessing/transferring the 
specimen (within study assay as defined by the protocol or future studies), evaluation of 
participant consent for the purpose provided, verification of specimen identifiers, and quality and 
quantity of the specimen are some of the items checked prior to authorization.  
 
If the purpose is for future studies, and the participant consents for storage for future use, the 
participant’s sample may be made available to the scientific research community per the ITN 
Sample Sharing Policy
 Any research conducted using stored 
samples for future use may also need appropriate regulatory approval, such as Institutional 
Review Board per the study consent. 
8. 
SAFETY MONITORING 
8.1 
OVERVIEW 
This study involves minimal intervention outside of the normal standard of care of the participant. 
For this study, “reportable” is used to denote “reportable” to the Institutional Review Board 

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(IRBs)/Ethics Committee (EC), Data Safety Monitoring Boards (DSMBs), and the study sponsor 
(NIAID).  
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 or other Sponsor. 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. 
For this study, reportable adverse events will be limited to those that meet one or more serious 
criteria and occur within 24 hours of the protocol-mandated blood draw. In addition, events that 
occur outside the 24 hour period that meet serious criteria will be reported if the site investigator 
deems a possible or definite association with a protocol mandated blood draw. The NIAID 
medical monitor will review each SAE and assess the relationship of the event to the study 
procedure. 
8.2 
 DEFINITIONS 
8.2.1 Adverse Event 
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). 
Only adverse events meeting serious criteria will be collected for this study (see Section 8.4). 
8.2.2 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)). 
We do not anticipate any reported SARs since this study is observational and there is no protocol 
specific study therapy regimen.  

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8.2.3 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or Sponsor (DAIT/NIAID), 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 DAIT/NIAID, its occurrence places the subject at 
immediate risk of death. It does not include an adverse event or suspected adverse 
reaction that, had it occurred in a more severe form, might have caused death. 
3. Inpatient hospitalization or prolongation of existing hospitalization. 
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 might not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they 
might jeopardize the subject and might require medical or surgical intervention to 
prevent one of the outcomes listed above. 
For this study, a SAE is defined as “any adverse event that meets one or more serious criterion 
and occurs within 24 hours of the protocol mandated blood draw for research specimens.” In 
addition, events that occur outside the 24 hour period that meet serious criteria should be reported 
if the site investigator deems a possible or definite association with a protocol mandated blood 
draw. 
The NIAID medical monitor will review each SAE and assess the relationship of the event to the 
study procedure. 
8.3 
GRADING AND ATTRIBUTION OF SERIOUS ADVERSE EVENTS 
8.3.1  Grading Criteria 
The study site will grade the severity of serious adverse events experienced by the study subjects 
that meet the study reporting criteria described in section 8.2 (above).  SAEs will be graded 
according to the criteria set forth in the National Cancer Institute’s Common Terminology 
Criteria for Adverse Events (CTCAE – Version 4.0) for all adverse events. 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 study investigator and has been deemed 
appropriate for the subject population to be studied in this protocol for all adverse events. 
Adverse events will be graded on a scale from 1 to 5 according to the following standards in the 
NCI-CTCAE manual: 

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a. Grade 1 = mild adverse event. 
b. Grade 2 = moderate adverse event. 
c. Grade 3 = severe and undesirable adverse event. 
d. Grade 4 = life-threatening or disabling adverse event. 
e. Grade 5 = death. 
Serious Adverse Events grading will be recorded on the appropriate SAE case report form. 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. 
8.3.2 Attribution Definitions 
The relationship, or attribution, of a serious adverse event to a study-mandated blood draw will 
initially be determined by the site investigator and recorded on the appropriate SAE CRF. Final 
determination of attribution will be determined by DAIT/NIAID. The relationship of an adverse 
event to study intervention will be determined using the descriptors and definitions provided in 
Table 1. Attribution of Serious Adverse Events.  
Table 1. Attribution of Serious 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.  
Related Categories 
2 
Possible 
The adverse event has a reasonable possibility to be related; there is 
evidence to suggest a causal relationship.  
3 
Definite 
The adverse event is clearly related.  
8.4 
COLLECTION AND RECORDING OF SERIOUS ADVERSE EVENTS 
8.4.1 Collection Period 
Serious adverse events will be collected from the time of first study blood draw until 30 days 
after the subject prematurely withdraws (without withdrawing consent), is withdrawn from the 
study, or completes the study. 
8.4.2 Collecting Serious Adverse Events 
Only adverse events that meet serious criteria will be collected for this study. A SAE that occurs 
within 24 hours after a protocol-mandated blood draw must be reported for this study. In addition, 
events meeting serious criteria that occur more than 24 hours after a protocol mandated blood 

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draw must be reported if the site investigator deems a possible or definite association with a 
protocol mandated blood draw. Such events can include, but are not limited to: 
• 
Infection at the phlebotomy site or infections where the phlebotomy site in 
considered a possible portal of entry. 
• 
Significant hemorrhage or bruising at the phlebotomy site. 
• 
Fainting associated with the phlebotomy procedure resulting in further injury. 
• 
Events meeting serious criteria that occur outside the 24 hour time parameters or are 
not listed above should be reported if the investigator deems a possible association 
with the protocol mandated blood draw for research specimens. 
Serious Adverse events may be discovered through any of the following methods: 
• 
Reviewing the information in the subject’s medical chart. 
• 
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 
a serious adverse event, as defined in Section 8.3, Grading and Attribution of Serious Adverse 
Events. 
8.4.3 Exceptions to Collection  
Non-study-related events should be reported only when they occur within 24 hours of a protocol 
mandated blood draw and meet serious criteria. (Please see Section 8.2.3, Serious Adverse 
Event). 
8.4.4 Recording Adverse Events 
Throughout the study, the investigator will record serious adverse events as described previously 
(Section 8.2, Definitions) on the appropriate AE/SAE form regardless of the relationship to study 
procedure. 
Once recorded, a serious adverse event will be followed until it resolves with or without sequelae, 
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. 
8.5 
REPORTING OF SERIOUS ADVERSE EVENTS 
8.5.1 Reporting of Serious Adverse Events to Sponsor (DAIT/NIAID)  
This section describes the responsibilities of the site investigator to report serious adverse events 
to the sponsor via the SACCC eCRF. Timely reporting of adverse events is required by 21 CFR 
and ICH E6 guidelines. 

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Site investigators will report all serious adverse events (see Section 8.2.2, Serious Adverse 
Event), regardless of relationship or expectedness within 24 hours of discovering the event. 
For serious adverse events, all requested information on the AE/SAE form should be provided. 
However, unavailable details of the event will not delay submission of the known information. As 
additional details become available, the adverse event / serious adverse event should be updated 
and submitted. 
8.5.2 Reporting of Adverse Events to IRBs/IECs 
All investigators shall report serious adverse events in a timely fashion to their respective 
IRBs/IECs in accordance with applicable local regulations and guidelines. 
8.6 
PREGNANCY REPORTING  
As no investigational agents or procedures are required in this study, pregnancies will not be 
recorded or considered a reportable serious adverse event. 
8.7 
REPORTING OF OTHER SAFETY INFORMATION  
An investigator shall promptly notify the site IRB as well as the SACCC when an “unanticipated 
problem involving risks to subjects or others” is identified, which is not otherwise reportable as a 
serious adverse event. 
8.8 
REVIEW OF SAFETY INFORMATION  
8.8.1 Medical Monitor Review 
The DAIT/NIAID Medical Monitor shall receive monthly reports from the SACCC compiling 
new and accumulating information on SAEs recorded by the study sites on appropriate eCRFs. 
In addition, the Medical Monitor shall review and make decisions on the disposition of the SAE 
reports received by the SACCC (See Section 8.5.1, Reporting of Serious Adverse Events to 
Sponsor). 
8.8.2 DSMB Review  
8.8.2.1 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 principal investigator and/or the DAIT/NIAID medical officer.  
After careful review of the data, the DSMB will make recommendations regarding study conduct 
and/or continuation. 

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9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 
9.1 
ANALYSIS SAMPLE 
The analysis sample will be comprised of all enrolled subjects that fulfill eligibility criteria and 
complete at least one mechanistic sample collection. Furthermore, clinical data and mechanistic 
specimens that have already been collected from enrolled subjects from previous ITN study 
participation may be used to supplement the ALLTOL specimens and data, as needed. 
In addition, the following subset analyses will be performed on all study endpoints: 
a. allograft type (liver vs kidney)  
b. previous ITN study participation vs non-ITN study-referral  
9.2 
ANALYSIS PLAN 
9.2.1 Primary Endpoint 
The primary endpoint is defined in section 3.3.2. 
A confounding factor may prevent measurement of normal and stable allograft function. 
Participants who return to normal and stable allograft function after resolution of the confounding 
factor and who otherwise meet the primary endpoint criteria will be adjudicated by a Primary 
Endpoint Adjudication Committee (see Study Definitions). 
The primary endpoint will be analyzed using Kaplan-Meier survival estimates and associated 
two-sided 95% confidence intervals, using a delayed-entry model controlling for left-truncation 
of survival times through adjusting for the time from achieving operational tolerance to the time 
of study enrollment. A participant will be considered to have lost operational tolerance if they 
experience rejection, have loss of stable allograft function (in the absence of confounding 
factors), or restart immunosuppression. The date of loss of operational tolerance will be the first 
date on which the subject meets any of the criteria for loss of operational tolerance. Subjects lost 
to follow-up will be censored at the time of occurrence of the event causing their loss to follow-
up. 
9.2.2 Secondary Endpoints 
The secondary endpoints are defined in section 3.3.3. 
• 
Proportion endpoints will be descriptively summarized using frequency tables with 
frequencies and percentages and 95% exact binomial confidence intervals. 
• 
Time-to-event endpoints will be assessed using Kaplan-Meier survival estimates and 
associated two-sided 95% confidence intervals from a delayed-entry model as described 
for the primary analysis. Statistical methods for interval-censored data will be applied for 
endpoints with unknown exact dates. Subjects who are lost to follow-up will be censored 
at the time of occurrence of the event. Furthermore, participants who lose operational 
tolerance will continue to be followed for the duration of the study. These participants 

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will be considered to have lost operational tolerance on the date of loss of stable allograft 
function (in the absence of confounding factors), reinitiation of immunosuppression or 
diagnosis of rejection, whichever comes first.  
• 
Continuous endpoints will be summarized using descriptive statistics (n, mean, standard 
deviation, median, minimum, maximum). 
• 
Categorical variables will be summarized using counts (n) and percentages (%). 
9.2.3 Description of Baseline Characteristics and Demographics 
Demographic and baseline characteristic data will be summarized with descriptive statistics (n, 
mean, standard deviation, median, minimum, and maximum for continuous variables; counts and 
percents for categorical variables). 
9.2.4 Medical History 
Medical history will be collected, including the existence of past and current chronic medical 
conditions, sensitization information (including previous transplants and cause of primary 
allograft loss), primary records for the transplant procedure (including donor source, cadaveric or 
living), and post-transplant evaluation and management (including initial immunosuppressive 
agents used, episodes of allograft dysfunction, and discontinuation of immunosuppressive 
agents). 
9.2.5 Use of Medications 
Medication use per section 5.2 and all immunosuppressive medication use will be collected.  
9.2.6 Study Completion 
The percent of participants who fail to complete the study, losses to follow-up, times to loss to 
follow-up, and reasons for discontinuation (AEs, other) will be presented. 
9.2.7 Adverse Events 
AEs and SAEs will be summarized using frequencies and proportions. 
9.3 
SAMPLE SIZE  
This is a prospective cohort trial with an objective to collect as many tolerant subjects together 
with their biological samples as possible. There is no minimum sample size, and all eligible 
subjects will be enrolled through January 31, 2021. We estimate that approximately 70 
participants may be enrolled from former ITN studies and from de novo referrals through 2021.  
9.4 
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL PLAN 
There will be no statistical analysis plan prepared for this exploratory study, but all analyses will 
be described in the final study report as appropriate. 

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10. 
ACCESS TO SOURCE DATA/DOCUMENTS 
The investigational sites participating in this study will maintain the highest degree of 
confidentiality permitted for the clinical and research information obtained from participants in 
this clinical trial. Medical and research records should be maintained at the investigational sites in 
the strictest confidence. However, as a part of the quality assurance and legal responsibilities of 
an investigation, the investigational sites must permit authorized representatives of the ITN, 
sponsor, and health authorities to examine (and to copy when required by applicable law) clinical 
records for the purposes of quality assurance reviews, audits, and evaluation of the study safety 
and progress. Unless required by the laws permitting copying of records, only the coded identity 
associated with documents or other participant data may be copied (and any personally 
identifying information must be obscured). Authorized representatives as noted above are bound 
to maintain the strict confidentiality of medical and research information that may be linked to 
identified individuals. The investigational sites will normally be notified in advance of auditing 
visits. 
11. 
QUALITY CONTROL AND QUALITY ASSURANCE 
The principal investigator is required to keep accurate records to ensure that the conduct of the 
study is fully documented. The principal investigator is required to ensure that all eCRFs are 
completed for every participant entered in the trial. 
The sponsor is responsible for regular inspection of the conduct of the trial, for verifying 
adherence to the protocol, and for confirming the completeness, consistency, and accuracy of all 
documented data. 
The eCRFs will be completed online via a web-based electronic data capture (EDC) system that 
has been validated and is compliant with Part 11 Title 21 of the Code of Federal Regulations. 
Some data requirements will be addressed outside the EDC using SAS® software. Data queries 
will be issued and resolved within the EDC system or SAS®. 
Study staff at the sites will enter information into the electronic CRFs, and the data will be stored 
remotely at a central database. Data quality will be ensured through the EDC system’s continuous 
monitoring of data and real-time detection and correction of errors. All elements of data entry 
(i.e., time, date, verbatim text, and the name of the person performing the data entry) will be 
recorded in an electronic audit trail to allow all changes in the database to be monitored and 
maintained in accordance with federal regulations.  
Study staff will enter data from a study visit on the relevant eCRFs within 3 days following the 
visit or the time when data become available. 

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12. 
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE 
12.1 
 STATEMENT OF COMPLIANCE 
This trial will be conducted in compliance with the protocol, current Good Clinical Practice 
(GCP) guidelines—adopting the principles of the Declaration of Helsinki—and all applicable 
regulatory requirements. 
Prior to study initiation, the protocol and the informed consent documents will be reviewed and 
approved by the sponsor and an appropriate ethics review committee or institutional review board 
(IRB). Any amendments to the protocol or consent materials must also be approved by the 
Sponsor and the IRB before they are implemented. 
12.2 
 INFORMED CONSENT 
The informed consent form is a means of providing information about the trial to a prospective 
participant and allows for an informed decision about participation in the study. All participants 
(or their legally acceptable representative) must read, sign, and date a consent form before 
participating in the study and undergoing any study-specific procedures. If a participant does not 
speak and read English, the consent materials must be translated into the appropriate language. 
The informed consent form must be updated or revised whenever important new safety 
information is available, whenever the protocol is amended, and/or whenever any new 
information becomes available that may affect participation in the trial. 
A copy of the informed consent will be given to a prospective participant for review. The study 
investigator or a qualified study coordinator on the investigator’s delegation of responsibility log 
will review the consent and answer questions. This may be done either in person or remotely. The 
participant will be informed that participation is voluntary and that he/she may withdraw from the 
study at any time, for any reason. 
Requirements for the assent and informed consent process will depend on the participant’s age. 
Sites will follow their institutional IRB policies to determine the appropriate assent and informed 
consent process for pediatric participants.  
12.3 
PRIVACY AND CONFIDENTIALITY 
A participant’s privacy and confidentiality will be respected throughout the study. Each 
participant will be assigned a unique sequential identification number by the SACCC. This 
number, rather than the participant’s name or other patient identifiers, will be used to collect, 
store, and report participant information. 
13. 
PUBLICATION POLICY 
The ITN policy on publication of study results will apply to this study. Authorized participants 
may find details regarding the policy statement on the ITN internet website at 
 

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14. 
REFERENCES 
1. 
NIDDK. Glomerular Filtration Rate (GFR) Calculators.  https://www.niddk.nih.gov/health-
information/communication-programs/nkdep/laboratory-evaluation/glomerular-filtration-rate-
calculators, Accessionned June 20, 2018. 
2. 
Roussey-Kesler G, Giral M, Moreau A, et al. Clinical Operational Tolerance after Kidney 
Transplantation. American Journal of Transplantation. 2006;6(4):736-746. 
3. 
Mazariegos GV, Reyes J, Marino IR, et al. Weaning of immunosuppression in liver transplant 
recipients. Transplantation. 1997;63(2):243-249. 
4. 
Lerut J, Sanchez-Fueyo A. An Appraisal of Tolerance in Liver Transplantation. American Journal 
of Transplantation. 2006;6(8):1774-1780. 
5. 
Benitez C, Londono MC, Miquel R, et al. Prospective multicenter clinical trial of 
immunosuppressive drug withdrawal in stable adult liver transplant recipients. Hepatology. 
2013;58(5):1824-1835. 
6. 
Feng S, Ekong UD, Lobritto SJ, et al. Complete immunosuppression withdrawal and subsequent 
allograft function among pediatric recipients of parental living donor liver transplants. JAMA : the 
journal of the American Medical Association. 2012;307(3):283-293. 
7. 
Pham MX, Teuteberg JJ, Kfoury AG, et al. Gene-Expression Profiling for Rejection Surveillance 
after Cardiac Transplantation. New England Journal of Medicine. 2010;362(20):1890-1900. 
8. 
Jarcho JA. Fear of Rejection — Monitoring the Heart-Transplant Recipient. New England Journal 
of Medicine. 2010;362(20):1932-1933. 
9. 
Einecke G, Reeve J, Sis B, et al. A molecular classifier for predicting future graft loss in late 
kidney transplant biopsies. The Journal of clinical investigation. 2010;120(6):1862-1872. 
10. 
Hricik DE, Nickerson P, Formica RN, et al. Multicenter validation of urinary CXCL9 as a risk-
stratifying biomarker for kidney transplant injury. Am J Transplant. 2013;13(10):2634-2644. 
11. 
Suthanthiran M, Schwartz JE, Ding R, et al. Urinary-cell mRNA profile and acute cellular 
rejection in kidney allografts. The New England journal of medicine. 2013;369(1):20-31. 
12. 
Newell KA, Asare A, Kirk AD, et al. Identification of a B cell signature associated with renal 
transplant tolerance in humans. The Journal of clinical investigation. 2010;120(6):1836-1847. 
13. 
Martínez-Llordella M, Lozano JJ, Puig-Pey I, et al. Using transcriptional profiling to develop a 
diagnostic test of operational tolerance in liver transplant recipients. The Journal of clinical 
investigation. 2008;118(8):2845-2857. 
14. 
Sagoo P, Perucha E, Sawitzki B, et al. Development of a cross-platform biomarker signature to 
detect renal transplant tolerance in humans. The Journal of clinical investigation. 
2010;120(6):1848-1861. 
15. 
Bohne F, Martinez-Llordella M, Lozano JJ, et al. Intra-graft expression of genes involved in iron 
homeostasis predicts the development of operational tolerance in human liver transplantation. The 
Journal of clinical investigation. 2012;122(1):368-382. 
 
 

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APPENDIX 1. SCHEDULE OF EVENTS  
 
 
Years (+/- 60 Days) 
Study Visit 
Screen 
1 
2 
3 
4 
5 
Visit Number 
0 
1 
2 
3 
4 
5 
General Assessments – Recipient 
Informed Consent 
X 
 
 
 
 
 
Medical and demographic history including transplant specifics 
X 
 
 
 
 
 
Contact information for local physician1 
X 
 
 
 
 
 
Inclusion/exclusion criteria2 
X 
 
 
 
 
 
HLA and blood typing (if available)3 
X 
 
 
 
 
 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
Clinical outcomes of interest review 
X 
X 
X 
X 
X 
X 
Review of all biopsies since last visit, if applicable 
 
X 
X 
X 
X 
X 
Review of all rejection episodes since last visit, if applicable 
 
X 
X 
X 
X 
X 
Verify participant has ongoing local follow-up care for 
transplant 
 
X 
X 
X 
X 
X 
General Assessments - Donor3 
Donor type 
X 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
HLA and blood typing (if available) 
X 
 
 
 
 
 
Laboratory Assessments 
Hematology - CBC with differential and platelets4 
X 
X 
X 
X 
X 
X 
Comprehensive Metabolic Panel5,6 
X7 
X 
X 
X 
X 
X 
Fasting Lipid Panel 
X 
X 
X 
X 
X 
X 
Urine albumin/creatinine ratio 
X 
X 
X 
X 
X 
X 
Urinalysis8 
X 
X 
X 
X 
X 
X 
Mechanistic Assessments 
Frozen PBMC – flow cytometry panel staining 
X 
X 
X 
X 
X 
X 
Frozen PBMC – cellular assays 
X 
X 
X 
X 
X 
X 
Serum – HLA alloantibodies, includes DSA, flow PRA 
X 
X 
X 
X 
X 
X 
Whole blood – RNA gene expression profiling 
X 
X 
X 
X 
X 
X 
Urine pellet – gene expression profiling 
X 
X 
X 
X 
X 
X 
Fecal - microbiome profiling 
X 
X 
X 
X 
X 
X 
                                                          
1 For local clinical care provider who is providing active follow-up care for the participant’s single organ liver or kidney allograft. 
2 Participants who complete their screening visit remotely must meet all eligibility criteria, with the exception of confirmed normal 
allograft function, prior to scheduling a sample collection visit. 
3 This is a retrospective collection of data from medical records, or a previous ITN trial if applicable. 
4 If not completed on the same date as the visit's mechanistic assessment, this assessment must be repeated at the time of the visit's 
mechanistic collection. 
5 Includes Na, K, Cl, HCO3, BUN, creatinine, calcium, glucose, albumin, total bilirubin, direct bilirubin, GGT, AST, ALT, and alkaline 
phosphatase. 
6 If there is a 25% increase in the value(s) collected during the scheduled annual visit compared to the participant’s baseline value, collect 
all liver function tests (for liver transplant recipients) or all serum creatinine levels (for kidney transplant recipients) since the last visit. 
7 Participants with abnormal allograft function tests will have their allograft function retested for confirmation (see section 6.3). Abnormal 
test results thought to be affected by confounding factors should be repeated once the confounding condition has resolved. If a repeat 
assessment is needed to confirm eligibility, the repeat value will be considered the participant’s baseline value (see section 3.1.2). 
8 Includes protein, glucose, WBC, and hematuria. 
9 Not indicated for remote visits. 

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