Impact of Two Prednisone-free Maintenance Immunosuppressive Regimens With Reduced Dose FK506+Everolimus vs. Standard Dose Tacrolimus (FK506)+ Mycophenolate Mofetil (MMF) on Subpopulation of T and B Cells, Renal Allograft Function and Gene Expression Profiles in Renal Allograft Biopsies at 12 Months Post-transplant. Prospective Single Center Study in Recipients of Renal Transplant Allograft.
Phase: NA
Conditions: End Stage Renal Failure With Renal Transplant
Sponsor: Northwestern University

 
Investigator’s Name: Lorenzo Gallon MD. 
Associated Professor of Medicine 
UNOS Medical Director  
Institution: Northwestern University Feinberg School of 
Medicine 
Address: 676 North St. Clair Suite 1900,  
Chicago, IL 60611 
Tel: 312-695-4457 
Email: l-gallon@northwestern.edu 
Version: 11-10-2016 
ePRISM ID: IIRP-1275 
 
 
Title of study 
Impact of two prednisone-free maintenance immunosuppressive regimens with 
reduced dose FK506+Everolimus vs. standard dose FK506+MMF on subpopulation 
of T and B cells, renal allograft function and gene expression profiles in renal allograft 
biopsies at 12, 24 and 36 months post-transplant. Prospective single center study in 
recipients of renal transplant allograft.  
 
Study purpose and rationale 
Immunosuppressive therapy with the calcineurin inhibitors (CNI) cyclosporine (CsA) 
and tacrolimus (Tac), has radically changed the field of organ transplantation. 
Ironically, although extensively and effectively used for kidney and other solid organ 
transplants, CsA and Tac cause significant adverse renal side effects: acute and 
chronic renal dysfunction, hemolytic-uremic syndrome, hypertension, electrolyte 
disturbances and tubular acidosis. Chronic nephrotoxicity from CNI has been 
implicated as a principal cause of post-transplant renal dysfunction. It is 
characterized by an irreversible and progressive tubular atrophy, interstitial fibrosis, 
and focal hyalinosis of small renal arteries and arterioles. Furthermore, by blocking 
IL2 production, this class of medications is also associated with a negative impact on 
generation of regulatory T cells, or (Tregs). (Tregs are an important subpopulation of 
helper T cells that has been associated with positive immunomodulation and donor 
specific hypo-responsiveness).  
 
In renal transplant recipients, complete avoidance of calcineurin inhibitors from the 
time of renal transplant surgery has been associated with increased incidence of 
acute cellular rejection. The combination of mTOR inhibitors with full dose CNI has 
been shown to be synergistically nephrotoxic, and has been associated with poor 
graft outcome. We and other investigators have tested conversion from CNI to mTOR 
inhibitors with promising results. We have shown that in a prednisone-free 
immunosuppression regimen, conversion from tacrolimus to mTor inhibitors is safe, 
and not associated with an increased risk of acute rejection. More importantly, we 
have shown that it is associated with a persistent increase of regulatory T cells (Data 
presented at ATC 09 and 2010). 
 
Recently, the A2309 study led to FDA-approval of Everolimus. The A2309 was a 
study designed to combine reduced dose CsA+Everolimus. Interestingly, the reduced 
exposure to CsA was not associated with an increased rate of ACR; renal allograft 
function was well maintained when compared to that of the control group. This study 

Investigator’s name: Lorenzo Gallon 
 
Page 2 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
study leads us to question the mechanism(s) behind the efficacy of a low dose CNI 
with an mTOR inhibitor in preventing acute allograft rejection. 
   
The present proposal is designed to investigate the mechanisms of the synergistic 
effect(s) of low dose CNI and mTOR inhibitors (Everolimus) in controlling alloreactive 
T and B cells while expanding Tregs.   
 
Our hypothesis, based on published data and our laboratory results (see preliminary 
data-Supportive documents), is that mTOR inhibitors allow expansion of Tregs and 
that low exposure to CNI is sufficient to control alloreactive T cells. Decreased 
exposure to CNI and concomitant increase of Tregs will then translate to improved 
renal allograft function and histology. 
 
Objectives  
The overall aim of the present study is to prospectively investigate the impact of two 
maintenance immunosuppressive regimens (both prednisone-free) on 
subpopulations of T and B cells and on renal allograft function at 12, 24 and 36 
months post-randomization. One regimen combines low CNI exposure (tacrolimus) 
with concentration controlled Everolimus, and the second combines standard dose 
CNI (Tacrolimus) with MMF. 
 
The primary endpoints of the study are to: 1) evaluate the impact of the two 
maintenance immunonusuppressive regimens on T cells subpopulations, including 
regulatory T cells and B cell subpopulations, at different time points post renal 
transplant, and2) determine the impact of the two regimens on renal allograft function 
at 12, 24 and 36 months post-randomization (post-transplant). Secondary endpoints 
of the study are to: 1) evaluate the impact of the two regimens on allograft 
immunohistopathology gene expression profiles and graft infiltrating cells (memory T 
cell (CD4+ or CD8+ CD45RO+), TREG (CD4+FoxP3+), memory B cell (CD19+CD27+)) at 
12 and 24 months in renal allograft biopsies, and 2) evaluate the impact of the 
regimens on acute rejection, graft loss and death at 12, 24 and 36 months post-
transplant. 
 
Population 
The study will be conducted at Northwestern University Feinberg School of 
Medicine/Northwestern Memorial Hospital, and subjects will be recruited during visits 
to the Comprehensive Transplant Center. We plan to enroll and randomize recipients 
and their respective donors until 40 recipients have started the study drug to which 
they were randomized. As of January 2014, 3 recipients randomized to the 
experimental arm and 1 randomized to the control arm were withdrawn prior to 
dosing due to surgical complications. Replacement pairs will be enrolled, bringing the 
prospective enrollment to a minimum of 44 donor/recipient pairs (at least 88 total 
subjects).   
 
Inclusion Criteria:  

Investigator’s name: Lorenzo Gallon 
 
Page 3 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
1) Adults between 18 and 70 years of age 
2) Either gender and of any ethnic background 
3) Single-organ recipients (kidney only) 
4) Able to understand the protocol and provide informed consent. 
5) Recipient of living donor kidney transplants 
6) PRA<20% 
 
Exclusion criteria: 
1) End Stage Renal Disease (ESRD) secondary to primary FSGS (focal segmental 
glomerulonephritis). 
2) Inability to fully understand the purpose of the study and the inability to sign the 
informed consent 
3) A significant or active infection 
4) Pregnant or nursing females 
5) History of severe hyperlipidemia not controlled with statins, subjects with 
Cholesterol > 400mg/dl 
6) Platelet count <100,000mm3, WBC < 2,000mm3 (or clinical practice) 
7) Any surgical, medical or psychiatric condition, other than the current transplant, 
which in the opinion of the investigator precludes enrollment into this trial 
 
Number of centers & subjects  
Northwestern University Feinberg School of Medicine /Northwestern Memorial 
Hospital. 
 
Investigational and reference therapy   
Everolimus. Dose: 0.75mg PO BID (concentration controlled). 
 
Study duration   
We expect the total duration for this study not to exceed 2 years from the date of the 
last subject’senrollment. 
 
Study design  
Renal Transplant Recipients: 
All recipients will receive the same induction immunotherapy at time of transplant.   : 
Campath (30mg IV x1) will be administered at the time of surgery. 
Methylprednisolone (500 mg IV) will be administered on the day of transplant and on 
days 1 (250 mg IV), and 2 (125 mg IV) post-transplant. No further steroids will be 
given post-transplant unless indicated by the following medical conditions: acute 
renal allograft rejection, renal diseases necessitating the use of steroids, and other 
systemic diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus 
(SLE), and asthma.  
 
Maintenance immunosuprression is administered according to the 1:1 randomization 
at time of enrollment:  20 subjects will begin standard dose tacrolimus and MMF 
(control arm) 

Investigator’s name: Lorenzo Gallon 
 
Page 4 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
 
Tacrolimus will be initiated according to our practice. The dose will be adjusted from 
day 3 on to achieve a target whole blood trough concentration of 8 ng/mL to 10 
ng/mL.  From month 2 until Month 6, the target tacrolimus trough level will be 
reduced to 6 ng/mL to 8 ng/mL.   After month 6, the target level will be reduced to 4 
ng/mL to 8 ng/mL.   
 
MMF dose will be initiated as 1 g b.i.d. (2 g/day).  Adjustments should be made for 
adverse events including but not limited to gastrointestinal intolerance and a 
decrease in WBC.  MMF trough or AUC shall not be used to adjust dosing.   
 
Twenty (20) subjects will begin low dose tacrolimus with concentration 
controlled everolimus (treatment arm) 
Tacrolimus will be initiated according to our practice.  In this treatment arm, the 
Tacrolimus dose will be adjusted from day 3 on, to a target whole blood trough 
concentration of 4 ng/mL to 7 ng/mL.  From month 2 until Month 6, the target 
Tacrolimus trough level will be 3 ng/mL to 6 ng/mL.  After month 6, the Tacrolimus 
dose should be adjusted in order to achieve a target trough level of 2 ng/mL to 5 
ng/mL.   
 
From day 5 on, the starting dose of Everolimus (0.75 mg bid) will be increased if the 
trough level is < 3 ng/mL, or reduced if the trough level is > 8 ng/mL.  
 
All drugs will be administered in an open label fashion. 
 
The following transplant recipient data will be collected at the time of randomization:. 
Demographics: Age at transplantation, sex and race. 
Causes of end-stage renal disease 
Transplant related information: donor age, histocompatibility and cross match data, 
viral serology, history of acute rejection and delayed graft function, use of ACEI 
and/or ARB, level of renal allograft function - estimated GFR ( e-GFR(12) using 
MDRD formula, proteinuria.  
 
Blood samples will be obtained prior to randomization from all renal transplant 
recipients for baseline T and B cell functional activity and characterization of the cell 
subpopulations by flow cytometry.  
 
-All recipients will be routinely followed at our Transplant center with post-transplant 
labs (SMA7, CBC, and relevant immunosuppression trough levels).  
 
Additional blood will be collected and analyzed for the study at 3, 6, 12, 24 and 36 
months post-randomization. If the final blood sample cannot be drawn at the 36 
month timepoint, it may be drawn up to 50 months post-transplant. These samples 
will be used to investigate the impact of the two maintenance immunonusuppressive 
regimens on subpopulation of T cells In comparison with baseline levels. Specifically, 

Investigator’s name: Lorenzo Gallon 
 
Page 5 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
we will look at regulatory T cells (Treg: Foxp3+CD4+ CD25bright+) that could have 
been generated via the two maintenance immunonusuppressive regimens, and 
correlate the generation of with their function.  
 
RNA from peripheral blood will be also collected and stored for post-hoc analyses of 
T and B-cell immunoresponse gene expression via gene arrays.  
 
Kidney biopsy samples will be collected with standard-of-care samples at time of 
transplant (back table), 3 months, 12 months and at 24 months post-transplant. 
These will help evaluateand compare the impacts of the two maintenance 
immunonusuppressive regimens on allograft immunohistopathology.  Gene 
expression profiles will also be analyzed.  These samples will be processed in the 
principal investigator’s research laboratory.   
 
Both groups of subjects will be followed through 36-months post-randomization. In 
addition to monitoring renal allograft function, we will evaluate the incidence of acute 
rejection, subject and graft survival, incidence of hypertension (HTN), malignancies, 
opportunistic infections and post-transplant diabetes mellitus (DM). 
 
Donors: 
Blood samples from donor subjects will also be obtained at either the time of 
randomization or post-surgery during standard-of-care follow-up appointments These 
donor leukocytes will be used as stimulator cells in studies of functional activity of 
recipient T-cells.  These samples will be processed in the principal investigator’s 
research laboratory.   
 
 
 
 
 

Investigator’s name: Lorenzo Gallon 
 
Page 6 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
Safety:  
Safety Endpoints: The primary safety endpoints will be assessed on the AEs and 
SAEs that are observed throughout the trial. 
For the purpose of this clinical trial, the National Cancer Institute Common 
Terminology Criteria for Adverse Events v3.0 (CTCAE), dated December 12, 2003, 
will be used to grade all adverse events. 
 
Recording of Adverse Events 
At each contact with the subject, the investigator/member of investigator’s team will 
seek information on adverse events by specific questioning and, as appropriate, by 
examination.  Information on all adverse events will be recorded in the source 
document, and also in the appropriate adverse event module of the case report form 
(CRF).  All clearly related signs, symptoms, and abnormal diagnostic procedures 
results should recorded in the source document, though should be grouped under 
one diagnosis. 
 
All adverse events occurring during the study period will be recorded.  The clinical 
course of each event should be followed until resolution, stabilization, or until it has 
been determined that the study treatment or participation is not the cause.  Serious 
adverse events that are still ongoing at the end of the study period must be followed 
up to determine the final outcome.  Any serious adverse event that occurs after the 
study period and is considered to be possibly related to the study treatment or study 
participation will be recorded and reported immediately. 
 
Stopping Rules 
Subjects developing post-transplant infections (i.e., UTI, CMV, HSV, EBV, HCV, 
HBV, HIV,PCP), that in the opinion of the investigator are detrimental to the subject 
and his/her participation in this research trial, will be stopped from participating in this 
study..  
 
Patient Authorization: This study is to be conducted according to US and international 
standards of Good Clinical Practice (FDA Title 21 part 312 and International 
Conference on Harmonization guidelines), applicable government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted 
independent Institutional Review Board (IRB), (Northwestern University Institutional 
Review Board), in agreement with local legal prescriptions, for formal approval of the 
study conduct.  The decision of the IRB concerning the conduct of the study will be 
made in writing to the investigator and a copy of this decision will be provided to the 
investigator/sponsor before commencement of this study.  The investigator will place 
a list of IRB members and their affiliate in the regulatory binder for the clinical trial. 
 
All subjects for this study will be provided with a printed consent form describing this 
study and providing sufficient information for subjects to make an informed decision 

Investigator’s name: Lorenzo Gallon 
 
Page 7 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
about their participation in this study. This consent form will be submitted with the 
protocol for review and approval by the IRB for the study.  The formal consent of a 
subject, using the IRB-approved consent form, will be obtained before the subject is 
submitted to any study procedures. This consent form will be signed by the subject 
and the investigator-designated research professional obtaining the consent.  The 
subject will be given a signed copy of the informed consent for their records. 
 
Statistical analysis:   
The primary endpoints of the study are graft function and subpopulations of T cells, 
including regulatory T cells and B cell subpopulations at 3, 6, 12, 24 and 36 months 
post kidney transplant. The secondary endpoints of the study include 1) incidence of 
acute rejection, graft loss, and death at 12,24 and 36 months post kidney transplant; 
2) allograft immunohistopathology and gene expression profiles at 12 and 24 months 
post kidney transplant. 
 
We will run univariate and bivariate analyses for all variables prior to our main 
analyses. We will evaluate continuous variables using t-tests or F-tests and 
categorical variables using c2. We will examine data distributions and test all 
variables for linear relationships or non-linear relationships.  
 
Descriptive statistics (means, standard deviation, frequency) and correlations (both 
Pearson and Spearman) among variables will be calculated for preliminary data 
assessment. 
Necessary transformation and imputations will be carried out based on the raw data 
distribution.  
 
As missing data are inevitable in a longitudinal study, we will determine whether 
missing data are MCAR (data are missing completely at random), MAR (data are 
missing at random), or NMAR (not missing at random). If missing data are MCAR or 
MAR, it is likely that the standard multivariate computations using PROC MI (multiple 
imputations) will not result in biased standard error estimates.   However, if missing 
data are NMAR, we will use the "pattern mixture" approach to compute a weighted 
average of the parameters that are associated with the missing data to estimate what 
the data would have been. 
 
For the primary objective, we hypothesize that subjects receiving low dose CNI with 
mTOR inhibitor (Everolimus) will exhibit significantly greater graft function and higher 
numbers of regulatory T cells as compared to the standard dose CNI+MMF group.  
 We will utilize both repeated-measures ANOVA and random mixed effect models to 
compare the trajectory change in graft function and proportion of regulatory T cells 
over 12, 24 and 36 months between the two experimental groups. The method of 
estimation will be maximum likelihood (ML).   
 

Investigator’s name: Lorenzo Gallon 
 
Page 8 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
A variety of covariance structures (first order regressive, compound symmetry, 
Toeplitz, variance components, unstructured) will be carefully examined and 
compared using the best fitting statistics, such as Akaike’s Information (AIC) and 
Bayesian Information Criteria (BIC). These statistics are likelihood functions and can 
be compared across models. If, in spite of randomization, distributions of subjects’ 
baseline characteristics are found to be different between the two experimental 
groups, these variables will be entered as covariates in the statistical analysis. 
 
For the secondary endpoints, we will use logistic regression, Kaplan-Meier method, 
and Cox proportional hazards models to analyze time-to-event outcomes (acute 
rejection, graft failure, and death). For allograft immunohistopathology and gene 
expression profile outcomes, we will first develop a comprehensive immune profile of 
each participant by longitudinal analysis of frequency and function of T and B cell 
subsets. We will examine how these correlate with immunohistopathology and 
alloantibody production using Pearson or Spearman correlations. We will compare 
correlation coefficients between the two experimental groups using Z -tests to 
determine the distinct effects of each treatment arm.  We will also compare the 
trajectory changes in cell populations and antibody production over 12, 24 and 36 
months using both repeated measures ANOVA and random mixed effect models. All 
tests will be two-sided, and an error rate of α <0.05 will be considered statistically 
significant. All data analyses will be performed using SAS 9.2 statistical software 
(SAS Inc., Cary, NC).      
 
Sample size and power   
We calculated our sample power for the two primary endpoints based on our clinical 
experience and small pilot data. Using a two-tailed α of 0.05, the anticipated effect 
sizes corresponding to each specific aim between the two treatment arms were 
calculated using a minimum of 80% statistical power and assumed 10% attrition at 12 
months.  
 
For the sample size of n= 20 in each group, the proposed study will have a power of 
0.82 and 0.99 to detect effect size of 1.0 and 2.0 for graft function and % regulatory T 
cell change, respectively, between the two treatment arms.  Because this is a pilot 
study, we do not consider the statistical power of secondary outcomes; It is 
anticipated that a much larger sample size will be needed in order to achieve 
statistical power for the secondary endpoints in this proposed study. 
 
Evaluation schedule:   
 
 
Supportive documents and preliminary data: 
 
Conversion from CNI to Mtor-based immunosuppression after kidney transplant 
changes the frequency and phenotype of Tregs 
 

Investigator’s name: Lorenzo Gallon 
 
Page 9 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
 
 
 
 
 
 
 
 
 
Abstract ATC 2010: 
Lorenzo Gallon, Yuming Yu, Nader Najafian, Xuemei Huang, Giovanna La Monica, James Mathew, Josh 
Miller, Joseph Leventhal, Luting Xu Medicine/Comprehensive Transplant Center, Northwestern University, 
Chicago; Medicine, Harvard University 
 
Subpopulation of T helper cells (Th1, Th17, and Treg) can play a major role in promoting rejection or can facilitate 
regulatory immune-mechanisms that can help the development of a pro-tolerant state towards the transplant. 
Little is known about the impact of immunosuppressive (IS) drugs on allospecific T cell subpopulations. Methods: 
A two-step culture system was used. Alloreactve memory CD4+ T cells were first generated in MLR culture with 
responding CD4+CD45RA+ nave T cells and allogeneic CD14+ monocytes. Memory CD4+ T cells, from the 
primary MLR, were enriched and re-stimulated by co-culturing with autologous CD14+ monocytes in the presence 
of antigenic stimulation of anti-CD3 to increase the frequency of Th1,Th17 and Treg cells. Two different IS agents, 
Tacrolimus (Tac) and Sirolimus (SRL) were added to the second culture alone or in combination at different 
concentrations. The effects on the generation of Th1, Th17, Treg cells from alloreactive memory T cells 
(CD4+CD45RO+) were then determined by intracellular cytokine staining. Results: After secondary stimulation, 
there was up to 10-fold increase, compared to primary stimulation, in the % of Th1, Th17 and Treg cells. Tac at 
low doses, significantly blocked the productions of IFN-  (Th1) and IL-17 (Th17), while SRL even at high 
concentration (10ng/ml or 20ng/ml) had minimal effect on IFN-  and IL-17 production (see top panels of figure). 
FOXP3 expression (Treg) was markedly increased in SRL compared to Tac (see bottom panel). When Tac and 
SRL were used in combination and at different concentrations, we found that Tac at 2-5 ng/ml with SRL at 2.5-
10ng/ml achieved the maximal effect in inhibiting the production of IFN-  and IL-17 while maintaining a high level 

Investigator’s name: Lorenzo Gallon 
 
Page 10 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
of FOXP3 expression. 
 
Conclusions: Tac and SRL have differential effects on subpopulation of T helper cells. These findings can help to 
guide the clinical use of IS drugs to promote Treg expansion while controlling Th1 and Th17 responses. 
Keywords: Immunosuppression; Sirolimus (SLR); Calcineurin; T cell activation 
Oral Session: Late-Breaking Basic Abstract Session (10:45 AM-12:15 PM) 
 
Selected references to support this application: 
 
1. 
Bennett WM. Insights into chronic cyclosporine nephrotoxicity. Int J Clin 
Pharmacol Ther 1996, 34:515. 
2. 
Bennett WM, DeMattos A, Meyer MM, Andoh T, Barry JM. Chronic 
cyclosporine nephropathy: the Achilles' heel of immunosuppressive therapy. 
Kidney Int 1996, 50:1089. 
3. 
Myers BD: Cyclosporine nephrotoxicity. Kidney Int 1986, 30:964. 
4. 
Puschett JB, Greenberg A, Holley J, McCauley J. The spectrum of 
cyclosporine nephrotoxicity. Am J Nephrol 1990, 10: 296. 
5. 
Myers BD, Ross J, Newton L, Luetscher J, Perlroth M. Cyclosporine-
associated chronic nephropathy. N Engl J Med 1984, 311:699. 
6. 
Goldstein DJ, Zuech N, Sehgal V, Weinberg AD, Drusin R, Cohen D. 
Cyclosporine-associated end-stage nephropathy after cardiac transplantation: 
incidence and progression. Transplantation 1997, 63:664. 
7. 
Hornberger J, Best J, Geppert J, McClellan M. Risks and costs of end-stage 
renal disease after heart transplantation. Transplantation 1998, 66:1763. 

Investigator’s name: Lorenzo Gallon 
 
Page 11 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
8. 
Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a 
nonrenal organ. N Engl J Med 2003, 349:931. 
9. 
Burdmann EA, Andoh TF, Yu L, Bennett WM. Cyclosporine nephrotoxicity. 
Semin Nephrol 2003, 23:465. 
10.  
Nankivell B., Alexander S. Rejection of the kidney allograft. New England 
Journal of Medicine 363:1451-1462 (2010). 
11. 
Kim IK, Bedi DS, Denecke C, Ge X, Tullius SG. Impact of innate and adaptive 
immunity on rejection and tolerance. Transplantation 86:889-94 (2008) 
12. 
Meier-Kriesche HU, Shold JD, Srinivas TR, Kaplan B. Lack of improvement in 
renal allograft survival despite a marked decrease in acute rejection rates over 
the most recent era. Am J Transplant 4:378-83 (2004) 
13 
Nankivell B, Borrows R, Fung C, et al. The natural history of chronic allograft 
nephropathy. N Engl J Med 349:2326:2333 (2003).  
14 
Pontrelli P, Rossini M, Infante B, et al. Rapamycin inhibits PAI-1 expression 
and reduces interstitial fibrosis and glomerulosclerosis in chronic allograft 
nephropathy. Transplantation 85: 125-134 (2008). 
15 
Lebranchu Y, Thierry A, Toupance O, Westeel P, Etienne I, Thervet E, Moulin 
B, Frouget T, Le Meur Y, Glotz D, Heng A, Onno C, Buchler M, Girardot-
Seguin S, Hurault de Ligny B. Efficacy on renal function of early conversion 
from cyclosporine to sirolimus 3 months after renal transplantation: concept 
study. Am J Transplant 9:1115:1123 (2009) 
16 
Servais A, Meas-Yedid V, Toupance O, Lebranchu Y, Thierry A, Moulin B, 
Etienne I, Presne C, Hurault de Ligny B, Le Pogamp P, Le Meur Y, Glotz D, 
Hayem C, Olivo Marin J, Thervet E. Interstitial fibrosis quantification in renal 
transplant recipients randomized to continue cyclosporine or convert to 
sirolimus.   Am J Transplant 9: 2552:2560 (2009) 
17 
Chhabra D, Grafals M, Cabral B, Leventhal J, Parker M, Gallon L. Late 
conversion of tacrolimus to sirolimus in a prednisone-free immunosuppression 
regimen in renal transplant patients. Clin Transplant 24 (2):199:206. 2010. 
18.  
Levitsky J, Gallon L, Miller J, Tambur A, Leventhal J, Flaa C, Huang X, Sarraj 
B, Wang E, Mathew JM. Allospecific regulatory effects of sirolimus and 
tacrolimus in the human mixed lymphocyte reaction. Transplantation 27;91(2): 
199-206, 2011 
19. 
Battaglia, M., et al. Rapamycin and interleukin-10 treatment induces T 
regulatory type 1 cells that mediate antigen-specific transplantation tolerance. 
Diabetes 55, 40-49 (2006). 
20. 
Battaglia, M., Stabilini, A. & Roncarolo, M.G. Rapamycin selectively expands 
CD4+CD25+FoxP3+regulatory T cells. Blood 105, 4743-4748 (2005). 
21. 
Nikolaeva, N., Bemelman, F.J., Yong, S.L., van Lier, R.A. & ten Berge, I.J. 
Rapamycin does not induce anergy but inhibits expansion and differentiation 
of alloreactive human T cells. Transplantation 81,445-454 (2006). 
22. 
Turnquist, H.R., et al. Rapamycin-conditioned dendritic cells are poor 
stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ 
T regulatory cells and promote organ transplant tolerance. J Immunol 178, 
7018-7031 (2007). 

Investigator’s name: Lorenzo Gallon 
 
Page 12 
Study name: Everolimus and Treg 
Version: 10-26-2017 
Everolimus 
ePRISM ID:  IIRP-1275 
23. 
Valmori, D., et al. Rapamycin-mediated enrichment of T cells with regulatory 
activity in stimulated CD4+ T cell cultures is not due to the selective expansion 
of naturally occurring regulatory T cells but to the induction of regulatory 
functions in conventional CD4+ T cells. J Immunol 177, 944-949 (2006). 
24. 
Baan, C.C., et al. Differential effect of calcineurin inhibitors, anti-CD25 
antibodies and rapamycin on the induction of FOXP3 in human T cells. 
Transplantation 80, 110-117 (2005). 
25. 
Gao, W., et al. Contrasting effects of cyclosporine and rapamycin in de novo 
generation of alloantigen specific regulatory T cells. Am J Transplant 7, 1722-
1732 (2007). 
26. 
Bestard O, Cruzado J, Mestre M, Caldes A, Bas J, Carrera M, Torras J, Rama 
I, Moreso F, Seron D, Grinyo J. Achieving donor-specific hyporesponsiveness 
is associated with FoxP3+ regulatory T cell recruitment in human renal 
allograft infiltrates. Journal of Immunology 179 4901:4909 (2007) 
27. 
Alvarez C, Opelz G, Garcia L, Susal C. Expression of regulatory T-cell-related 
molecule genes and clinical outcomes in kidney transplant recipients. 
Transplantation 87:857-863 (2009) 
28. 
Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, 
Aiello S, Cassis L, Gotti E, Gaspari F, Cattaneo D, Perico N, Remuzzi G. 
Regulatory T cells and T cells depletion: role of immunosuppressive drugs. 
JASN 18: 1007-1018 (2007) 
29. 
Ruggenenti P, Perico N, Gotti E, et al. Sirolimus versus cyclosporine therapy 
increases circulating regulatory T cells, but does not protect renal transplant 
patients given alemtuzumab induction from chronic allograft injury. 
Transplantation, 84: 956-964 (2007) 
30. 
Wang Y, Camirand G, Lin Y, Froicu M, Deng S, Shlomchik W, Lakkis F, 
Rothstein D. Regulatory T cells require mammalian target of rapamycin 
signaling to maintain both homeostasis and alloantigen-driven proliferation in 
lymphocyte-replete mice. Journal of Immunology 186:000-000 (2011) 
31. 
Valujskikh A. The challenge of inhibiting alloreactive T cell memory. Am J 
Transplantation 6: 647-651 (2006) 
32. 
Valujskikh A, Baldwin III W, Fairchild R. Recent progress and new 
perspectives in studying T cell responses to allografts. Am J Transplantation 
10: 1117-1125 (2010) 
33. 
Pearl J, Parris J, Hale D, Hoffmann S, Bernstein W, McCoy K, Swanson S, 
Mannon R, Roederer M, Kirk A. Immunocompetent T cells with a memory-like 
phenotype are the dominant cell type following antibody-mediated T-cell 
depletion. Am J Transplantation 5:465-474 (2005) 
34. 
Araki K, Turner A, Shaffer V, Gangappa S, Keller S, Bachmann M, Larsen C, 
Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Nature 
460:108-112 (2009) 
35. 
Ferrer I, Wagener M, Robertson J, Turner A, Araki K, Ahmed R, Kirk A, Larsen 
C, Ford M. Cutting edge: Rapamycin augments pathogen-specific but not graft-
reactive CB8+ T cell responses. The Journal of Immunology 185:2004-2008, (2010)