Safety and Preliminary Efficacy of Donor-derived Regulatory Dendritic Cell (DCreg) Infusion and Immunosuppression Withdrawal in Living Donor Liver Transplantation
Phase: PHASE1
Conditions: Living Donor Liver Transplantation
Sponsor: Angus W. Thomson PhD DSc

 
Clinical Protocol - Liver 
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C. Clinical Protocol - Liver 
Table of Contents 
Clinical Protocol - Liver ........................................................................................................................... 1 
PROTOCOL SYNOPSIS ....................................................................................................................... 3 
1. 
OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE ............................. 10 
1.1. OBJECTIVES ............................................................................................................................ 10 
1.2. SPECIFIC AIMS ........................................................................................................................ 10 
1.3 BACKGROUND and RATIONALE .......................................................................................... 10 
1.4 SIGNIFICANCE ......................................................................................................................... 23 
2. 
RESEARCH DESIGN AND METHODS .................................................................................... 24 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS .................................................. 24 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN .................................................................. 25 
2.3 STUDY DRUG ........................................................................................................................... 37 
2.4 STUDY ENDPOINTS ................................................................................................................ 43 
2.5 STATISTICAL ANALYSIS ....................................................................................................... 44 
3. 
HUMAN SUBJECTS ................................................................................................................... 46 
3.1 SUBJECT POPULATION .......................................................................................................... 46 
3.2 INCLUSION CRITERIA ............................................................................................................ 47 
3.3 EXCLUSION CRITERIA ........................................................................................................... 47 
4. 
IRB APPROVAL AND FDA AMENDMENTS .......................................................................... 48 
5. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES ............................................ 48 
5.1 RECRUITMENT METHODS: ................................................................................................... 48 
5.2 INFORMED CONSENT PROCEDURES .................................................................................. 49 
6. 
POTENTIAL RISKS AND BENEFITS ....................................................................................... 49 
6.1 POTENTIAL RISKS ................................................................................................................... 49 
6.2 ALTERNATIVE TREATMENTS .............................................................................................. 53 
6.3 POTENTIAL BENEFITS ........................................................................................................... 53 
6.4 DATA SAFETY MONITORING ............................................................................................... 53 
6.5 RISKS MANAGEMENT PROCEDURES ................................................................................. 58 
7. 
COSTS AND PAYMENTS .......................................................................................................... 60 
7.1 COSTS ......................................................................................................................................... 60 
7.2 PAYMENTS ............................................................................................................................... 60 
8. 
QUALIFICATIONS AND SOURCE OF SUPPORT .................................................................. 61 
8.1 QUALIFICATIONS OF THE INVESTIGATORS .................................................................... 61 

 
Clinical Protocol - Liver 
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8.2 SOURCES OF SUPPORT .......................................................................................................... 63 
9. 
REFERENCES FOR SECTION C ............................................................................................... 63 
 
 
 

 
Clinical Protocol - Liver 
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PROTOCOL SYNOPSIS 
 
Protocol Title: 
Safety and Preliminary Efficacy of Donor-derived Regulatory 
Dendritic Cell (DCreg) Infusion and Immunosuppression Withdrawal 
in Living Donor Liver Transplant (LDLT) Recipients 
Short Title: 
DCreg in Living Donor Liver Transplantation 
Protocol 
Number: 
STUDY19020267 (formerly PRO16110490) 
NCT Number: 
NCT03164265 
Version # and 
Date: 
7.2 (May 2, 2023) 
Clinical 
Phase: 
Phase I/II 
Investigational 
Drugs: 
Allogeneic (Donor-derived) Regulatory Dendritic Cells (DCreg) 
Trial Site: 
University of Pittsburgh and UPMC 
IND Sponsor: 
Angus W. Thomson, PhD 
Investigator: 
Abhi Humar, MD 
Sub-
Investigators: 
Angus W. Thomson, PhD 
Chris Hughes, MD 
Amit Tevar, MD 
Swaytha Ganesh, MD 
Fadi G. Lakkis, MD 
Michele Molinari, MD 
A. Jake Demetris, MD 
Diana Metes, MD 
Paul Szabolcs, MD 
Warren Shlomchik, MD 
Adriana Zeevi, PhD 
Study 
Monitor: 
Angus W. Thomson, PhD 
Research 
Facilities: 
Thomas E. Starzl Transplantation Institute (STI), University of 
Pittsburgh and UPMC 
Immunologic Monitoring and Cellular Products Laboratory, 
University of Pittsburgh Cancer Institute (UPCI) 
Clinical 
Laboratories: 
UPMC Clinical Laboratories 
UPMC Pathology & Tissue Typing 
The Institute for Transfusion Medicine (ITxM), UPMC 

 
Clinical Protocol - Liver 
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Creative Testing Solutions lab in Phoenix, AZ 
 
Study 
Objectives: 
Primary Objective: To determine the safety and preliminary 
efficacy of donor-derived regulatory dendritic cell (DCreg) infusion to 
achieve immunosuppression withdrawal in living donor liver 
transplant (LDLT) recipients 
Secondary Objectives:  
1. To evaluate the influence of donor-derived DCreg infusion and 
immunosuppression withdrawal on donor specific antibody (DSA) 
production  
2. To conduct a preliminary assessment of the impact of donor-
derived DCreg infusion and immunosuppression withdrawal on the 
alloimmune response  
3. To determine the influence of immunosuppression withdrawal on 
renal function, quality of life, and cardiovascular risk factors 
 
Study 
Hypothesis: 
Donor-derived DCreg, generated ex vivo and administered 
prospectively to living donor liver transplant recipients treated with 
conventional immunosuppression (IS), will be safe and induce 
immunological changes reflective of donor-specific hypo-
responsiveness, allowing for a high likelihood of being able to 
successfully wean immunosuppression by approximately 24 months 
post-transplantation.  
 
Study Design: 
Phase I/II, single center, prospective, open-label, non-
controlled, non-randomized, interventional, cohort study in 
which low risk living donor liver transplant (LDLT) recipients will 
receive a single infusion of donor-derived DCreg with concurrent 
mycophenolic acid (MPA) therapy (1/2 dose) 1 week prior to 
transplantation.  All patients will be maintained on standard of care 
IS (MPA and Tacrolimus [Tac]) for the 1st 12 months after 
transplantation.  Participants will then be evaluated for TAC weaning 
at 1 yr after transplantation.  In cases where prednisone is 
substituted for MPA, prednisone must be discontinued prior to Tac 
weaning.Those who meet the criteria of no rejection, permissive 
LFTs, and permissive liver biopsy results* will proceed to Tac 
weaning. Successfully weaned participants who remain rejection-
free will undergo 3 years of follow-up after the last dose of 
immunosuppression.  They will undergo a liver biopsy at 1 yr and 3 
yrs after immunosuppression withdrawal.  Participants who are 
removed from the study protocol at any time will return to standard 

 
Clinical Protocol - Liver 
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of care but will continue to be followed by the study team and may 
undergo a liver biopsy at the end of the study (4.5 yrs after 
transplantation). 
 
* Permissive LFTs are defined as ALT, AST and total bilirubin < 2.5 
times the upper limit of normal. A permissive biopsy finding is based 
on 2016 Comprehensive Update of the Banff Working Group on 
Liver Allograft Pathology (the criteria detailed in Table 8, Demetris et 
al. 2016) [1], displayed in Section 2.2a4 of this protocol. 
 
Study 
Schedule: 
Day -28 to -15: donor leukapheresis 
Day -14 to -7: DCreg preparation 
Day -7: DCreg infusion 
Day -7 to 0: half dose MPA* (500mg bid)  
Day 0: liver transplant, no induction 
Day 0 to 7: rapid steroid taper 
Day 0 to 12 months: SOC MPA*and Tac 
Month 12: liver biopsy, stop MPA^ 
Month 12 to 18-24: wean Tac to off^ 
Year 2.5-3 (1 year after end of weaning): liver biopsy 
Year 4.5-5 (3 years after end of weaning): liver biopsy 
* an equivalent dose of either mycophenolate mofetil (MMF; 
CellCept®) or enteric coated Myfortic® may be used 
^This is an anticipated weaning schedule. MPA will be weaned per 
SOC but must be discontinued prior to Tac weaning.  
In cases where prednisone is substituted for MPA, prednisone must 
be discontinued prior to Tac weaning. 
Study 
Schematic: 
 
 

 
Clinical Protocol - Liver 
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Planned 
Sample Size:  
Up to 15 Recipient/Donor Pairs: 
All patients will receive the same dose of DCreg (2.5 – 10.0 million 
cells/kg body wt) 
Study 
Duration: 
7 years total (2 years of recruitment + 5+0.5 years of follow-up of 
recipients) 
 
Major 
Inclusion 
Criteria:  
Donor 
1. Able to understand and provide informed consent  
2. Male or female between the ages of 18 and 55  
3. Meet all standard institutional and UNOS criteria for liver donation 
4. For females of childbearing potential, a negative urine or serum 
pregnancy test  
5. Negative for HIV (5th generation Test and NAT), HTLV-1, HTLV-
2;* 
6. Negative for hepatitis C (antibody and NAT), hepatitis B (surface 
antigen and NAT)* 
*does not preclude donors from undergoing leukapheresis but cells 
may not be infused into recipient. 
 
Recipients 
1. Low risk recipient approved for LDLT, irrespective of gender, 
race, or ethnic background.  Low risk is defined by absence of 
exclusion criteria (below). 
2. Between ages 18 and 75 years 
3. Undergoing de novo (first) liver transplant  
4. Female subjects of childbearing potential must have a negative 
pregnancy test upon study entry. 
5. Agreement to use contraception; according to the FDA Office of 
Women’s Health (http://www.fda.gov/birthcontrol), there are a 
number of birth control methods that are more than 80% effective. 
Female participants of child-bearing potential must consult with their 
physician and determine the most suitable method(s) from this list to 
be used from the time that study treatment begins until 1 year after 
completion of immunosuppression withdrawal. 

 
Clinical Protocol - Liver 
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Major 
Exclusion 
Criteria: 
1. Positive T or B cell cross-match prior to DCreg infusion 
2.  Repeat liver transplant 
3.  Prior other solid organ transplant 
4.  Significant co-morbid conditions such as severe heart or lung 
disease 
5.  Following etiology of liver disease: Primary Sclerosing 
Cholangitis (PSC), autoimmune, Primary Biliary Cirrhosis (PBC) 
6.  If prior history of Hepatitis B or C (HBV or HCV) infection.  
Hepatitis B or C Virus (HBV or HCV) viral load positive at the 
time of transplantation (successfully treated HBV or HCV 
patients are not excluded) 
7.  Positive antigen-antibody immunoassay for HIV-1/2 
8.  Creatinine > 1.6 mg/dl prior to transplantation 
 
Study 
Endpoints: 
Primary Endpoints:  
1. Safety: Safety will be determined by assessing the proportion of 
subjects who experience the following events:  
i) CTCAE Grade 4 or higher infusion reaction 
ii) CTCAE Grade 4 or higher infection 
iii) Malignancy other than non-melanoma skin cancer or HCC 
recurrence 
iv) Rejection resulting in recipient death or retransplantation 
v) Biopsy-proven severe acute rejection 
vi) Any grade chronic rejection 
vii) Non-surgical graft loss 
viii) Recipient death 
 
2. Preliminary Efficacy:  Proportion of patients able to achieve 
staged immunosuppression withdrawal with operational tolerance 1 
year after complete immunosuppression cessation based on liver 
biopsy criteria detailed in Table 9 of the 2016 Comprehensive 
Update of the Banff Working Group on Liver Allograft Pathology 
(Demetris et al. 2016)[1] as shown in Section 2.2b.item 2 of this 
protocol. 
 
Secondary Endpoints:  
1. DSA levels will be measured early (<6 wks) and late (>6 wks) after 
transplantation  
 
2. In vitro studies conducted in the Human Immunology Laboratory at 
the STI will address secondary objective #2. 

 
Clinical Protocol - Liver 
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(a)  Impact of DCreg infusion on recipients’ immune response to 
donor alloantigens as measured by following parameters: 
i) 
Frequency, phenotype and function (in donor-specific 
assays) of conventional CD4 and CD8 T cell 
populations: naïve T cells (Tn), central memory T cells 
(Tcm), effector memory T cells (Tem), stable effector 
memory T cells (TemRA), and follicular helper T cells 
(Tfh). 
ii) 
Frequency and phenotype of CD4 regulatory T cell 
populations (Treg) 
(b)  We will also assess longevity of infused donor DCreg in 
circulation of recipient by identifying donor HLA on peripheral 
blood dendritic cells. 
 
3. Recipient interview and medical records reviews will address the 
secondary objective #3 as follows: 
 
(a) Change in renal function (defined as estimated GFR 
calculated by CKD-EPI: http://www.qxmd.com/calculate-
online/nephrology/ckd-epiegfr) will be calculated from 1 year 
post-transplantation to 1 year following immunosuppression 
weaning. 
(b) Quality of Life will be measured by the SF-36 at 1 year post-
transplantation and at the end of the study, and the change in 
both measures will be calculated. 
(c) Cardiovascular factors assessed include incidence of 
hypertension necessitating medication, post-transplantation 
diabetes, 
hyperlipidemia, 
hypercholesterolemia 
as 
documented from medical record review at yearly post-
transplant visits. 
 

 
Clinical Protocol - Liver 
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Study 
Stopping 
Rules 
The incidence of specific events will be continuously monitored 
throughout the study to determine if any of their observed subject-
based incidence rates exceed a threshold. A safety related 
suspension of enrollment will occur, pending expedited review of all 
pertinent data by the institutional review board (IRB) and the 
independent Data Safety Monitoring Board (DSMB) for this study 
based on the following: 
 
Recipient Stopping Rules (Phase I - prior to initiation of Tac weaning): 
i)  Any CTCAE Grade 4 or higher infusion reaction 
ii)  4 patients with CTCAE Grade 4 or higher infection 
iii)  2 patients with malignancy other than non-melanoma skin 
cancer; or HCC recurrence 
iv)  Any rejection resulting in recipient death or retransplantation 
v)  Any biopsy-proven severe acute rejection  
vi)  Any grade chronic rejection  
vii)  2 patients with non-surgical graft loss 
viii)  3 recipient deaths 
 
Recipient Stopping Rules (Phase II – Tac weaning through year 5 ± 
0.5): 
i)  3 patients with CTCAE Grade 4 or higher infection 
ii)  4 patients with malignancy other than non-melanoma skin 
cancer; or HCC recurrence 
iii)  Any rejection resulting in recipient death or retransplantation 
iv)  Any biopsy-proven severe acute rejection 
v)  2 patients any grade chronic rejection  
vi)  2 patients with non-surgical graft loss 
vii)  2 recipient deaths 
 
 

 
Clinical Protocol - Liver 
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1. 
OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
 
1.1. OBJECTIVES 
 
Primary: 
To determine the safety and preliminary efficacy of donor-derived regulatory dendritic 
cell (DCreg) infusion to achieve immunosuppression withdrawal (defined as gradual 
withdrawal of all immunosuppression) in living donor liver transplant (LDLT) recipients 
 
Secondary: 
1. To evaluate the influence of donor-derived DCreg infusion and immunosuppression 
withdrawal on donor specific antibody (DSA) production  
2. To conduct a preliminary assessment of the impact of donor-derived DCreg infusion 
and immunosuppression withdrawal on the alloimmune response  
3. To determine the influence of immunosuppression withdrawal on renal function, 
quality of life, and cardiovascular risk factors 
 
 
 
1.2. SPECIFIC AIMS 
 
We hypothesize that donor-derived DCreg, generated ex vivo and administered prospectively to living 
donor liver transplant recipients treated with conventional immunosuppression (IS), will be safe and 
induce immunological changes reflective of donor-specific hypo-responsiveness, allowing for a high 
likelihood of being able to successfully wean and withdraw immunosuppression by approximately 24 
months post-transplantation.  
 
Specific Aims:   See Objectives above (Section 1.1) 
 
1.3 BACKGROUND AND RATIONALE 
1.3a. Introduction: 
Long-term immunosuppression after solid organ transplantation is associated with life-threatening side 
effects that include infection, malignancy, diabetes and increased risk of cardiovascular disease. In 
addition, use of calcineurin inhibitors (CNI) causes nephrotoxicity and a significant rate of chronic renal 
insufficiency (CRI) at 5 years after orthotopic liver transplantation (OLT).  Many patients with CRI after 
OLT require renal replacement therapy (dialysis or renal transplantation) and are at increased mortality 
risk [2-7].  A large study based on the Scientific Registry of Transplant Recipients  (SRTR) and the 
Centers for Medicare and Medicaid Services (CMMS) ESRD Program database showed an ESRD 
incidence of 15 per 1000 patient years, implying that in 20 years, ~30% of patients require renal 
replacement therapy [8].  Patients demonstrating progressive loss in functional nephron mass are also 
at risk of progressing to chronic kidney disease (CKD), which is known to confer > 4 fold increase in 
mortality risk.  Immunosuppression (CNI) withdrawal, particularly before significant renal injury has set 
in, may preserve renal function and prevent morbidity and mortality associated with CKD/CRI and renal 

 
Clinical Protocol - Liver 
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replacement therapy.  Immunosuppression withdrawal may also mitigate the increased risk of infection, 
malignancy, 
diabetes, 
and 
cardiovascular 
disease 
directly 
associated 
with 
conventional 
immunosuppressive medications. 
 
Our goal is to develop a novel, safe cell therapy approach that, when used in conjunction with 
liver transplantation would facilitate the safe complete withdrawal of immunosuppression without 
jeopardizing allograft function or histology.  Recent evidence suggests that, by exploiting inherent 
mechanisms of immune regulation, it may be possible to achieve this goal. Rare, naturally-occurring 
regulatory immune cells, either innate or adaptive, critically regulate immunity, promote antigen (Ag)-
specific T cell hyporesponsiveness and prevent adverse immune reactions in the healthy steady-state. 
In addition to Treg, being evaluated for cell-based therapy in organ transplantation, a compelling rationale 
has emerged for clinical testing of regulatory DC modified ex vivo to promote their regulatory 
properties (DCreg) [9-12]. Thus, in rodents, infusion of donor-derived DCreg before transplantation, 
including their combination with conventional IS, promotes indefinite (> 100 day) organ allograft survival. 
More importantly and uniquely, using a robust, clinically-relevant, non-human primate (NHP) model with 
minimal IS, we have shown that infusion of DCreg, one week before transplantation, significantly and 
safely prolongs renal allograft survival, with no evidence of host sensitization [13].  Equally significant, is 
our demonstration that prolongation of allograft survival is associated with selective attenuation of donor-
reactive Tmem responses [13, 14], an important barrier to improving long-term graft survival and 
achieving operational tolerance [15, 16]. 
 
1.3b. Development of and rationale for DCreg therapy  
DC are highly-specialized, bone marrow-derived APC that induce or regulate innate and adaptive 
immunity [9, 17-20]. DCreg play a crucial role in maintaining self tolerance in the healthy steady-state 
[21-23]. Over the past 20 yr, innovative continuously NIH-funded research led by the Dr. Thomson’s (IND 
Sponsor) laboratory has revealed that these DCreg subvert naïve and memory T cell responses by 
various mechanisms [9, 10, 24-26]. Importantly, DCreg can induce or restore T cell tolerance in many 
animal models of autoimmune disease [27-30] or organ transplant rejection [9, 10, 31, 32]. In 
transplantation, both allogeneic DCreg and donor Ag-pulsed host (autologous) DCreg are effective [9, 
10, 33]. Our work has also confirmed that adoptive transfer of allogeneic (donor-derived) DCreg can 
safely regulate T cell responses in clinically-relevant NHP models, including MHC mis-matched organ 
allograft recipients [13, 14, 34], an important bridge to clinical testing. There is also well-documented 
evidence that adoptive transfer of human DCreg (in vitro-generated autologous DC) via local 
administration can control specific T cell responses to model Ags (flu matrix peptide and KLH) in healthy 
volunteers [35, 36]. These important insights have driven recent development of clinical grade human 
DCreg [37-43], testing in phase I autoimmune disease trials in which autologous DCreg have been 
administered locally, in type-1 diabetes [42], or rheumatoid arthritis (RA) [43, 44], and intraperitoneally in 
refractory Crohn’s disease  [45] with results that emphasize the feasibility, safety and potential efficacy 
of DCreg therapy.  
Our laboratory has played major roles in the discovery, characterization and therapeutic application of 
DCreg, especially in transplantation [32, 46-56]. Thus we were first to show that these regulatory innate 
immune cells, that are deficient in expression of MHC and co-stimulatory molecules and in production of 

 
Clinical Protocol - Liver 
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pro-inflammatory cytokines, could subvert antigen-specific T cell responses, both in vitro and in vivo. We 
summarize below evidence from rodent, NHP and human studies that supports the safety and, in the 
case of pre-clinical models, the efficacy of DCreg in organ transplantation. 
1.3b2. Rodent data: We were first to demonstrate, in rodent models, the ability of DCreg of donor origin, 
when adoptively transferred to prospective MHC mismatched pancreatic islet or heart allograft recipients, 
to markedly prolong transplant survival [46, 47]. Pre-transplant administration (d.-7) of donor-derived 
DCreg, alone or with low doses of immunosuppressive agents, can also induce donor-specific transplant 
tolerance [31, 49, 50, 57-59]. The route of administration, dosage, dose regimen and duration and timing 
of dosing (single i.v. infusion of up to 5 million donor DCreg/kg, 7d before transplantation) proposed in 
our clinical trial, are supported by experiments in rodents and NHP [13] (see below).  We and others 
further showed that the combination of these donor-derived DCreg with low doses of conventional or 
experimental immunosuppressive agents (in particular co-stimulation blocking agents) could induce 
donor-specific organ transplant tolerance [49, 50, 57, 58]. The observations are summarized in Table 1. 
It is also important to note that use of ‘standard of care’ (SOC) immunosuppressive agents together with 
DCreg promotes long-term allograft survival in rodents (Table 2). This is of direct relevance to the use of 
SOC immunosuppressive therapy in our proposed clinical trial. These compelling demonstrations of the 
efficacy of DCreg therapy in rodent transplant models, validated by numerous groups in the US and 
Europe [60], provided strong justification to evaluate DCreg therapy in NHP transplant recipients.  
 
Table 1:  Promotion of indefinite heart allograft survival in rodents by donor regulatory dendritic cells 
DC 
Source 
Species 
DC treatment 
When 
administered 
Additional 
treatment 
Route of 
injection 
MST 
Ref 
MoDC 
rat 
GM-CSF 
d+14/15 
none 
iv 
>160d 
Hayamizu 
K, 1998 [61] 
BMDC 
mouse 
GM-CSF + 
TGFβ 
d -7 
Anti-CD40L 
mAb 
iv 
>100d 
(40%) 
Lu L, 1999 
[57] 
BMDC 
mouse 
Low GM-CSF 
d -7 
none 
iv 
>100d 
Lutz MB, 
2000 [31] 
BMDC 
mouse 
NF-κB +  
rAd CTLA4Ig 
d -7 
none 
iv 
>100d 
(40%) 
Bonham 
CA, 2002 
[49] 
BMDC 
rat 
GM-CSF +  
IL-4 
d -7 
ALS 
iv 
>200d 
(50%) 
DePaz HA, 
2003 [59] 
BMDC 
mouse 
Low GM-CSF 
d -7 
Anti-CD54 
mAb + 
CTLA4Ig 
iv 
>100d 
Wang Q, 
2003 [50] 
BMDC 
rat† 
GM-CSF + IL-4 + 
dexamethasone 
d-10 
CTLA4Ig + 
cyclosporine 
iv 
>100d 
Mirenda V, 
2004 [58] 
ALS: anti-lymphocyte serum; BMDC: bone marrow-derived dendritic cells; i.v.: intravenous; MoDC: monocyte-
derived DC 
†Renal transplant 
 
 

 
Clinical Protocol - Liver 
13 
 
 
 
 
 
 
 
 
 
 
1.3b2. Non-human primate (NHP) data:  Primate transplantation models are regarded as important 
predictors of the safety and efficacy of innovative immunosuppressive/tolerogenic regimens since the 
NHP immune system more closely resembles that of humans than mice, and since as in humans (but 
not in mice), memory T cells present an important and difficult to overcome barrier to the induction of 
donor-specific tolerance [64-67]. In our recently-published work [13](Section J, Publication 1), we have 
used a robust, MHC-mismatched, life-sustaining rhesus macaque renal transplant model to evaluate for 
the first time, the safety and efficacy of donor-derived DCreg therapy. DCreg were generated from CD14 
immunobead-isolated monocytes in a single leukapheresis product of the prospective kidney donor 
monkey in rhu GM-CSF and IL-4. During the 7d culture of these cells, VitD3 and IL-10 was added to 
promote the maturation-resistant DCreg phenotype. The question posed by these studies was whether 
DCreg of donor origin, infused prospectively (once only) 7 days prior to transplant, could safely prolong 
graft survival using a minimal immunosuppressive regimen of co-stimulation blockade and mTOR 
(mechanistic target of rapamycin) inhibition. Our data (Figure 1 and summarized in Table 3) clearly show 
that 1) no adverse effects were encountered, 2) no evidence of host sensitization was detected, as 
determined by circulating alloantibody levels, 3) graft survival was prolonged significantly in the group 
receiving DCreg compared to graft recipients that did not receive the cell infusion, 4) weight loss and 
proteinuria were less marked in DCreg-infused monkeys and 5) evidence was obtained and now 
published, based on Eomes and CTLA4 expression, of significant, donor–specific attenuation of Tmem 
responses, both in peripheral blood (Figure 2) and the transplant [13, 14].  
Table 2:  Evidence that use of standard-of-care immunosuppressive agents (corticosteroid, MMF, 
CNI) together with DCreg promote long-term allograft survival in rodents 
Agent                         
Type of allograft (species) 
Reference 
MMF                         
pancreatic islet (mouse) 
Adorini L, 2003 [62] 
Dexamathasone     
renal (rat) 
Mirenda V, 2004  [58] 
Tacrolimus               
composite tissue (rat) 
Eun SC, 2013 [63] 
Cyclosporine           
composite tissue (rat) 
Ikeguchi R, 2008 [55] 
 
Renal (rat) 
Mirenda V, 2004 [58] 

 
Clinical Protocol - Liver 
14 
  
  
Table 3:  Conclusions from NHP studies by Investigator’s group (Ezzelarab et al, Am J Transplant 2013; 
[13]) 
Pre-transplant (d -7) infusion of donor-derived DCreg (3-106/kg) in combination with a minimal 
immunosuppressive regimen in rhesus monkey MHC-mismatched renal allograft recipients (Belatacept X 10-56 
days + Rapamycin X 6 months) 
• 
Safe; no adverse effects 
• 
No host sensitization, as determined by circulating alloAb levels 
• 
Significant, marked prolongation of renal allograft survival 
• 
Weight loss and proteinuria is less marked in DCreg-infused monkeys 
• 
Attenuation of donor-reactive memory T cells after transplantation - potential basis of biomarker analysis 
for monitoring anti-donor memory cell responses 
 
Figure 1.  DCreg infusion prolongs MHC-mismatched renal allograft survival in rhesus monkeys. (A), Costimulation 
blockade/rapamycin-based immunosuppression protocol. Monkeys received DCreg (3-10 x 106/kg i.v.) (n=6) or no cells (n=6) on day -7 
relative to kidney transplantation on day 0. Each group of monkeys was given CTLA4Ig (25 mg/kg i.v.), either from day -7 to day 10 
post-transplant (n=4 per group) or from day -7 to day 56 (n=2 per group). Intramuscular rapamycin was commenced on day -2 and 
whole blood trough levels maintained at 10-15 ng/ml (day 0-day 28); 5-10 ng/ml (day 29-day 150) and 1-5 ng/ml (day 151-day 180). 
Immunosuppressive treatment was stopped at day 180. (B), serum creatinine levels in control (n=6) and DCreg-treated animals (n=6) at 
various times post transplant. 

 
Clinical Protocol - Liver 
15 
 
 
1.3b3. Human Data:  Various pharmacologic agents and cytokines have been used to generate GMP 
grade human DCreg for prospective clinical use in chronic inflammatory disease [37-39, 42-44] and the 
safety of locally-administered, autologous (unpulsed or autoAg-pulsed) monocyte-derived DCreg in type-
1 diabetes [42] or RA [43, 44] and of DCreg administered intraperitoneally in refractory Crohn’s disease 
[45] has been documented. Moreover, limited but relevant experience with infusion of allogeneic (donor-
derived) regulatory macrophages (Mreg) into human renal transplant recipients exists [68].  This 
experience in humans is summarized below: 
 
(i) DCreg generation 
Various agents that have been used by different groups to generate GMP grade human DCreg for 
prospective clinical use in chronic inflammatory disease are described in Table 4. 
 
 
Figure 2: (A) DCreg infusion enhances PD-1 and CTLA4 expression on donor-reactive 
CD4+Tmem  in renal-allograft recipient monkeys. Incidences of PD1+CTLA4+ populations in ex-
vivo-stimulated CD95+ CD4+Tmem from representative control and DCreg-treated monkeys (n=4 
monkeys analyzed/group). Recipient PBMC obtained 28 d after transplantation, were cocultured with 
either donor or third party stimulators for 5 d.). (B) DCreg infusion spares reductions in donor-
specific Eomeslo CTLA4hi CD8+ Tmem in renal allograft recipient monkeys (from ref [14]). 
A 
B 

 
Clinical Protocol - Liver 
16 
 
ILT3/4; immunoglobulin-like transcript 3/4. 
*Already tested in phase I clinical trials 
(ii) Studies using autologous DCreg 
Type-1 diabetes 
Table 4. Agents used to generate human regulatory dendritic cells (DCreg) for (prospective) clinical use 
Agent(s) Used 
Reported Properties of DCreg 
Antigen 
(if used) 
Actual or 
Intended 
Application 
Author/ 
Reference 
*Antisense 
oligonucleotides 
targeting 
CD40, 
CD80 and CD86 
− 
Surface CD40, CD80 and 
CD86 low 
− 
IL-12 and TNF low 
− 
(IL-10 and IL-4, slight increase 
in serum levels in vivo) 
No Ag used 
Type-1 
Diabetes 
Giannoukakis, 
et al [42] 
PHASE I CLINICAL TRIAL 
 
 
 
*BAY 11-7082 
− 
CD40 low 
− 
CD86 relatively high 
− 
MHC Class II high 
− 
Low T cell stimulatory capacity 
Four 
citrullinated 
peptides 
Rheumatoid 
Arthritis 
Thomas, et al 
[44] 
PHASE I CLINICAL TRIAL 
 
 
 
Dexamethasone 
 
VitaminD3 
 
TLR-agonist (e.g. 
LPS or MPLA) 
− 
MHC Class II high 
− 
CD80 and CD86 intermediate 
− 
CD40 and CD83 low 
− 
IL-10 and TGF-β high 
− 
IL-12, IL-23, TNF low 
− 
Capable of CCR-dependent 
migration 
Synovial 
fluid 
containing 
joint-
associated auto-Ag 
Rheumatoid 
Arthritis 
Hilkens, et al 
[69] 
VitaminD3 
 
Cytokine cocktail 
(TNFα, PGE2, IL-
1β) 
− 
CD40, CD80 and CD86 low 
− 
IL-12 low 
− 
IL-10 high 
− 
Down-modulated T cell 
activation 
Seven 
myelin 
peptides 
Multiple 
Sclerosis 
Raϊch-Regué, et 
al [41] 
IL-10 
 
Cytokine cocktail 
(TNFα, PGE2, IL-
1β) 
− 
CD80, CD86, CD40 and CD83 
low 
− 
ILT3 and ILT4 up-regulated 
− 
IL-10 very high 
− 
IL-13 and IFN-γ low 
− 
Low T cell stimulatory capacity 
No Ag used 
Unspecified 
Boks, et al [39] 

 
Clinical Protocol - Liver 
17 
The first human clinical trial of DCreg was undertaken by colleagues at the University of Pittsburgh [42] 
in adult, type-1 diabetic patients. In this trial, autologous DCreg were treated ex vivo with anti-sense 
oligonucleotides targeting co-stimulatory molecules, a strategy tested initially in mice in collaboration with 
the Principal Investigator of this preIND application [70]. DCreg generated in this way induce T cell 
unresponsiveness and/or apoptosis in non-obese diabetic (NOD) mice and can prevent or reverse 
autoimmune diabetes. The principal outcome of the phase I clinical trial was that the autologous DCreg, 
administered subcutaneously, were shown to be safe and well-tolerated, as none of the patients 
experienced any adverse effects.  The DCreg that we propose to test in liver transplant patients will be 
generated from prospective live liver donors in the same GMP facility (the FACT-accredited Immunologic 
Monitoring and Cellular Products Laboratory, University of Pittsburgh Cancer Institute). Its former 
Director, Lisa Butterfield, PhD, Professor of Medicine and Immunology, has collaborated with us very 
closely in generating the human DCreg (huDCreg) data that we have incorporated in this application. Dr. 
Butterfield and this facility have extensive experience of generating GMP-grade DC for cancer therapy 
trials [50, 71-73]. 
 
Rheumatoid arthritis 
Also in the field of autoimmunity, a phase I open-label study of DCreg in rheumatoid arthritis (RA) has 
been performed by Thomas et al at the University of Queensland, Australia [43, 44]. In this trial, 
autologous DCreg were generated by treatment with an inhibitor of NFκB signaling, to prevent their 
maturation. In the Pittsburgh diabetes trial, the DCreg were not loaded with Ag, whereas in the RA trial, 
the autologous DCreg were pulsed with putative autoantigen (citrullinated peptides). Patients received a 
single dose intradermally. As in the autoimmune diabetes trial at the University of Pittsburgh, the vaccine 
was well-tolerated and no adverse effects were observed. In some cases, laboratory indices of severe 
disease symptoms appeared to improve. This study demonstrates that autologous DCreg loaded with 
disease-appropriate Ag are safely tolerated, with no notable adverse effects. While neither of these phase 
I trials demonstrated measurable efficacy, the demonstration of autologous DCreg safety in humans is 
an important step in further development of DCreg therapy.  A further clinical trial using autologous 
autoantigen-pulsed DCreg for the treatment of RA is has been developed by Hilkens and Isaacs at 
Newcastle University in the UK, (ClinicalTrials.gov identifier: NCT01352858). In their protocol, monocyte-
derived DC are modified pharmacologically with dexamethasone and VitD3, and treated additionally with 
a TLR4 agonist - clinical grade MPLA [37, 74]. The TLR4 ligand is used to stimulate the DC to process 
and present Ag efficiently on MHC class II.  As with the DCreg we propose to use in our proposed trial, 
these DCreg are characterized by high levels of IL-10 production upon stimulation and low levels of IL-
12, IL-23 and TNFα production.  As the DCreg can present Ag in association with low co-stimulatory 
molecules, they have the ability to induce hyporesponsiveness in Ag-experienced memory T cells, whilst 
polarizing naïve T cells towards anti-inflammatory cytokine production. 
In their most recent report, the Newcastle University UK group [75] have assessed the safety of inta-
articular autologous DCreg (tolerogenic DC) in patients with inflammatory arthritis and an inflamed knee 
in an unblinded, randomised, controlled, dose escalation Phase I trial. DCreg were differentiated from 
CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of 
three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an 
inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of 

 
Clinical Protocol - Liver 
18 
disease in the target knee within 5 days of treatment. Feasibility was assessed by successful DCreg 
manufacture and acceptability via patient questionnaire. Potential effects on disease activity were 
assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment 
Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood. 
There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present 
in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined 
day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of 
observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in 
peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the 
protocol by participants was high. The authors concluded that intra-articular DCreg administration 
appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 
DCreg but no systemic clinical or immunomodulatory effects were detectable. 
 
 
 
Multiple sclerosis 
A recent ex vivo study by Raïch-Regué et al [41] has shown that myelin peptide-pulsed human DC can 
induce anergy in myelin-specific T cells obtained from relapsing-remitting multiple sclerosis (MS) patients. 
The authors differentiated DCreg from monocytes of both healthy and MS patients, and found that, when 
treated with VitD3, they showed decreased expression of co-stimulatory molecules and reduced IL-12 
production, as well as enhanced IL-10 secretion. These DCreg induced stable, autoAg-specific 
hyporesponsiveness in T cells from MS patients, and exhibited an anti-inflammatory cytokine profile, 
even when re-challenged with LPS. Boks et al [39] have described human DCreg generated with IL-10 
that make high levels of IL-10, are low T cell stimulators, and optimal for functional Treg induction. 
 
Refractory Crohn’s disease (CD) 
In a phase-I, single-center, sequential-cohorts, dose-range study [45], autologous DCreg (= “tolerogenic 
DC”) were generated ex vivo from monocytes and administered by sonography-guided intraperitoneal 
(ip) injection. Six sequential refractory-CD cohorts were established: the first 3 cohorts received a single 
ip injection of DCreg at escalating doses [2 x 10(6)/5 x 10(6)/10 x 10(6)] and the last 3 cohorts received 
3 biweekly ip injections at the same escalating doses. Safety was sequentially evaluated. Patients were 
assessed from week 0 to 12 and followed up for 1-year period for safety. Nine patients were included. 
No adverse effects were detected during DCreg injection or follow-up. Three patients withdrew from the 
study due to CD worsening. Crohn's Disease Activity Index [CDAI] decreased from 274 [60] {mean 
(standard deviation [SD])} to 222 [113] [p = 0.3]; one [11%] patient reached clinical remission [CDAI < 
150] and two [22%] clinical response [CDAI decrease ≥ 100]. Crohn's Disease Endoscopic Index of 
Severity [CDEIS] decreased from 18 [5] to 13 [8] [p = 0.4]; lesions improved markedly in three patients 
[33%]. Quality of life (inflammatory bowel disease questionnaire [IBDQ]) changed from 125 [27] to 131 
[38] [p = 0.7]; remission [IBDQ at Week 12 ≥ 170] was reached in one [11%] case and response [IBDQ 

 
Clinical Protocol - Liver 
19 
score increase ≥ 16] in two [22%]. Ip administration of autologous DCreg appeared safe and feasible in 
refractory CD patients.  
Taken together, these human studies demonstrate that DCreg are capable of inducing stable 
hyporesponsiveness in antigen-reactive T cells and indicate the promise of DCreg therapy.  
 
(iii)  Allogeneic regulatory macrophages (Mreg) in renal transplantation 
Limited but relevant experience with injecting allogeneic (donor-derived) regulatory myeloid cells into 
humans exists.  In several recent publications [76-78], the University of Regensburg group have reported 
on renal transplant recipients infused once with donor-derived Mregs, either 7 d before or after transplant. 
In their most recent report [68], the group reported on two renal transplant recipients infused once with 
donor-derived regulatory macrophages (Mregs) (7 – 8 million/Kg i.v.) 7 days prior to transplantation.  Both 
patients were minimized to low-dose tacrolimus monotherapy within 24 week of transplantation and 
subsequently maintained excellent graft function. After central venous administration, most Mregs 
remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, 
and bone marrow within 30 hours after infusion. By 1 year post-transplant, both patients displayed 
patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, 
both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the 
range consistent with nonrejection.  No adverse effects were observed with 1 year follow-up. This report 
closely resembles what we propose here, i.e., to administer similar numbers of donor-derived DCreg, 7 
days before live donor liver transplant in combination with CNI, MPA and steroid.  Therefore, this report, 
albeit in a limited number of subjects, supports the feasibility and safety of our proposed trial. 
 
1.3b4. Summary 
Our extensive work and that of others has shown convincingly that donor-derived DCreg are a promising 
novel regulatory innate immune cell therapy option for inhibition of allograft rejection. They offer the 
potential to promote or restore Ag-specific tolerance and to reduce patients’ dependence on non-specific 
immunosuppressive drugs. The success of DCreg generation in vitro, including our own data on 
generation of human DCreg (Section D, IND #16945), and their safety and efficacy in animal models, 
including (as we have shown) a clinically-relevant, NHP life-sustaining transplant model, supports the 
initiation of clinical testing in living donor liver transplantation. 
 
1.3c.  Clinical Experience with Immunosuppression Withdrawal in Liver Transplant Recipients. 
1.3c1. Experience in patients not on immunosuppression through non-compliance or out of 
medical necessity 
Published reports have described that a subset of liver transplant recipients who discontinue 
immunosuppression because of noncompliance or for medical reasons per physician’s instructions (e.g. 
severe infection or PTLD) continue to have normal graft function, suggesting that they are operationally 
tolerant.  Although immunosuppression withdrawal was associated with episodes of acute rejection, 
these were mild and reversible often without the need to administer steroid boluses.  Graft loss due to 
immunosuppression withdrawal was rare.  Table 5 (below), from Lerut et al. [79] summarizes outcomes 

 
Clinical Protocol - Liver 
20 
in liver transplant recipients who were withdrawn from immunosuppression for variable periods of time 
for a variety of medical reasons. 
 
 
Table 5: Temporary immunosuppression withdrawal in liver transplantation (Leruit et al., Table 1 [79]) 
 
 
1.3c2. Elective weaning of immunosuppression (clinical trials) in patients on standard of care 
immunosuppression 
(1) Clinical experience prior to 2006 with elective weaning of immunosuppression (6m to >5yrs) after liver 
transplantation has been reviewed by Lerut et al [79].  Cumulatively, immunosuppression withdrawal was 
attempted in 304 patients (mixed pediatric and adult population) at multiple centers worldwide with 
withdrawal success rates ranging between 6 and 33%, averaging ~20%.  Acute rejection rates after 
immunosuppression withdrawal ranged between 12 and 76% while chronic rejection rates were between 
0 and 6%.  Acute rejection episodes were generally mild and almost universially reversible.  Graft loss 
due to rejection was observed in only 2 adult patients. 
  
 
 
 

 
Clinical Protocol - Liver 
21 
Table 6: Complete elective immunosuppression withdrawal in liver transplantation (Leruit et al., Table 2 
[79]) 
 
(2) A recent prospective, single-arm, multicenter clinical trial conducted by Sanchez-Fueyo and 
colleagues[80] attempted immunosuppression withdrawal in 98 adult liver transplant recipients > 3yrs 
after transplantation.  Immunosuppression was withdrawn over a 6 – 9 month period.  Out of the 98 
patients, 57 (58%) rejected and were placed back on immunosuppression (non-tolerant group), while 41 
(42%) were successfully weaned (tolerant group).  In non-tolerant patients, all rejection episodes were 
mild and reversible. In 21 cases, rejection was reversed with reinstitution of basal immunosuppression in 
addition to higher CNI levels. In the remaining patients, reinstitution of baseline immunosuppression was 
combined with low-dose steroids (20mg over 4-6 weeks; 30 patients), moderate-dose steroids (40-60 mg 
over 4-6 weeks; 4 patients), and steroid boluses (1 patient). Ultimately, normal graft function was restored 
in all cases.  All tolerant recipients (defined as absence of rejection and normal graft function 1 year after 
discontinuation of immunosuppression) remained rejection free up to 3 years following drug withdrawal.  
Three-yr follow-up biopsies in this group did not show any progressive clinically significant histological 
damage. Tolerance was independently correlated with time since transplantation.  For patients between 
3 and 5.7 years post-transplant at the time of initiating weaning, only 12% achieved the primary endpoint 
of immunosuppression withdrawal. In contrast, patients between 5.7 and 10.6 years post-transplant 
experienced a success rate of 38%, and in those >10.6 years post-transplant, the success rate was 80%.  
Therefore, the higher than expected overall success rate in the Sanchez-Fueyo trial is likely attributable 
to patients who had been stable for many years (> 5 - 6 years) after transplantation, as compared to the 
historical clinical experience reviewed by Lerut et al. in Table 5, above. 

 
Clinical Protocol - Liver 
22 
(3) In 2016, the NIH Immune Tolerance Network reported, in abstract form, a prospective, randomized 
trial designed to determine clinical benefit of early post-transplant immunosuppression withdrawal in 
stable, adult liver transplant recipients who did not receive induction therapy and were maintained on 
calcineurin inhibition [81]. Immunosuppression weaning from CNI montherapy was started between 12 
and 24 months after transplantation. Complete immunosuppression withdrawal was achieved in 13% of 
those eligible for the weaning protocol (10 of 77 patients).  Of those who failed withdrawal, 30 had biopsy-
proven rejection and 37 had abnormal liver tests; all resolved with resumption of CNI therapy with or 
without antimetabolites (MPA or AZT).  Only 13 patients (19%) required corticosteroids.  Outcomes of 
immunosuppression weaning/withdrawal were similar for patients with primary diagnoses of HCV and 
non-immune, non-viral liver failure. 
(4) Also potentially relevant is the prospective, multicenter, open-label, single-group, pilot study by Feng 
et al examining complete, late immunosuppression withdrawal in 20 pediatric recipients of parental living 
donor liver transplants (LDLR) (ITN ref 16) [82].  Patients who received liver transplants for viral hepatitis 
or autoimmune disease were excluded.  Entry requirements included stable liver function on 
monotherapy at time of weaning initiation.  Immunosuppression withdrawal was performed over a 
minimum of 36 weeks.  Applying careful selection criteria, 12 patients (60%) of this group achieved 
operational tolerance defined as an absence of rejection and normal graft function 1 year after 
discontinuation of immunosuppression.  Of the 8 patients who did not reach the primary endpoint of 
complete immunosuppression withdrawal, one was due to protocol violation, two due to acute rejection, 
and 5 due to indeterminate rejection.  The seven patients with rejection were treated with reinitiation or 
increase in immunosuppression – all returned to baseline allograft function.  As in the Sanchez-Fueyo 
study, the best correlate with complete immunosuppression withdrawal was weaning initiation time after 
transplantation (8.4 years for the operationally tolerant group vs.  6.1 years for the non-tolerant group). 
 
1.3c3. Elective weaning of immunosuppression (clinical trial) in patients who received 
experimental immunomodulatory therapy in addition to standard of care immunosuppression 
In a pilot study by Todo et al [83] performed in Japan, adoptive transfer of an ex vivo-generated regulatory 
T-cell-enriched cell product was conducted in 10 consecutive adult patients early post-LDLT. Cells were 
generated using a 2-week coculture of recipient lymphocytes with irradiated donor cells in the presence 
of anti-CD80/86 monoclonal antibodies.  All patients underwent splenectomy at the time of 
transplantation, received a single dose of cyclophosphamide (40 mg/kg) on POD #5, and were infused 
with the regulatory T cell preparation on POD #13.  All patients were initiated on MPA, steroids and CNI 
immunosupression at the time of transplantation. MPA and steroids were discontinued by 1 month and 
CNI weaning was begun at 6 months after transplantation.  CNI dose was reduced every 3 months, and 
completely discontinued over a period of 18 months. Seven patients who completed successful weaning 
and cessation of immunosuppressive agents were drug free for 16-33 months at time of publication: 4 
patients have been drug free for more than 24 months. The 3 recipients who failed weaning had 
autoimmune liver diseases and developed mild rejection during weaning and then resumed conventional 
low-dose immunotherapy.  In the successfully weaned (operationally tolerant) recipients, serial liver 
biopsies at time of weaning, at complete discontinuation of immunosuppression, 1 yr after 
discontinuation, and 2 years after discontinuation showed insignificant histological changes ranging from 
no abnormalities to low grade immune activity.  Two patients had either none to mild or mild to moderate 
fibrosis in the post-weaning biopsies.  The latter had concomitant mild chronic HCV hepatitis [83].   

 
Clinical Protocol - Liver 
23 
 
1.3c4. Summary 
Immunosuppression withdrawal in liver transplant recipients is feasible and safe but success rates, 
defined as absence of rejection and normal graft function 1 year after discontinuation of 
immunosuppression, are determined by time of initiation of weaning after transplantation and by whether 
experimental immunomodulatory therapy was administered at the time of transplantation or not.  In adult 
recipients, success rate is ~10-15% after early withdrawal and ~20-40% after late withdrawal.  
Immunomodulation with splenectomy, cyclophosphamide and Treg infusion in a pilot LDLT study enabled 
early immunosuppression withdrawal in 70% of patients.  In all studies, acute rejections caused by 
immunosuppression weaning or withdrawal were mild and reversible and graft loss due to rejection was 
very rare (< 1%).  Patients whose primary etiology of liver failure was autoimmunity were at increased 
risk of rejection during or after weaning.  Immunosuppression withdrawal appears to be safe in patients 
whose primary etiology of liver failure is HCV, but presence of active HCV hepatitis predisposes to liver 
fibrosis. 
 
1.4 SIGNIFICANCE 
 
Development of safe and effective approaches that improve long-term graft survival while reducing the 
burden of conventional immunosuppressive drugs, will have a significant impact on the health, as well as 
costs, of thousands of organ transplant recipients. Extensive pre-clinical studies conducted by Dr. 
Thomson (IND Sponsor) and others in rodents and humanized mouse models are persuasive regarding 
the potential of regulatory immune cell therapy to enhance long-term allograft survival and in many 
instances, promote donor-specific tolerance [84]. The case for evaluation of DCreg generated ex vivo is 
particularly compelling [9, 11, 12]. First, DC are inherently tolerogenic, uniquely well-equipped, 
professional Ag-presenting cells (APC) that potently regulate innate and adaptive immunity [19, 85]. 
Second, in many animal studies, DCreg adoptively transferred to allograft recipients before transplant 
induce Ag-specific tolerance and promote indefinite graft survival [9]. Moreover, this therapeutic effect 
does not appear to depend on the in vivo persistence of intact DCreg [86-88]. Independence of efficacy 
and underlying regulatory mechanisms on persistence of intact donor DCreg may be a distinct 
advantage over other cell therapy approaches, where costly repeated infusion of large numbers of 
cells and their sustained viability/stability/replication may be required to achieve a therapeutic effect. 
Third, an important attribute of DCreg is their ability to regulate preformed Tmem responses [74, 89, 90], 
that, due to molecular mimicry, cross-react with HLA Ags [66], representing a major barrier to long-term 
graft survival in humans [15, 16, 91, 92]. Fourth, in normal humans, adoptive transfer of monocyte-derived 
DCreg induces Ag-specific inhibition of effector T cell function [35, 36]. Fifth, using minimal IS in a robust, 
NHP model, we have reported that a single infusion of 3.5-10.106/kg donor-derived DCreg pre-transplant 
safely prolongs renal allograft survival, without host sensitization [13]. Importantly, this effect is 
associated with attenuation of donor-specific, alloreactive Tmem responses [13, 14]. 
The DCreg approach could potentially be readily applied in the clinic since a single infusion of a relatively 
small number of DCreg (3.5-10.106/kg) in NHP is sufficient to achieve a salutary therapeutic effect. 
Therefore, neither expensive expansion of the cell product over several weeks (compared with Treg), nor 
repeated infusion appears necessary. It is also likely that DCreg will have broader clinical application to 

 
Clinical Protocol - Liver 
24 
encompass recipients of organ transplants from deceased donors as rodent studies have shown that 
delay of DCreg infusion to 7 or 14 d after transplant is still effective in prolonging graft survival [55, 93], 
providing ample time to prepare DCreg from deceased donors. 
The possibility that DCreg administration could enable early withdrawal of immunosuppression after liver 
transplantation carries the potentially great advantage of sparing patients the cardiovascular, infectious, 
neoplastic, and renal side effects of long-term immunosuppression, particulary with CNI.  Liver 
transplantation provides a particular opportunity to achieve this goal because liver grafts are more 
tolerated by the recipient’s immune system than other solid organ grafts, with immunosuppression 
withdrawal possible in ~20% of patients overall (in the absence of any experimental, immunomodulatory 
therapy) compared to <5% in renal transplant recipients.  Increasing the proportion of liver transplant 
recipients who can be withdrawn from immunosuppression through the administration of DCreg around 
the time of transplantation could reap significant health benefits to this patient population. 
 
2. 
RESEARCH DESIGN AND METHODS 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
 
We will conduct a single center, open-label, phase I/II study to test the safety and preliminary efficacy of 
a single infusion of donor-derived DCreg in de novo living donor liver transplant recipients who will receive 
mycophenolic acid (MPA) and tacrolimus (Tac) without antibody induction. Note that any time MPA is 
referenced in this protocol equivalent doses of either mycophenolic mofetil (MMF; CellCept®) or enteric 
coated Myfortic® are acceptable. DCreg will be prepared from monocytes obtained by leukapheresis from 
prospective (non-mobilized) living liver donors and infused into their respective recipients, 7 d before 
transplantation.  ½ dose MPA will be administered concomitant with DCreg infusion. In eligible patients, 
Tac weaning will begin around month 12 (month 11.5 to month 13, pending liver biopsy results) and 
continue through at least month 18; Tacrolimus weaning is expected to take less than 12 months and will 
not exceed 55 weeks. In cases where prednisone is substituted for MPA, prednisone must be 
discontinued prior to Tac weaning.Follow-up will continue for 3 years following the last dose of 
immunosuppression. Study duration will be 7 yrs (2 yrs accrual + 5+0.5 yrs follow-up). During follow-up, 
clinical data along with peripheral blood (serum and PBMC) and both SOC protocol and for-cause 
allograft biopsies will be collected and analyzed. Our study schematic is shown below: 
 

 
Clinical Protocol - Liver 
25 
 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN 
Participant recruitment is described in Section C5.1. Briefly, patients and donors will be recruited by 
their transplant physicians. As shown in the timeline below, we will enroll the first 4 patients 3 months 
apart giving us up to 9 months of follow-up post-DCreg infusion, including DSA monitoring.  We are 
confident that a 9-month period should give sufficient time for observing DSA or other early or 
intermediate adverse events of DCreg. This initial slow pacing of recruitment builds in time for a 
thorough evaluation of safety on a patient-by-patient basis. Assuming no safety concerns, the 
remaining patients will be recruited on a rolling basis.  
 

 
Clinical Protocol - Liver 
26 
  
 
2.2a Study Procedures:   
 
An overview of the study procedures is shown in flow chart 1. 
 
 
 
Clinical protocol timeline per participant
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
Pt 1 
R
L
T
X
-
-
-
-
-
-
-
X
Pt 2
R
L
T
X
-
-
-
-
-
-
X
Pt 3
R
L
T
X
-
-
-
-
-
-
-
Pt 4
R
L
T
X
-
-
-
-
-
-
Pt 5
R
L
T
X
-
-
-
-
-
-
Pt 6
R
L
T
X
-
-
-
-
-
Pt 7
R
L
T
X
-
-
-
-
Pt 8
R
L
T
X
-
-
-
Pt 9
R
L
T
X
-
-
Pt 10
R
L
T
X
-
Pt 11 
R
L
T
X
Pt 12
R
L
T
X
Pt 13
R
L
T
X
Pt 14
R
L
T
X
Pt 15
R
L
T
X
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
Pt 1 
X
-
-
-
-
-
-
Pt 2
X
-
-
-
-
-
-
Pt 3
X
X
-
-
-
-
-
-
Pt 4
X
X
-
-
-
-
-
-
Pt 5
-
X
X
-
-
-
-
-
-
Pt 6
-
-
X
X
-
-
-
-
-
Pt 7
-
-
-
X
X
-
-
-
-
Pt 8
-
-
-
-
X
X
-
-
-
Pt 9
-
-
-
-
-
X
X
-
-
Pt 10
-
-
-
-
-
-
X
X
-
Pt 11 
-
-
-
-
-
-
-
X
X
Pt 12
-
-
-
-
-
-
-
X
Pt 13
-
-
-
-
-
-
-
X
Pt 14
-
-
-
-
-
-
-
X
Pt 15
-
-
-
-
-
-
-
X
R = Identify & Recruit,
1
2
3
4
5
6
7
8
9
10
11
12
L= Leukapheresis, 
Pt 1 
T = Generation of DCreg product, Infusion & Transplant, 
Pt 2
 = Wean Tac, 
Pt 3
X = Biopsy
Pt 4
Pt 5
- = dashes represent the additional time that may be needed for weaning and ultimately the end of the study due to an extended weaning period
Pt 6
-
Pt 7
-
-
Pt 8
-
-
-
Pt 9
-
-
-
-
Pt 10
-
-
-
-
-
Pt 11 
-
-
-
-
-
-
Pt 12
X
-
-
-
-
-
-
Pt 13
X
-
-
-
-
-
-
Pt 14
X
-
-
-
-
-
-
Pt 15
X
-
-
-
-
-
-
Year 7
Year 1
Year 2
Year 3
Year 4
Year 5
Year 6
Flow chart 1: Study Overview 

 
Clinical Protocol - Liver 
27 
  
 
2.2a1. Screening: Donors and recipients undergo rigorous screening as part of standard of care prior to 
being deemed eligible for a living-donor transplant. Therefore, a separate screening visit specifically for 
the purposes of this study will not typically be needed. When a transplant physician indicates that a 
patient may be a candidate for the study, the study coordinator or other appropriate member of the study 
team will review the patient’s medical record to verify eligibility (a waiver of consent for screening 
procedures will be requested) prior to consent being obtained. The informed consent procedures are 
detailed in Section C5.2. 
 
All donors are tested for HIV (5th generation test and NAT), CMV IgG, CMV IgM, Treponema pallidum, 
HCV (antibody and NAT), HBV (surface antigen and NAT) as part of our standard of care. For donors 
enrolled in this study, we will ensure that the sample is drawn for testing within 7 days of leukapheresis. 
At the time blood is drawn for SOC testing, samples will be drawn for additional testing that is not part of 
our standard of care (HTLV-1, HTLV-2,). Testing will be performed in the UPMC clinical laboratories and 

 
Clinical Protocol - Liver 
28 
at Creative Testing Solutions lab in Phoenix, AZ.  Results of all testing will be obtained and reviewed. We 
will review the test results prior to leukapheresis, when possible.  Positive test results do not make the 
donor ineligible for the leukapheresis procedure itself. However, positive test results, with the exception 
of CMV, would deem the cells unusable. If the test results are obtained prior to the leukapheresis 
procedure, the procedure will be cancelled. If the donor has already undergone the leukapheresis 
procedure, the lab will be notified to destroy their donated cells.  
 
Positive CMV testing in the donor will not preclude administration of the donor cell product but may require 
prophylaxis of the recipient with valganciclovir.  This will be done per our institution's standard protocol 
for checking CMV status:  
1. Check CMV recipient and donor CMV serostatus on all patients. All recipient tests should be 
checked pretransplant. If the result is equivocal in the recipient, repeat the test in 2 weeks. An 
equivocal serology in the donor should be considered positive whereas repeat equivocal 
serology in the recipient is assumed to be negative. 
2. Perform surveillance for CMV viremia with quantitative PCR weekly post-transplant until the 
week 12. Begin preemptive therapy with valganciclovir at a dose of 900 mg PO bid or 
ganciclovir IV 5mg/kg q12h (if GI absorption of oral medications is questionable, such as in 
patients with significant vomiting or diarrhea, or those critically ill in ICU) for the following: 
a. For high risk “mismatched” patients, i.e. CMV serology D+/R-, begin therapy for any 
positive quantitative number in the PCR test. 
b. For lower-risk patients, i.e. recipients who are seropositive (R+) pretransplant, start 
therapy if quantitative PCR is ≥ 500 copies/ml. 
3. Valganciclovir or ganciclovir dose should be adjusted for renal dysfunction. Calculate the 
estimated creatinine clearance and follow the dosing recommendation as below: 
 
Creatinine clearance (ml/min) 
Dose of PO valganciclovir 
Dose of IV ganciclovir 
60 or greater 
900 mg PO bid 
5 mg/kg IV q12 
40-59 
450 mg PO bid 
2.5 mg/kg IV q12 
25-39 
450 mg PO daily 
2.5 mg/kg IV q24 
10-24 
450 mg PO every other day 
1.25 mg/kg IV q24 
Less than 10 (on hemodialysis) 
Use IV ganciclovir 
1.25 mg/kg IV qHD 
 
4. Check repeat CMV PCR 1-2 weeks after the antiviral therapy is started, then weekly thereafter. 
There is no need to check the viral load more frequently. Continue the treatment until there are 
2 negative PCR tests performed at least one week apart.  
 
The Donor and Recipient will have blood samples collected for Pre-transplant sensitization as measured 
by T and B cell complement-dependent cross-match and DSA within 30 days prior to scheduled 
leukapheresis (results will be collected from SOC medical record) and within 5-6 d after DCreg infusion 
(within 72 hr prior to transplant). This will be done at an outpatient lab of UPMC by a trained professional 
of UPMC. 
 
2.2a2. Donor leukapheresis: Prospective living liver donors will undergo 3 – 5 hr outpatient 
leukapheresis at the 7 West Outpatient Transplant Unit by the Institute for Transfusion Medicine (ITxM) 

 
Clinical Protocol - Liver 
29 
without growth factor mobilization ~28 to 15 d prior to scheduled transplantation of the recipient. The 
donor will be asked to withhold caffeine for about 24 hour prior to the leukapheresis.  If they take any 
diuretics or any blood pressure medications, they may be asked to hold those on the day of the 
leukapheresis procedure. Monocytes obtained by elutriation will be cryopreserved until 14 d before 
transplantation. A second donor leukapheresis will be performed if the number of monocytes obtained is 
not sufficient for generating the required DCreg number (see Section D, IND# 16945). Up to a 75ml blood 
sample will be collected from the donor at the time of leukapheresis, processed, and stored for use in 
immunological studies as stimulators (Objective 2) or targets of recipient cells. Donor Leukapheresis will 
be performed by a trained staff member of the ITxM as per their standard operating procedures. Donors 
will sign a separate consent form when they have leukapheresis. This consent form will explain details of 
the leukapheresis, any medications they will receive before and during the leukapheresis, and any risks 
associated with the leukapheresis. 
 
2.2a3. DCreg infusion: DCreg will be prepared over 7 d culture (Section D, IND# 16945). At the end of 
the culture period, they will be suspended in in 200 ml plasmalyte A + 5% HSA and infused into the liver 
recipient once, 7 d before transplant (D -7) via a peripheral venous line (if PICC line is present, this may 
be used) over 0.5 - 1 hr.  The i.v. line will be flushed with 50 ml normal saline over 10 – 15 min after cell 
infusion.  Refer to Section 2.3.2 for further details. 
 
The most significant safety concern related to DCreg dosing is the increased risk of recipient sensitivity 
against donor antigens at higher doses. However, sensitization has less drastic consequences for liver 
transplant recipients than for kidney transplant recipients. For example, sensitization does not deem 
potential liver recipients ineligible for transplantation as it does for potential kidney recipients. Therefore, 
in evaluating the risk:benefit ratio for this protocol, we determined that it would be most appropriate to 
maximize the potential benefit by administering a potentially therapeutic dose of DCreg rather than using 
a dose-escalation approach as we proposed for IND# 16945 which uses an identical product in kidney 
transplant recipients. 
 
2.2a4. Ongoing Assessments 
 
Standard Labs: Standard Labs (drawn as SOC) will be monitored for patient safety at approximately 
days 1, 7, 14, 30 and at approximately months 3, 6, 9 and 12 post-transplant, and then monthly from 
month 12 to approximately year 3. Liver function tests to include AST, ALT and total Bili will be drawn 
every 2 weeks +/- 1 week beginning at month 12 through year 2 and then approximately monthly until 
the end of the study. Since these tests are included in the SOC labs, subjects will have laboratory tests 
for research once a month, as SOC labs drawn from month 12 to the end of the study will be utilized in 
place of a research blood draw.  Standard labs include: CBC w/ diff, complete metabolic panel 
(including LFTs), GGTP/ALP, serum cr, eGFR.  Results from SOC lipid profiles will be collected when 
completed. Research blood draws will be done at an outpatient lab of UPMC by a trained professional 
of UPMC or at Quest Diagnostics. 
Prograf (Tac) levels:  All Prograf levels will be monitored post-transplant per SOC. 
Physical Exam/ Vital signs:  Per SOC, a physical exam will be performed and vital signs obtained at 
approximately days 1, 7, 14, and 30 and at approximately 3, 6, 9, 12, and 18 months post-transplant. 

 
Clinical Protocol - Liver 
30 
Then, physical Exam/ Vital signs will be monitored after 18 months at approximately every 6-12 months 
per SOC. The exams will be performed by a trained professional at UPMC. 
Research Blood Draws: Peripheral blood for IMMUNOLOGICAL IMPACT, DSA and DCreg tracking 
will be collected from recipients at approximately day -7. Blood for pre-transplant T and B cell 
crossmatch, DSA and DCreg tracking will be collected from recipients within 72 hours prior to 
transplantation and blood for DCreg tracking only will be collected on day of transplant.  We will then 
collect peripheral blood for IMMUNOLOGICAL IMPACT, and DSA at approximately 1, 3, 6, 12, 18 (end 
of weaning), 30 (1 year post-weaning), and 54 (3 years post-weaning) months after transplantation and 
at time of for-cause biopsies.  Donors will be asked to provide up to a 75 ml of peripheral blood at the 
time of leukapheresis. This will be done at an outpatient lab of UPMC by a trained professional of 
UPMC.  
Research Urine Collection: Spot Urine will be collected from the recipient at approximately days 28 
and 90 and then again at approximately months 6, 12, 24, 36, and 48 post-transplant. This will be done 
at an outpatient lab of UPMC by a trained professional of UPMC and will be processed in UPMC 
laboratories. 
Telephone Consultation: Beginning at approximately month 12 through the end of the study after 
results received and reviewed for standard labs and prior to any change in immunosuppression 
withdrawal taper a telephone consultation will be performed by the research coordinator with the 
recipient to discuss lab results, adverse events, non- immunosuppression medication changes, and 
new immunosuppression dosing. If no change in immunosuppression is planned, qualified clinical staff 
may conduct the telephone consultation per SOC. 
Liver and Lymph Node Tissue Collection: On the day of the transplant surgery, post hepatectomy, a 
sample of liver waste tissue and some lymph nodes will be obtained for research. This will help 
investigate secondary hepatic organ cellular infiltrates, specifically the presence and location of infused 
DCreg and their possible interaction with recipient T cells. An on-call transplant pathologist will also be 
present in the OR to collect the samples to assess for any possible cancerous tumors. Since these 
samples will obtained post hepatectomy and also reviewed by a pathologist in the OR prior to being 
collected for research there is no added risk to the subject. If there are no lymph nodes removed with 
the native hepatectomy then the transplant surgeon will remove a string of up to 4 abdominal lymph 
nodes. An on-call transplant pathologist will be in the OR to review the samples for any cancerous 
tumors prior to providing to the research team. 
Implantation Liver Biopsy: An implantation liver biopsy is to be performed within approximately 60 
minutes post reperfusion. The study pathologist will provide a baseline reading. 
Post- Transplant Liver Biopsy: All post- transplant liver allograft biopsies will be performed at a 
UPMC facility by trained UPMC staff. During these liver biopsies, 2 core liver tissue samples will be 
taken, one for clinical purposes and the second for research purposes. At years 1 and approximately 
2.5 (1 year post-weaning), this will be done as part of SOC; at approximately year 4.5 (3 years post-
weaning) this will be done as part of the research protocol. For subjects who return to standard of care 
(on immunosuppression at end of study), the year 4.5 biopsy will be optional.  These protocol-
mandated biopsies will be read by both the study pathologist and local pathologist. The study pathology 
read will be used for the purposes of analysis and determination of eligibility for immunosuppression 
withdrawal, and for assessment of the primary endpoint. The local pathology read will be used for the 

 
Clinical Protocol - Liver 
31 
purposes of clinical management. The study pathology read will be available to the investigators for 
consideration at their discretion. For-cause biopsies will also be read by both the study pathologist and 
local pathologist. The local pathology read will be used for clinical management for all for-cause 
biopsies. The study pathology read of for-cause biopsies will be used for the purposes of analysis in 
conjunction with any clinical treatment to determine study outcomes such as reported rates of rejection. 
 
2.2a5. Concurrent Immunosuppression (IS) and Post-Weaning Management: 
1. D -7 to D 0 (Pre-transplant IS): MPA (500 mg twice daily) will be initiated on the day of 
DCreg infusion (d -7). The first dose will be administered prior to cell infusion. MPA may be 
replaced with an equivalent dose of enteric coated Myfortic® when necessary.  
 
2. D 0:  a) Steroid (i.v. solumedrol 500 mg) will be administered after reperfusion; b) MPA will 
be administered at a dose of 1000 mg twice-daily starting immediately after surgery. MPA 
may be replaced with an equivalent dose of enteric coated Myfortic® when necessary.  
3. D 1 to D 7 (Rapid Steriod Taper) 
a. Corticosteroids: Per SOC (Policy LT-IMMUN-1111) corticosteroids will be tapered 
over 7 days post-transplantation. Recipients will receive i.v. solumedrol (80 mg b.i.d.) 
on d1 post-transplant. The dose will be tapered through day 4. On day 5, solumedrol 
will be discontinued and oral prednisone (20 mg) will be initiated once daily. The 
dose will be tapered through day 7. Prednisone will be discontinued on day 8. 
b. MPA: Per SOC, all recipients will receive MPA 1000 mg twice-daily, or an equivalent 
dose of Myfortic®, starting immediately after surgery; the dose will be adjusted based 
on white cell count and other clinical side effects according to SOC.  
c. Tacrolimus: Per SOC, all recipients will receive oral tacrolimus (0.5- 1.0 mg/kg twice-
daily); the dose will be adjusted to achieve a trough level of 8-10 ng/ml. 
4. D 7 to M 12   
a. MPA: Per SOC, all recipients will receive MPA 1000 mg twice-daily, or an equivalent 
dose of Myfortic®, post-transplant; the dose will be adjusted based on white cell count 
and other clinical side effects according to SOC. MPA will be weaned per SOC. If 
MPA is not discontinued before or at month 12, participants will not be eligible for 
tacrolimus weaning. 
b. Tacrolimus: Per SOC, all recipients will receive oral tacrolimus.  
5. M 12 to M 18-24 (Tacrolimus weaning) 
Recipients will be eligible for tacrolimus weaning if they remain rejection-free during             
MPA weaning and have permissive LFTs (ALT, AST, Total bilirubin < 2.5 times the upper 
limit of normal) and permissive SOC liver biopsy at 12 months. In cases where 
prednisone is substituted for MPA, prednisone must be discontinued prior to Tac 

 
Clinical Protocol - Liver 
32 
weaning.Permissive liver biopsy is defined according to Table 7 below (from Demetris et 
al, [1]).  
 
Patients who fail eligibility for weaning for any of the above reasons, will return to SOC 
and will be followed up for the rest of the duration of the study. 
Note: Patients who have been switched from tacrolimus to everolimus (rapamycin) for 
any reason during the first year after transplantation will not have their rapamycin 
weaned but will be followed for the duration of the study. 
For recipients deemed eligible for tacrolimus weaning, weaning will occur in 3 to 6 week 
intervals with each subsequent reduction based on LFT stability (see flow chart 2) over 
the prior 3 to 6 week interval. Tacrolimus weaning is expected to take less than 12 
months and will not exceed 55 weeks. No single reduction should exceed 50% of the 
daily dose except the final reduction. Withdrawal will proceed as follows: 
• 
Phase 1-Daily Dosing (Number of intervals will vary depending on starting 
dose): Reduce total daily dose to 50-75% of previous dose until a dose of 0.5 
mg/day is achieved 
 
• 
Phase 2-Less than daily dosing (5 intervals): Initial reduction to 5 doses per 
week; subsequent intervals eliminate 1 dose (i.e. day) per week 
In cases where abnormal LFTs are not noted, but a for-cause liver biopsy is conducted, 
decision making will start at (2) on the flow chart. 
 
Table 7: Baseline or pre-weaning biopsy findings conducive to minimization of IS (Demetris, et 
al, Table 8, [1]) 

 
Clinical Protocol - Liver 
33 
 
6. End of Weaning to YR 5 ± 0.5 (post-weaning period) 
 
Recipients will remain off immunosuppression until end of study period unless liver 
dysfunction is identified by increase in permissive LFTs > or = 2.5x normal or a biopsy 
finding that causes concern.  In such cases, we will follow decision flow chart 3. 
  
 
 
 
 

 
Clinical Protocol - Liver 
34 
 
7. Treatment of Acute Rejection:  
Recipients who are diagnosed with rejection during or post immunosuppression withdrawal 
will be evaluated by the clinical team and, per flow charts 2 and 3, treated for rejection, 
placed back on the clinically indicated dose of immunosuppression and considered a trial 
failure.  
8. Antimicrobial Prophylaxis: All patients will receive prophylaxis for PCP, CMV, EBV and 
fungal infection according to SOC. 
 
Rationale for concurrent immunosuppressive regimen: 
In our proposed trial, transplant recipients will receive combination immunosuppressive agents according 
to SOC at our Institute, with two exceptions: (a) MPA will be initiated 7d before transplant, at the time of 
donor DCreg infusion, instead of on the day of transplant. The pre-transplant dose of MPA will be half 
the standard post-transplant dose, 500 mg twice daily, due to increased drug bioavailability in patients 
with low GFR, and (b) after MPA weaning, which is standard of care, CNI weaning will commence. The 
rationale for initiating MPA before transplant, at the time of donor DCreg infusion, is to further minimize 
the remote chance of host sensitization against donor Ags that could potentially be caused by donor 
DCreg infusion.  The utility and safety of pre-transplant IS in reducing sensitization has been documented 

 
Clinical Protocol - Liver 
35 
in patients receiving unmodified DST [94-98].  Furthermore, MPA augments and maintains the regulatory 
function of DC [99, 100].   
Antibody (Ab)-induction therapy is used in only a minority of LDLT recipients.  Nevertheless, the rationale 
for not using induction Ab treatment in patients enrolled in this trial is to avoid potential targeting of DCreg 
infused before transplantation or dampening of immunoregulatory pathways triggered by DCreg.  We 
established in NHP that such an approach (pre-transplant IS at the time of DCreg infusion and avoidance 
of lymphocyte-depleting agents) is safe and effective. We have opted, however, not to use a 
costimulation-blockade and/or rapamycin-based immunosuppressive regimen such as that employed in 
our NHP study, because of the unproven efficacy of these agents in liver transplantation compared to 
SOC IS with MPA, tacrolimus, and steroids. In cases where prednisone is substituted for MPA, 
prednisone must be discontinued prior to Tac weaning.Since our proposed clinical trial is a safety 
and preliminary efficacy trial, we have chosen to adhere to a safe and proven IS regimen that does not 
interfere with DCreg action. Infusion of DCreg together with SOC IS (MPA, tacrolimus, and prednisone) 
as proposed in this clinical trial, is justified on the basis of published rodent studies (Section C1.3). 
 
2.2b. Study Visits and Assessments: 
 
1. For the Primary Objective of SAFETY, the following will be assessed in the graft recipients  
 
i) 
Adverse events (AE) rate related to DCreg infusion to the time of transplant (up to 7 
days after infusion). 
ii) 
Pre-transplant sensitization as measured by T and B cell complement-dependent cross-
match prior to or at the time of leukapheresis and within 5-6 d after DCreg infusion 
(within 48 hr prior to transplant).  
iii) 
Incidence of clinically suspected rejection, biopsy-proven acute clinical or sub-clinical 
rejection (BPAR) rate as documented from medical record review at approximately 1, 3, 
6, 12, 18, 24, 30, 36, 42, 48 & 54 months post-transplant. 
iv) 
Allograft failure or patient death as documented from medical record review at 
approximately at 12, 18, 24, 30, 36, 42, 48 & 54 months post-transplant. 
v) 
Incidence rates of opportunistic infection (CMV, EBV, PCP, systemic fungal, bacterial, 
and mycobacterial) and malignancy (including PTLD) as documented from medical 
record review at 1, 3, 6, 12, 18, 24, 30, 36, 42, 48 & 54 months post-transplant. 
 
 
2. For the Primary Objective of PRELIMINARY EFFICACY, the following will be assessed in the 
graft recipients: 
Absence of rejection within 1 year after complete immunosuppression cessation based on liver biopsy 
criteria detailed in Table 8, below (from the 2016 Comprehensive Update of the Banff Working Group 
on Liver Allograft Pathology, Demetris et al. 2016 [1]). 
 

 
Clinical Protocol - Liver 
36 
 
 
3. For the Secondary Objective related to DSA, we will measure donor-specific antibody (DSA) titer 
and isotype using single Ag bead assay at approximately 1, 3, 6, 12, 18, 30 and 54 months post-
transplant. DSA measurement requires typing for human leukocyte antigen (HLA) in both the recipient 
and the donor. This will be done at an outpatient lab of UPMC by a trained professional of UPMC and 
will be processed in UPMC laboratories. 
 
 
4. For the Secondary Objective of IMMUNOLOGICAL IMPACT, we will collect up to a 75 ml of 
peripheral blood from recipients for immunological studies at approximately 1, 3, 6, 12, 18, 30, and 54 
months after transplantation and at time of for-cause biopsies.  Donors will be asked to provide up to a 
75 ml of peripheral blood at the time of leukapheresis. 
 
5. For the Secondary Objective of CHANGES OVER TIME, we will collect the following data from 
questionnaires, recipient interviews and/or medical record reviews:  
i) Change in renal function (defined as estimated GFR calculated by CKD-EPI: 
http://www.qxmd.com/calculate-online/nephrology/ckd-epiegfr) will be calculated from 1 year post-
transplantation to 1 year following immunosuppression weaning. 
 
ii) Quality of Life will be measured by the SF-36 at 1 year post-transplantation and at the end of the 
study, and the change in scores will be calculated. These surveys will be conducted in a private 
clinic setting by the trained professional at UPMC. 
 
iii) Cardiovascular factors assessed include incidence of hypertension necessitating medication, 
post-transplant diabetes, hyperlipidemia, hypercholesterolemia as documented from medical record 
review at yearly post-transplant visits. 
 
2.2b1. Follow-up Exams and Labs (Donors): 
Table 8: Follow-up biopsy findings suggesting that the patient is unlikely to benefit from minimal or 
absent immunosuppression (Demetris, et al, Table 9, [1]) 

 
Clinical Protocol - Liver 
37 
AE related to leukapheresis of the donor (e.g., anaphylaxis, hypersensitivity reactions, thrombophlebitis, 
embolism, infection, cardiovascular events, and death) will be assessed by donor report 12 to 48 hours 
and again at 7 days (+ 1 day) after leukapheresis procedure. 
 
 
2.2b2.  Table of all study visits and assessments (Recipients only, based on minimum weaning 
schedule):  
 
 
 
 
2.3 STUDY DRUG 
 
2.3.1. Study Drug Preparation and Dispensing  
 
Monocyte elutriation from donor leukapheresis products and preparation of GMP DCreg will be performed 
at 
the 
UPCI 
Immunologic 
Monitoring 
and 
Cellular 
Products 
Laboratory 
(IMPCL) 
(http://www.upci.upmc.edu/imcpl/cpl.cfm) under the direction of Theresa Whiteside, PhD. The lab is CAP-
inspected, CLIA-certified, FACT-accredited, and maintains a Facilities Master File with the FDA. Dr. 
Whiteside is a clinical immunologist and has extensive experience in evaluating human immune-

 
Clinical Protocol - Liver 
38 
mediated diseases. The IMCPL lab supports not only cancer-related DC vaccine trials, but also immune 
response-inhibiting DC, that have been safety-tested at our Center in type-1 diabetes [42]. We have 
already generated DCreg from cryopreserved monocytes in 3 large scale-up cultures from healthy 
volunteers and verified the viability, purity, phenotype and stability of the final cell product. See Section 
D: Chemistry, Manufacturing and Control Information (CMC), from IND# 16945) for details of the DCReg 
preparation. 
 
As noted in IND# 16945, Section D: CMC Information, section 3.2.4, the cells are formulated per SOP-
0310 (Section D Appendix 5, IND# 16945) with a target amount of donor DCreg per kg in ml of 2 - 5% 
HSA loaded into a transfer pack that is labeled per CMC section 5.2 (also Item E. Labeling). The product 
is hand-delivered to the patient treatment area. 
 
All patients will receive the same dose of DCreg (2.5 – 10.0 million cells/kg body wt). The IMPCL will be 
able to provide an estimate that there are enough cells to achieve that dose range about 24 hours after 
they receive the leukapheresis product. They will not know the exact number of DCreg cells until the day 
of infusion. If the final formulation contains fewer than 2.5 million cells/kg body wt the recipient will not 
receive the infusion and that subject will be replaced. 
 
2.3.2. Drug Administration  
On Day -7, the prospective liver transplant recipient will receive an infusion of donor-derived DCreg.  This 
infusion will be performed in the 7W Patient Service Center of the Thomas E. Starzl Transplantation 
Institute or in the Montefiore Clinical and Translational Research Center (MUH-CTRC) located on the 6th 
floor of Montefiore Hospital. Both allow for close monitoring of patients and is equipped to manage 
possible medical emergencies. 
 
In the morning of Day -7, prior to DCreg infusion, the prospective transplant recipient will take MPA 
500mg.  From this day until the day of transplant, the patient will continue to receive MPA 500mg twice 
daily (1/2 of a standard dose) to minimize the low potential risk of sensitization against donor 
alloantigen caused by the infusion of donor-derived DCreg. Subjects will be provided a bottle with 7-day 
supply of MPA and will be asked to return empty bottle when they come in on day of transplant. 
 
Upon admission, patients will undergo a physical examination and a peripheral venous line will be 
inserted, if no PICC line is present. Thirty to sixty minutes prior to the infusion, the patient will receive a 
prophylactic dose of acetaminophen 500mg-1000mg PO to mitigate possible febrile reactions to the 
DCreg infusion.  Additionally, a prophylactic dose of diphenhydramine 25-50mg PO or IV will also be 
given to prevent possible urticaria or pruritus. If the transplant recipient is a female of child bearing 
potential and has an incompatible RH factor with the donor, RhoGAM, a prescription medicine may be 
given by intramuscular injection to prevent Rh immunization, a condition in which an individual with Rh-
negative blood develops antibodies after exposure to Rh-positive blood. The DCreg suspension will be 
infused over 30-60 minutes on an infusion pump.  After the infusion is completed, a 50ml normal saline 
flush will be administered over 10-15 minutes via an infusion pump.   
 
During the entire time the patient is in the unit (7W or MUH-CTRC), study team members (including a 
doctor and nurse) will be present or immediately available for any questions or complications that may 

 
Clinical Protocol - Liver 
39 
arise. The patient will not be discharged until post-infusion blood test results have been reviewed and the 
study team determines that it is safe for the patient to leave. 
 
 
 
Monitoring 
 
Cardiac Monitoring. Upon admission to 7W, the patient will undergo a baseline 12-lead EKG. Prior 
to the administration of the DCreg, the patient will be placed on a cardiac monitor, and will be 
continuously monitored through the infusion and for approximately one hour post infusion completion.  
 
Vital Signs. Vital signs (temperature, blood pressure, respirations, pulse, pulse oximetry) will be 
checked at baseline (prior to transfusion), approximately every 15 minutes during the infusion and for 
one hour post infusion completion.  Vital signs will then be monitored approximately every 30 minutes 
for the next three hours for a total of 4 hours of monitoring post infusion completion. 
 
Blood tests  
The following blood tests will be conducted at baseline and approximately one hour post-infusion: 
• 
CBC and platelets 
• 
PT, PTT, INR 
• 
Chemistry panel to include LIVER and KIDNEY function tests 
 
Additional blood will be drawn for future laboratory studies as follows: 
• 
Baseline/Pre-Infusion: 65 mL blood in 6 green top tubes and 5 mL blood in 1 red top tube for 
Immunologic studies, DSA and DCreg tracking 
• 
Within approximately 1 hour post-infusion: 10 mL green top for DCreg tracking   
 
Premature Discontinuation of Investigational Agent 
DC reg therapy will be discontinued for any participant for any of the following reasons: 
• 
Hypersensitivity reaction 
• 
Grade 4 Adverse Event  
• 
Cytokine release syndrome 
There are circumstances in which subjects who provide informed consent might not receive the study 
drug or might receive less than a full dose of the study drug. The amount of study drug received will be 
recorded on the participants’ case report form. 
If prospective liver transplant recipient develops minor adverse event during DCreg infusion [defined as 
those with CTCAE ≤ 3] infusion will be temporarily discontinued for 30 minutes. The remaining infusion 
will be administered after 30 minutes at a slower rate and infusion time will be doubled. Additional 
doses of acetaminophen 500-1000mg PO and diphenhydramine 25-50mg PO or IV may be 
administered to alleviate symptoms of minor adverse reaction. If the symptoms recur the infusion will be 
discontinued, no further DCregs will be administered.  Patient will be monitored during the entire period 
including one-hour post infusion.   
 

 
Clinical Protocol - Liver 
40 
If it is discovered that the dose of the DCreg infusion will be less than 2.5 million cells/kg body wt., the 
subject will not receive the infusion or any other study medication and will be discharged. 
 
2.3.3. Dose Selection 
The cell dose range proposed in our study (2.5 – 10 million/kg) corresponds to the range in which both 
safety and therapeutic efficacy were demonstrated in our NHP renal transplant model (see Section 
1.3b2).  In the NHP study, animals received 3 – 10 million/kg donor-derived DCreg i.v. once, 7 days prior 
to transplantation. We did not observe any adverse events related to cell infusion or any evidence of host 
sensitization based on measurement of circulating alloantibody. We did observe significant prolongation 
of graft survival, reduction in weight loss and proteinuria, and attenuation of Tmem responses in monkeys 
that received DCreg. In this particular NHP study, minimal immunosuppression was given and was 
tapered and discontinued by 6 months post-transplantation, as described in Section 1.3b2 and Figure 1. 
 
No human data regarding infusion of allogeneic DCreg exist. However, the Regensburg group in 
Germany used a related donor monocyte-derived (allogeneic) regulatory immune cell type, Mreg, which 
they infused into > 20 renal transplant recipients (see Section 1.3b4). In their most recent report, they 
infused 8 million cells/kg and 7.1 million cells/kg i.v., respectively into two patients. No adverse events 
related to cell infusion, allosensitization, or graft rejection were observed [68].  These doses of allogeneic 
Mreg infused in humans are within our proposed DCreg dose range of 2.5 – 10 million DCreg per kg.  
 
Limited tracking studies in the two Regensburg patients showed that after central venous administration, 
Indium 111-labeled Mregs were seen by SPECT imaging to traffic from the pulmonary vasculature via 
the blood to liver, spleen, and bone marrow within 30 hours after infusion [68]. Tracking was not 
performed later than 30 hours.  
 
2.3.4. Treatment Period 
The experimental treatment consists of transplant recipients receiving a single infusion of donor-derived 
DCreg, 7 days prior to transplantation followed by weaning of CNI from months 12 to 18 post-
transplantation.  
 
2.3.5. Medication Compliance 
The study drug dosage regimen consists of a single infusion of DCreg. Thus, research subject adherence 
to the regimen is not a concern. There are circumstances in which subjects who provide informed consent 
might not receive the study drug or might receive less than a full dose of the study drug. The amount of 
study drug received will be recorded on the participants’ case report form. 
 
Subjects who: 
a.      Are enrolled but do not receive DCreg infusion will be replaced. No study intervention or follow-up 
will occur. 
b.      Receive a partial DCreg infusion (less than 2.5 million cells per kg weight) will be replaced. 
Additionally, they will be followed on the reduced follow up Recipient SOE (2.2b2. See footnote) and will 
not be eligible for weaning.   
c.      Receive DCreg infusion but do not receive a liver transplant per protocol will be replaced. 
Additionally, they will be followed on the reduced follow up Recipient SOE (2.2b2. See footnote) and will 

 
Clinical Protocol - Liver 
41 
not be eligible for weaning.  These subjects may receive a transplanted liver from their original donor or 
a different donor at a later date. 
d.      Receive DCreg infusion and are transplanted per protocol will follow the recipient SOE (2.2b2). If 
they subsequently decline to participate in the weaning, for reasons unrelated to adverse events, they 
will not be replaced but will be followed up according to reduced follow up Recipient SOE (2.2b2. See 
footnote). 
 
Study follow-up is commensurate with routine clinical follow-up. Transplant patients, in general, tend to 
be highly motivated, making non-compliance with recommended follow-up unlikely. All study patients will 
be followed up at the Starzl Transplantation Institute, UPMC, for the entire study period.   
 
2.3.6. Medication Storage and Accountability 
DCreg will be formulated and loaded into a labeled transfer pack within 24 hours of infusion. The 
transfer pack will be stored at 4o C and hand-delivered to the patient treatment area. Each DCreg 
preparation is specific to a donor/recipient pair and only used for the intended recipient. Any excess 
cells from remaining after the trial closure are either destroyed or used for research, per the trial PI. 
 
2.3.7 Concomitant Medications 
Immunosuppressive Therapy: Transplant recipients will receive FDA-approved, combination 
immunosuppressive medications according to SOC at the Starzl Transplantation Institute (STI) with two 
exceptions: (a) MPA will be initiated 7 days prior to transplantation, at the time of donor DCreg infusion, 
instead of on the day of transplantation. The pre-transplant dose of MPA will be half the standard post-
transplant dose, 500 mg twice daily, due to increased bioavailability of the drug in patients with low GFR, 
and (b) after MPA weaning, which is standard of care, CNI weaning will commence. In cases where 
prednisone is substituted for MPA, prednisone must be discontinued prior to Tac weaning. 
 
Outline of the proposed immunosuppressive therapy and weaning is as follows: 
1. D -7 to D 0 (Pre-transplant IS): MPA (500 mg twice daily) will be initiated on the day of 
DCreg infusion (d -7). The first dose will be administered prior to cell infusion. MPA may be 
replaced with an equivalent dose of enteric coated Myfortic® when necessary.  
 
2. D 0:  a) Steroid (i.v. solumedrol 500 mg) will be administered after reperfusion; b) MPA will 
be administered at a dose of 1000 mg twice-daily starting immediately after surgery. MPA 
may be replaced with an equivalent dose of enteric coated Myfortic® when necessary.  
3. D 1 to D 7 (Rapid Steriod Taper) 
a. Corticosteroids: Per SOC (Policy LT-IMMUN-1111) corticosteroids will be tapered over 7 
days post-transplantation. Recipients will receive i.v. solumedrol (80 mg b.i.d.) on d1 post-
transplant. The dose will be tapered through day 4. On day 5, solumedrol will be 
discontinued and oral prednisone (20 mg) will be initiated once daily. The dose will be 
tapered through day 7. Prednisone will be discontinued on day 8. In cases where 
prednisone is substituted for MPA, prednisone must be discontinued prior to Tac 
weaning. 

 
Clinical Protocol - Liver 
42 
b. MPA: Per SOC, all recipients will receive MPA 1000 mg twice-daily, or an equivalent dose 
of Myfortic®, starting immediately after surgery; the dose will be adjusted based on white 
cell count and other clinical side effects according to SOC.  
c. Tacrolimus: Per SOC, all recipients will receive oral tacrolimus (0.5- 1.0 mg/kg twice-daily); 
the dose will be adjusted to achieve a trough level of 8-10 ng/ml. In cases where 
prednisone is substituted for MPA, prednisone must be discontinued prior to Tac 
weaning. 
4. D 7 to M 12   
a. MPA: Per SOC, all recipients will receive MPA 1000 mg twice-daily, or an equivalent dose 
of Myfortic®, post-transplant; the dose will be adjusted based on white cell count and other 
clinical side effects according to SOC. MPA will be weaned per SOC. If MPA is not 
discontinued before or at month 12, participants will not be eligible for tacrolimus weaning. 
In cases where prednisone is substituted for MPA, prednisone must be 
discontinued prior to Tac weaning. 
b. Tacrolimus: Per SOC, all recipients will receive oral tacrolimus.  
5. M 12 to M 18-24 (Tacrolimus weaning) 
Permissive liver biopsy is defined according to Table 8 from Demetris et al, displayed in 
section 2.2a4 item 6 [1].  
Patients who fail eligibility for weaning for any of the above reasons, will return to SOC 
and will be followed up for the rest of the duration of the study. 
Note: Patients who have been switched from tacrolimus to everolimus (rapamycin) for 
any reason during the first year after transplantation will not have their rapamycin 
weaned but will be followed for the duration of the study. 
For recipients deemed eligible for tacrolimus weaning, weaning will occur in 3 to 6 week 
intervals with each subsequent reduction based on LFT stability (see flow chart 2) over 
the prior 3 to 6 week interval. Tacrolimus weaning is expected to take less than 12 
months and will not exceed 55 weeks. No single reduction should exceed 50% of the 
daily dose except the final reduction. Withdrawal will proceed as follows: 
• 
Phase 1-Daily Dosing (Number of intervals will vary depending on starting 
dose): Reduce total daily dose to 50-75% of previous dose until a dose of 0.5 
mg/day is achieved 
 
• 
Phase 2-Less than daily dosing (5 intervals): Initial reduction to 5 doses per 
week; subsequent intervals eliminate 1 dose (i.e. day) per week 
In cases where abnormal LFTs are not noted, but a for-cause liver biopsy is conducted, 
decision making will start at (2) on flow chart 2. 
 
6. M 18 to YR4.5 (post-weaning period) 
 
Recipients will remain off immunosuppression until end of study period unless liver 

 
Clinical Protocol - Liver 
43 
dysfunction is identified by increase in permissive LFTs > or = 2.5x normal or a biopsy 
finding causes concern.  In such cases, we will follow decision flow chart 3. 
 
7. Treatment of Acute Rejection:  
Recipients who are diagnosed with rejection during or post immunosuppression withdrawal 
will be evaluated by the clinical team and, per flowcharts 2 and 3, treated for rejection, placed 
back on the clinically indicated dose of immunosuppression and considered a trial failure.  
8. Antimicrobial Prophylaxis: All patients will receive prophylaxis for PCP, CMV, EBV and 
fungal infection according to SOC. 
  
2.3.8 Rescue Medications 
See treatment of acute rejection above 
 
2.4 STUDY ENDPOINTS 
2.4.1 Primary Endpoints:  
(a) Safety: Safety will be determined by assessing the proportion of subjects who experience the 
following events:  
i)  
CTCAE Grade 4 or higher infusion reaction 
ii)  
CTCAE Grade 4 or higher infection 
iii)  
Malignancy other than non-melanoma skin cancer or HCC recurrence 
iv)  
Rejection resulting in recipient death or retransplantation 
v)  
Biopsy-proven severe acute rejection 
vi)  
Any grade chronic rejection 
vii)  Non-surgical graft loss 
viii)  Recipient death 
(b) Preliminary Efficacy:  Proportion of patients able to achieve staged immunosuppression withdrawal 
with operational tolerance 1 year after complete immunosuppression cessation based on liver biopsy 
criteria detailed in Table 9 of the 2016 Comprehensive Update of the Banff Working Group on Liver 
Allograft Pathology (Demetris et al. 2016)[1] as shown in Section 2.2b.item 2 of this protocol. 
2.4.2 Secondary Endpoints: 
1. DSA levels will be measured early (<6 wks) and late (>6 wks) after transplantation  
2. In vitro studies conducted in the Human Immunology Laboratory at the STI will address secondary 
objective #2. 
(a) Impact of DCreg infusion on recipients’ immune response to donor alloantigens as measured by 
following parameters: 
i) Frequency, phenotype and function (in donor-specific assays) of conventional CD4 and CD8 
T cell populations: naïve T cells (Tn), central memory T cells (Tcm), effector memory T cells 
(Tem), stable effector memory T cells (TemRA), and follicular helper T cells (Tfh). 
ii) Frequency and phenotype of CD4 regulatory T cell populations (Treg) 
 

 
Clinical Protocol - Liver 
44 
(b) We will also assess longevity of infused donor DCreg in circulation of recipient by identifying donor 
HLA on peripheral blood dendritic cells. 
 
3. Recipient interview and medical records reviews will address the secondary objective #3 as follows: 
 
(a) Change in renal function (defined as estimated GFR calculated by CKD-EPI: 
http://www.qxmd.com/calculate-online/nephrology/ckd-epiegfr) will be calculated from 1 year 
post-transplantation to 1 year following immunosuppression weaning. 
 
(b) Quality of Life will be measured by the SF-36 at 1 year post-transplantation and at the end of 
the study, and the change in both measures will be calculated. 
 
(c) Cardiovascular factors assessed include incidence of hypertension necessitating medication, 
post-ttransplantation diabetes, hyperlipidemia, hypercholesterolemia as documented from 
medical record review at yearly post-transplant visits. 
2.5 STATISTICAL ANALYSIS 
The statistical methods are focused on defining stopping rules and justifying the sample size of n = 15 
DCreg recipients based on providing sufficient power to identify rates elevated by at least 10% for each 
of the endpoints, while maintaining a reasonable type I error, i.e. achieving a relatively low probability of 
stopping the trial when a given endpoint is in truth at the expected background rate. The analysis 
population will be all 15 patients treated with DCreg.  
(i) For the stopping rules and associated power and type I error rates, we calculated probabilities of 
correctly stopping if the true rate was elevated by at least 10% or falsely stopping the trial (if the assumed 
background rate was the true rate) separately for each AE using a simple binomial distribution with the 
assumed probabilities.  
First Year – pre-weaning: 
CTCAE grade 4 or higher infusion reaction: With an expected rate near 0% (used 0.1%), the 
stopping rule was set at X≥1 (i.e. any events). Using this stopping rule, under these 
assumptions, the rate of false rejections is only 1.2%. The power is over 90% (93.1%) for 
detecting a rate of 20%. 
 
CTCAE grade 4 or higher infection: With an expected rate of 15%, the stopping rule was set 
at X≥4. Using this stopping rule, under these assumptions, the rate of false rejections is 9.2%. 
The power is quite low for either a 10 or 20% increased rate, but we have about 80% power 
(86.6%) for detecting a rate of 45% (or 30% higher than the expected 15%). 
 
Malignancy other than non-melanoma skin cancer or HCC recurrence: With an expected 
rate of 4%, the stopping rule was set to X≥2. The false significant error rate was about 8% but 
with reasonable to high power (82 to 95%) for detecting significant differences with increases of 
10 and 20% (to 14% and 24%) in the event rate.  
 

 
Clinical Protocol - Liver 
45 
Rejection resulting in recipient death or re-transplantation:  With an expected rate of 0.5%, 
the stopping rule was set at X≥1. Using this stopping rule, under these assumptions, the rate of 
false rejections is 5.8%. The power is quite high for either a 20 or 30% increased rate with 
>90% power (93 and 99). 
 
Biopsy-proven severe acute rejection: With an expected rate of 1%, the stopping rule was set 
at X≥1. Using this stopping rule, under these assumptions, the rate of false rejections is 
somewhat high at 11.4%. The power is reasonable (75.3%) for detecting an increase of 10% 
and is over 90% (94 and 99%) for increases of 20% and 30%.  
 
Any grade chronic rejection: With an expected rate of 1%, the stopping rule was set at X≥1. 
Using this stopping rule, under these assumptions, the rate of false rejections is somewhat high 
at 11.4%. The power is reasonable (75.3%) for detecting an increase of 10% and is over 90% 
(94 and 99%) for increases of 20% and 30%. 
 
Non-surgical graft loss: With an expected rate of 5%, the stopping rule was set to X≥2. The 
false significant rate was about somewhat high at 11.8% and the power was low for detecting a 
10% increase (to 15%) at 55.7%, but the power was high for the alternatives of a 20% or 30% 
increase, with 84% and 96% power, respectively.  
 
Recipient death: With an expected rate of 10%, the stopping rule was set to X≥3. The false 
significant error rate was somewhat high at 11.1% but with about 75% power for detecting a 
20% increase and over 90% power (92%) for detecting a 30% increase in the death rate.  
 
Year 2-5 ± 0.5: 
CTCAE grade 4 or higher infection: With an expected rate of 7.5%, the stopping rule was set 
at X≥3. Using this stopping rule, under these assumptions, the rate of false rejections is 5.6%. 
The power is quite low for either a 10 or 20% increased rate, but we have over 80% power 
(88.6%) for detecting a rate of 37.5% (or 30% higher than the expected 7.5%). 
 
Malignancy other than non-melanoma skin cancer or HCC recurrence: With an expected 
rate of 12%, the stopping rule was set at X≥4. Using this stopping rule, under these 
assumptions, the rate of false rejections is 4.6%. The power is quite low for either a 10 or 20% 
increased rate, but we have over 80% power (81.5%) for detecting a rate of 42% (or 30% higher 
than the expected 12%). 
 
Rejection resulting in recipient death or re-transplantation:  With an expected rate of 0.5%, 
the stopping rule was set at X≥1. Using this stopping rule, under these assumptions, the rate of 
false rejections is 5.8%. The power is quite high for either a 20 or 30% increased rate with 
>90% power (93 and 99). 
 
Biopsy-proven severe acute rejection: With an expected rate of 1%, the stopping rule was set 
at X≥1. Using this stopping rule, under these assumptions, the rate of false rejections is 

 
Clinical Protocol - Liver 
46 
somewhat high at 11.4%. The power is reasonable (75.3%) for detecting an increase of 10% 
and is over 90% (94 and 99%) for increases of 20% and 30%.  
 
Any grade chronic rejection: With an expected rate of 2%, the stopping rule was set at X≥2. 
Using this stopping rule, under these assumptions, the rate of false rejections is 2.3%. The 
power is quite low for either a 10% increased rate, but we have near 80% power (77.8%) for 
detecting an increase of 20% and over 90% power (93.5%) for detecting an increase of 30% 
above the expected rate. 
 
Non-surgical graft loss: With an expected rate of 5%, the stopping rule was set to X≥2. The 
false significant rate was about somewhat high at 11.8% and the power was low for detecting a 
10% increase (to 15%) at 55.7%, but the power was high for the alternatives of a 20% or 30% 
increase, with 84% and 96% power, respectively.  
 
Recipient death: With an expected rate of 5%, the stopping rule was set to X≥2. The false 
significant rate was about somewhat high at 11.8% and the power was low for detecting a 10% 
increase (to 15%) at 55.7%, but the power was high for the alternatives of a 20% or 30% 
increase, with 84% and 96% power, respectively.  
 
For the endpoint of preliminary efficacy, we will compare the proportion of recipients who are 
successfully weaned off immunosuppression after receiving the DCreg infusion to the expected number 
of recipients who could be weaned off immunosuppression without any additional intervention per 
historical data (15%). Our sample of 12 recipients will give us 80% power to detect a significant difference 
between true proportions of 15% and 67%.   
(ii) For the secondary endpoints, the analysis will focus on descriptive and graphical summaries of the 
data given the small sample size. We will not focus on p-values and significance tests but will instead 
aim to identify trends worth further investigation in a larger trial.   
 
3. 
HUMAN SUBJECTS 
 
3.1 SUBJECT POPULATION 
The study population will be living donor transplant recipients between the ages of 18 and 75 managed 
at the University of Pittsburgh Medical Center (UPMC) and their adult donors. Enrollment into the study 
is defined as providing informed consent by the recipient for study participation and by the donor for 
leukapheresis as per University of Pittsburgh IRB policies. To achieve N=15 recipients who will receive 
the DCreg infusion, we anticipate initially enrolling 18 recipient/donor pairs. 
 
3.1.1 Inclusion of Women and Minorities 
Women will be included in the proposed research. In the cases of liver transplantation approximately 
70% of recipients are males and 30% are females. Therefore, we anticipate the enrollment to mirror these 
numbers. 
 

 
Clinical Protocol - Liver 
47 
Minorities will be included in the proposed research. Based on demographic distribution of the patient 
population at UPMC, the racial categories include approximately 75% white, 15% African American, and 
10% other racial groups. Therefore, we anticipate a similar representation of minorities in this study. 
 
3.1.2 Inclusion of Children 
Children under age 18 will not be included in the proposed research because the standard of care for 
such children is different than the standard of care for adults.  
 
3.1.3 Inclusion of Prisoners and Other Special Classes of Subjects 
Special classes of subjects such as fetuses, pregnant women, prisoners, or others who may be 
considered vulnerable populations will not be included. 
 
3.2 INCLUSION CRITERIA 
 
Donors 
1. Able to understand and provide informed consent  
2. Male or female between the ages of 18 and 55  
3. Meet all standard institutional and UNOS criteria for liver donation 
4. For females of childbearing potential, a negative urine or serum pregnancy test  
5. Negative for HIV (5th generation Test and NAT), HTLV-1, HTLV-2;* 
6.   Negative for hepatitis C (antibody and NAT), hepatitis B (surface antigen and NAT)**does not 
preclude donors from undergoing leukapheresis but cells may not be infused into recipient. 
 
Recipients 
1. Low risk recipient approved for LDLT, irrespective of gender, race, or ethnic background.  Low 
risk is defined by absence of exclusion criteria (below). 
2. Between ages 18 and 75 years 
3. Undergoing de novo (first) liver transplant  
4.  Female subjects of childbearing potential must have a negative pregnancy test upon study 
entry. 
5.  Agreement to use contraception; according to the FDA Office of Women’s Health 
(http://www.fda.gov/birthcontrol), there are a number of birth control methods that are more than 
80% effective. Female participants of child-bearing potential must consult with their physician 
and determine the most suitable method(s) from this list to be used from the time that study 
treatment begins until 1 year after completion of immunosuppression withdrawal. 
 
3.3 EXCLUSION CRITERIA 
 
Recipients 
1. Positive T or B cell complement cross-match prior to infusion 
2. Repeat liver transplant 
3. Other solid organ transplant 

 
Clinical Protocol - Liver 
48 
4. Significant co-morbid conditions such as severe heart or lung disease 
5. Following etiology of liver disease: PSC, autoimmune, PBC 
6. If prior history of Hepatitis B or C (HBV or HCV) infection.  Hepatitis B or C Virus (HBV or HCV) 
viral load positive at the time of transplantation (successfully treated HBV or HCV patients are not 
excluded) 
7. Positive antigen-antibody immunoassay for HIV-1/2 
8. Creatinine > 1.6 mg/dl prior to transplantation 
 
4. 
IRB APPROVAL AND FDA AMENDMENTS 
 
The Investigator will obtain, from the University of Pittsburgh Institutional Review Board (IRB), 
prospective approval of the clinical protocol and corresponding informed consent form(s); modifications 
to the clinical protocol and corresponding informed consent forms, and advertisements (i.e., directed at 
potential research subjects) for study recruitment.    
 
The only circumstance in which a deviation from the current IRB-approved clinical protocol/consent 
form(s) may be initiated in the absence of prospective IRB approval is to eliminate an apparent immediate 
hazard to the research subject(s).  In such circumstances, the Investigator will promptly notify the 
University of Pittsburgh IRB of the deviation. The Investigator should also notify the sponsor of this event.   
 
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 CFR Parts 50 and 
21 CFR 56, and in conformance with applicable International Conference on Harmonization (ICH) 
Guidelines on Good Clinical Practice (CGP).  
 
In the event that the University of Pittsburgh IRB requires, as a condition of approval, substantial changes 
to this Phase I/II clinical protocol, or in the event of the Investigator’s decision to modify the previously 
accepted clinical protocol the Sponsor will submit (i.e., in advance of implementing the change) a Protocol 
Amendment to the IND describing any change to the Phase I/II clinical protocol that significantly affects 
the safety of the subjects.  For changes that do not affect critical safety assessments, the revisions to the 
clinical protocol will be addressed in the Annual Report to the IND.  
 
5. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES 
 
5.1 RECRUITMENT METHODS:  
Adult recipients who have been approved for living donor transplantation and will undergo primary de 
novo liver transplantation will be recruited, along with their donors, by their transplant physician/s. If the 
patient is under the care of a physician who is not an investigator on the study, then the caring physician 
will ask the patient for permission to be approached by a member of the study team.  
 
As shown in Section C2.2, we will enroll the first 4 patients 3 months apart giving us up to 9 months of 
follow-up post-DCreg infusion, including DSA monitoring.  We are confident that a 9-month period should 
give sufficient time for observing DSA or other early or intermediate adverse events of DCreg. This initial 

 
Clinical Protocol - Liver 
49 
slow pacing of recruitment builds in time for a thorough evaluation of safety on a patient-by-patient basis. 
Assuming no safety concerns, the remaining patients will be recruited on a rolling basis.  
Subjects who: 
a. Are enrolled but do not receive DCreg infusion will be replaced. No study intervention or follow-up 
will occur. 
b. Receive DCreg infusion but do not receive a liver transplant per protocol will be replaced. 
Additionally, they will be followed on the reduced follow up Recipient SOE (2.2b2. See footnote) 
and will not be eligible for weaning.  These subjects may receive a transplanted liver from their 
original donor or a different donor at a later date. 
c.  Receive DCreg infusion and are transplanted per protocol will follow the recipient SOE (Appendix 
3). If they subsequently decline to participate in the weaning, for reasons unrelated to adverse 
events, they will not be replaced but will be followed up according to reduced follow up Recipient 
SOE (2.2b2. See footnote). 
 
5.2 INFORMED CONSENT PROCEDURES  
As noted in Section C2.2a1, a waiver of consent to review the medical records for screening purposes 
will be requested. The study coordinator or other appropriate member of the study team will review the 
patient’s medical record to verify eligibility once eligibility is verified and the patient agrees to being 
contacted by a member of the study team, the informed consent process will begin. 
If the potential subjects are in the clinic, the study team will explain the study to them at that time. If they 
are not in clinic, the study team will contact them via telephone and provide a brief description of the 
study. If the potential subjects are interested in participating, they (both recipient and donor) will be 
brought in to clinic to continue the informed consent discussion. Once all elements of informed consent 
have been addressed, they will be given ample time to both ask questions and think about the study. If 
the potential participant/s does not make a decision about participating during this visit, another visit will 
be scheduled to give each individual enough time to make an informed decision about participating. 
Written informed consent will be obtained prior to any research interventions and at least 4 weeks prior 
to transplant.  
Vulnerable patients, including those who do not have the decision-making capacity to provide consent, 
will not be approached for this Phase I/II safety study.  
Subjects will be provided with publicly available reports regarding the study outcomes upon request. 
 
6. 
POTENTIAL RISKS AND BENEFITS 
 
6.1 POTENTIAL RISKS 
6.1.1 Risk of Experimental Drug Intervention 
Risks to recipient of DCregs: 
 
1. 
Adverse events related to injection of investigational cell therapy include pain, redness, bleeding 
and swelling at site of injection; fever, headache, infection, cell embolism, and allergic reactions 
such as rash or bronchospasm; cytokine release syndrome 

 
Clinical Protocol - Liver 
50 
2. 
Sensitization against donor alloantigens 
3. 
Allograft rejection secondary to sensitization 
4. 
Graft loss due to rejection 
5. 
Opportunistic infection secondary to immunosuppression not specific to donor alloantigens 
6. 
Malignancy secondary to immunosuppression not specific to donor alloantigens 
7. 
Patient death 
 
Note that anticipated risks 2 – 7 have not been observed in animal studies or in limited human studies 
using a related myeloid immune cell type. In our NHP studies, graft rejection was observed in NHPs 
receiving minimal immunosuppression or completely weaned off of immunosuppression, but rejection 
was delayed markedly compared with controls.  
 
Risk to recipient of standard medications and procedures related to the infusion: 
1. Acetaminophen 
Acetaminophen is considered to be safe and effective in the recommended doses, according to the ‘Drug 
Facts’ label. When taken incorrectly, however, acetaminophen can cause liver damage. The risk of liver 
damage may be increased in people who drink more than three alcoholic drinks every day, take more 
than the recommended dose (overdose), or take any additional drugs that also contain acetaminophen 
at the same time. 
 
2. Diphenhydramine   
Common side effects of diphenhydramine are an increase in the thickness of lung secretions and 
drowsiness.    
Infrequent side effects of diphenhydramine include blood disorder, extra heartbeat, hallucination, 
seizures, blurred vision, inability to empty bladder, chronic trouble sleeping, confusion, diarrhea, difficult 
or painful urination, dizziness, dry mouth, dryness of the nose, easily angered or annoyed, excessive 
sweating, fast heartbeat, head pain, heart throbbing or pounding, hives, involuntary quivering, itching, 
loss of appetite, nervous nightmares, over-excitement, problems with eyesight, rash, ringing in the ears, 
sensation of spinning or whirling, stomach cramps, sun-sensitive skin, and throat dryness. 
 
3. Risks of Normal Saline Flush: 
Common side effects of Normal Saline include fever, injection site swelling, redness, or infection. 
Uncommon but serious side effects of Normal Saline include fast heartbeat, fever, rash, joint pain, or 
shortness of breath.    
 
4. Risks of Mycophenolate Mofetil (MMF): 
Common side-effects are diarrhea, vomiting, and decrease in the number of red and/or white blood cell.  
Rarely, patients may experience gastrointestinal bleeding. Cases of progressive multifocal 
leukoencephalopathy, a rare, often fatal, infection of the brain have been reported in people taking MMF. 
 
5. Risks of Mycophenolic Acid (MPA): 
Common side-effects are constipation, diarrhea, nausea, vomiting, and decrease in the number of red 
and/or white blood cell.  Rarely, patients may experience gastrointestinal bleeding. 

 
Clinical Protocol - Liver 
51 
 
6.1.2 Risk of Study Procedures 
 
1. All subjects (recipients and donors): 
a. Risk of Blood Draw: 
The risks of having blood taken are discomfort, bleeding, fainting, small blood clot or swelling to the vein 
and area surrounding where the blood is drawn, bruising where the needle enters the skin and infection. 
There is also a very small chance of infection at the needle puncture site. 
 
b. Genetic Testing/DNA Analysis:   
The risk of a breach of confidentiality is rare and occurs in less than 1% (less than 1 out of 100 people) 
and could potentially impact future insurability, employability, or reproduction plans, or has a negative 
impact on family relationships and/or result in paternity suits or stigmatization  
 
c. Medical Record collection:    
There is a rare risk of breach of confidentiality.    
  
2. Risks to donor: 
a. Leukapheresis: 
The interventional study will involve leukapheresis of the donors to harvest the DCreg. Leukapheresis is 
not a novel procedure, and standard methods will be used. Risks associated with the leukapheresis 
procedure include weakness, nausea or feeling faint as a result of the decrease in blood volume; 
tenderness/bruising at the needle site; localized infection at needle puncture site; and the risks associated 
with the transfusion of blood components, if necessary in response to other symptoms/risks.  
Complications associated with such transfusions may include: Fatal hemolytic transfusion reaction 
caused by administration of incompatible blood. Although each blood component is screened, there is a 
possibility of transmitting infectious diseases or agents such as hepatitis, AIDS, cytomegalovirus, 
bacteria, malaria and other rare diseases. Generally, these risks do not apply to transfusion of albumin. 
Alloimmunization: The production of antibodies against donor red cells, white blood cells or platelet 
antigens. Allergic reactions, fever or fever-like reactions and circulatory overload may also occur. After 
therapeutic plasma exchange or red blood cell exchange, the platelet count of white blood cell count may 
be decreased. There may be decreases in red blood cell count or coagulation factors after repeated, 
frequent therapeutic plasma exchanges or white blood cell or platelet depletions. Laboratory parameter 
will be monitored regularly.  
Possible anticoagulant discomfort: Sodium citrate, an anticoagulant, is added to the blood to prevent 
clotting. Sodium citrate is metabolized by the body. Although it is not toxic it can sometimes cause 
temporary symptoms such as tingling/ numbness of the lips and/or fingers, muscle cramping, chilliness, 
feelings of anxiety and rarely convulsions during treatment.  
Premature termination of procedure: Since the removal of blood and return of blood is accomplished 
through the use of needles and tubing, it is possible that clotting could occur in the needles or tubing and 
this may lead to the termination of the individual procedure. Loss of red blood cells due to leakage or 
breakage of the plastic tubing or containers may occur and thus prevent the return of the red blood cells 
to the patient.  
Hetastarch (a red cell sedimenting agent) may be used for cell depletion procedures. Hetastarch has 

 
Clinical Protocol - Liver 
52 
been reported to produce general hypersensitivity reactions such as wheezing and certain allergic skin 
conditions. Side effects that have been reported include but not limited to: headache, vomiting, temporary 
weight gain, insomnia, fever and mild flu- like symptoms, edema, dizziness, chest pain, chills, anxiety 
and increased heart rate. It is uncertain whether these side effects are attributable to the hetastarch, the 
procedure, additional medications the patient may be receiving or some combination of these factors. 
Hypersensitivity reactions can occur even after Hetastarch has been discontinued.   
 
b. Risk of HIV testing:   
Infrequent Risks:  Risk of Breach of Confidentiality   
 
3. Risks to recipient: 
a. Risk of Withdrawal of Immunosuppression:  
Rejection: 
The most serious risk is rejection of transplanted liver.  Rejection can occur at any point during the study. 
The medications used to treat rejection have additional risks and side effects. 
It is very likely that treatment will end an acute rejection episode. The liver can fully recover from rejection, 
without long-term consequences.  The chance that an acute rejection episode is either difficult to treat or 
cannot be treated is low, estimated at <1.0%.   
 
Risk of Chronic Rejection: 
There is a small chance (<2%) of developing chronic rejection. 
 
b. Risks of Liver Biopsy: 
The risk of death from liver biopsy is extremely low, ranging from 0.1% to 0.01%. The most frequent 
problem from a liver biopsy is bleeding. The risk of bleeding ranges from 0.35% to 0.5%.   
The risk of liver biopsy includes:   
- Bleeding  
- Low blood pressure 
- Shoulder pain 
- Pain at the needle insertion site 
- Accidental puncture of other organs (lung, kidney colon and gallbladder) 
- Infection 
 
c. Risk of Lymph Node Removal: 
           - Bleeding           - Infection 
           - Tissue/ Organ Injury: Although uncommon, adjacent organs and tissues may be injured as a 
result of your surgery. This includes kidneys, colon, bowel, vascular structures, nerves, muscles, lungs, 
spleen, liver, pancreas and gallbladder.  On rare occasions, further surgery may be required to address 
these unexpected injuries. 
             -  Lymphocele: Lymphatic fluid can rarely collect in the area where the lymph nodes were 
removed, which may require drainage of the fluid or further surgery. 
 
d. Risk of Survey: 
Rarely, people experience psychological discomfort during questioning   
 

 
Clinical Protocol - Liver 
53 
e. Risk of EKG/ Cardiac Heart Monitor:   
Rarely, the adhesive pads used to attach the leads for recording can cause skin irritation.  
 
6.2 ALTERNATIVE TREATMENTS 
 
There are no FDA approved alternatives to donor-derived DCreg therapy. Patients who refuse to 
participate will be treated per standard of care. The alternative to CNI weaning is to continue on CNI 
therapy per standard of care. Patients who enroll in the study and receive the DCreg infusion, but refuse 
CNI weaning, will be treated per SOC and followed until year 4.5. 
 
6.3 POTENTIAL BENEFITS 
 
It is possible that the individual patients in this study will see no benefits from their participation. However, 
by giving what we expect to be a therapeutic dose of DCreg, we anticipate that some recipients will be 
successfully weaned from their immunosuppressive therapy. This will allow those recipients to avoid the 
risks of long-term immunosuppression (infection, malignancy, hypertension, diabetes, increased risk of 
cardiovascular disease, renal failure). 
  
6.4 DATA SAFETY MONITORING  
 
6.4.1 Data Safety Monitoring Board 
 
An independent Data and Safety Monitoring Board (DSMB) will be created to review this study.  After 
initial approval and at periodic intervals (to be determined by the committee) during the course of the 
study, the DSMB responsibilities are to: 
 
1. Review the research protocol, informed consent documents and plans for data and safety 
monitoring; 
2. Evaluate the progress of the study, including periodic assessments of data quality and 
timeliness, participant recruitment, accrual and retention, participant risk versus benefit, 
adverse events, unanticipated problems, performance of the trial sites, and other factors that 
can affect study outcome; 
3. Consider factors external to the study when relevant information becomes available, such as 
scientific or therapeutic developments that may have an impact on the safety of the 
participants or the ethics of the study; 
4. Review clinical center performance, make recommendations and assist in the resolution of 
problems reported by the PI; 
5. Protect the safety of the study participants; 
6. Report on the safety and progress of the study; 
7. Make recommendations to the PI, and if required, to the FDA or funding agency concerning 
continuation, termination or other modifications of the study based on the observed beneficial 
or adverse effects of the treatment under study; 
8. Monitor the confidentiality of the study data and the results of monitoring;  

 
Clinical Protocol - Liver 
54 
9. Assist the PI by commenting on any problems with study conduct, enrollment, sample size 
and/or data collection. 
 
The DSMB will include experts in solid organ transplantation, biostatistics and clinical trial design, and 
ethics.  Members will consist of persons independent of the investigators who have no financial, scientific, 
or other conflict of interest with the study.  Written documentation attesting to absence of conflict of 
interest will be required. 
 
The University of Pittsburgh Office of Clinical Research, Health Sciences will provide the logistical 
management and support of the DSMB.  A safety officer (chairperson) will be identified at the first 
meeting.  This person will be the contact person for serious adverse event reporting.  Procedures for this 
will be discussed at the first meeting. 
 
The first meeting will take place before initiation of the study to discuss the protocol, approve the 
commencement of the study, and to establish guidelines to monitor the study.  The follow-up meeting 
frequency of the DSMB will be determined during the first meeting.  An emergency meeting of the DSMB 
will be called at any time by the Chairperson should questions of patient safety arise.   
 
6.4.2 Data Safety Monitoring Plan 
 
Monitoring of safety and data quality in the proposed study will be the responsibility of all personnel on 
the project, with primary responsibility and supervision by the Investigator.  The Institutional Review 
Board will approve the Statement of Informed Consent for the study and provide institutional oversight of 
data and safety issues. The study protocol will be approved prior to recruiting or obtaining consent from 
any participants. Moreover, the study will be reviewed at a minimum of annual basis (or more frequently 
as deemed necessary) by the IRB committee. Each participant will sign the Informed Consent Form 
described above prior to participating in the study. To ensure participant safety, once participants are 
enrolled in the study, study staff will immediately report all adverse and serious adverse events to one of 
the Investigators. The Investigator will, per standardized procedures, report them to the IRB for their 
review. With regard to monitoring of data quality and protected health information, all required personnel 
proposed for this project will have the required human subjects and confidentiality training, which includes 
information about maintaining data integrity and security. Confidentiality will be guarded using established 
procedures such as storing data in locked cabinets within locked offices or locked data rooms, coding by 
study identification numbers rather than any personally identifying information to avoid revealing the 
identity of subjects, and aggregating data across participants. The key linking names and study 
identification numbers will be kept separately from the data sets with limited access by study personnel. 
Only study personnel will have access to the data sets on protected servers. In order to maintain the 
highest standard of data entry quality, all data will be double-entered, with discrepancies highlighted so 
that they can be reviewed by the project coordinator. Oversight of all aspects of data management will 
occur with the Investigator. 
 
 
Data Monitoring Plan. The proposed study will use the FDA definition of adverse events (AE) and serious 
adverse events (SAE). Any SAE, which is unexpected and related to study intervention, will be reported 

 
Clinical Protocol - Liver 
55 
immediately to the IRB and will be followed by an additional letter detailing the nature of the SAE. In the 
event that a participant either withdraws from the study or the investigators decide to discontinue a 
participant due to a SAE, the participant will be monitored by the study investigators until (a) a resolution 
is reached (e.g., the problem has resolved or stabilized with no further change expected), (b) the SAE is 
determined to be clearly unrelated to the study intervention, or (c) the SAE results in death. As noted in 
Section C2.3.5, all subjects will be followed for 4.5 years. Outcomes of SAEs will be regularly reported to 
the IRB and the sponsor. A summary of the SAEs that occurred during the previous year will be included 
in the annual progress report as well as in the annual IRB renewal. 
 
6.4.3 Parameters to be Monitored 
 
The following progress will be monitored throughout the course of the research to ensure the safety of 
subjects as well as the integrity and confidentiality of their data. 
 
• 
An evaluation of the progress of the research study, including subject recruitment and 
retention, and an assessment of the timeliness and quality of the data. 
 
• 
A review of collected data (including adverse events, unanticipated problems, and subject 
withdrawals) to determine whether there is a change to the anticipated benefit-to-risk 
assessment of study participation and whether the study should continue as originally 
designed, should be changed, or should be terminated. 
 
• 
An assessment of external factors or relevant information (eg. pertinent scientific literature 
reports or therapeutic development, results of related studies) that may have an impact on 
the safety and study participants or the ethics of the research study. 
 
• 
A review of study procedures designed to protect the privacy of the research subjects and 
the confidentiality of their research data. 
 
The severity of adverse changes in physical signs or symptoms will be classified according to CTCAE as 
follows: 
 
• 
Grade 1 (Mild): asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated. 
 
• 
Grade 2 (Moderate): minimal, local or noninvasive intervention indicated; limiting age-
appropriate ADL (Activities of Daily Living). 
 
• 
Grade 3 (Severe): medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self care ADL. 
 
• 
Grade 4 (Life-threatening): consequences; urgent intervention indicated. 
 
• 
Grade 5 (Death): event is a direct cause of death. 

 
Clinical Protocol - Liver 
56 
 
6.4.4 Frequency of Monitoring 
 
The Investigator will review subject safety data as it is generated. The Investigator, sub-investigators, 
and the research staff will meet on at least a quarterly basis to re-evaluate study goals, subject 
recruitment, data coding and retention, documentation and identification of adverse events, complaints 
and confidentiality of subjects. There will be an evaluation of the progress of the research study, including 
assessments of data quality, time lines, participant recruitment, accrual, and retention. The Investigator 
will also review the outcome and adverse event data to determine whether there is any change to the 
anticipated benefit-to-risk ratio of study participation and whether the study should continue as originally 
designed or should it be re-evaluated and changed. 
 
6.4.5 Reportable Adverse Events 
 
For this study, a serious adverse event is any untoward clinical event that is thought by either the 
investigator or the sponsor to be related to the study and results in any of the following outcomes:  
 
1. Death 
2. A life-threatening adverse event  
3. Inpatient hospitalization or prolongation of an existing hospitalization   
4. A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions 
5. A congenital anomaly or birth defect 
6. Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the patient, or subject, and may require medical, or surgical intervention to 
prevent one of the serious outcomes listed above. 
 
If clinically important and unexpected adverse experiences or clinically important study-related adverse 
experiences occur, they will be recorded on the adverse event case report form.  
 
Since adverse event reporting begins in the pre-transplant period at the time of the DCreg infusion, any 
adverse events occurring prior to transplant and attributed to the subject’s underlying liver disease will 
not be recorded for the study.      
 
6.4.6 Adverse Events Reporting Timeline 
 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug or 
procedures must be reported to the IRB within 24 hours of discovery of the incident with subsequent 
follow-up submission of a detailed written report. 
 
The FDA will be notified by telephone or facsimile transmission of a human adverse event that is fatal or 
life-threatening no later than 7 calendar days after receiving the respective human adverse event 
information, followed by the subsequent submission of a written IND Safety Report. 

 
Clinical Protocol - Liver 
57 
 
Serious and unexpected adverse events associated with the use of the study drug or procedures will be 
reported to the IRB with subsequent follow-up submission of a detailed written report in accordance with 
the respective policies and procedures of the IRB. Written IND Safety Reports will be submitted to the 
FDA as soon as possible and, in no event, later than 15 calendar days following the investigator-sponsor’s 
receipt of the respective adverse event information. 
 
A summary report of the findings will be prepared and submitted to the regulatory agencies. 
 
6.4.7 Withdrawal of Subjects and Stopping Criteria: 
 
 
6.4.7a  Withdrawal of Subjects 
Adverse Events: In this safety study, adverse events are study endpoints. Therefore, any patient who 
enrolls in the study and receives an infusion will be followed for a full 4.5 years (see 2.3.5 Medication 
Compliance). If a patient experiences an adverse event during the infusion, or after infusion but prior to 
transplant, the DSMB will review the event to determine whether it is study-related and will provide 
guidance regarding whether or not the patient should be replaced. Recipients who are diagnosed with 
rejection during or post immunosuppression withdrawal will be evaluated by the clinical team and, per 
flow charts 2 and 3, treated for rejection, placed back on the clinically indicated dose of 
immunosuppression and considered a trial failure. 
 
Other Criteria: None 
 
6.4.7b. Discontinuation of the Clinical Trial 
Since this is a Phase I/II study, safety is a primary endpoint. Therefore, stopping rules were previously 
discussed in detail in sections 2.4 and 2.5 and are summarized again below. Stopping rules imply that 
the trial shall be halted, not terminated, pending determination of the cause of the adverse event by the 
DSMB. Patients undergoing follow-up at the time of discontinuation will continue to be followed.  
Stopping Rules: 
The stopping rules are based on the incidence of the adverse events (AE) listed below, graded according 
to 
Common 
Terminology 
Criteria 
for 
Adverse 
Events 
(CTCAE, 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html). The incidence of specific events will be continuously 
monitored throughout the study to determine if any of their observed subject-based incidence rates 
exceed a threshold. A safety related suspension of enrollment will occur, pending expedited review of all 
pertinent data by the IRB and the DSMB for this study based on the following: 
 
Recipient Stopping Rules (Phase I - prior to initiation of Tac weaning): 
i)  
Any CTCAE Grade 4 or higher infusion reaction 
ii)  4 patients with CTCAE Grade 4 or higher infection 
iii)  2 patients with malignancy other than non-melanoma skin cancer; or HCC recurrence 
iv)  Any rejection resulting in recipient death or retransplantation 
v)  Any biopsy-proven severe acute rejection  
vi)  Any grade chronic rejection  
vii)  2 patients with non-surgical graft loss 

 
Clinical Protocol - Liver 
58 
viii)  3 recipient deaths 
 
Recipient Stopping Rules (Phase II – Tac weaning through year 5 ± 0.5): 
i)  
3 patients with CTCAE Grade 4 or higher infection 
ii)  4 patients with malignancy other than non-melanoma skin cancer; or HCC recurrence 
iii)  Any rejection resulting in recipient death or retransplantation 
iv)  Any biopsy-proven severe acute rejection 
v)  2 patients any grade chronic rejection  
vi)  2 patients with non-surgical graft loss 
vii)  2 recipient deaths 
If study is halted in part or total, enrolled subjects will be informed via a letter from the investigator.  All 
subjects will continue to be followed per study protocol. Since this is a one-time infusion of experimental 
drug (DCreg), we do not anticipate the need for a new consent form for continuing subjects unless 
changes to the weaning protocol or follow up schedule will affect the subjects. All sub-investigators will 
be informed by the investigator in a face-to-face meeting. 
 
6.5 RISKS MANAGEMENT PROCEDURES 
 
6.5.1  Protection Against Risks   
 
General Risks of Study Protocol and Procedures 
All research interventions/activities will be conducted in private patient care areas. The collection of 
sensitive information about subjects is limited to the amount necessary to achieve the aims of the 
research, so that no unneeded sensitive information is being collected. 
 
Case Report Forms (CRFs) will be deidentified and stored in a password and firewall protected 
database only available to personnel involved in the study. Several of these electronic CRFs will serve 
as original source documents.  Access privileges and passwords will limit data entry and viewing 
capabilities based on each person’s role in the study. In some cases paper-based medical records will 
also serve as source documents. These will not be deidentified but will be kept locked in a secure 
location within the study coordinator's office and will be only accessible to personnel involved in the 
study who need access to such information. 
 
All specimens collected during the study will be stripped of subject identifiers and stored according to a 
similar coding protocol as described above. These specimens will be stored safely in the Human 
Immunology Research Core of the Thomas E. Starzl Transplantation Institute, which is overseen by Drs. 
Fadi Lakkis and Diana Metes, co-investgators on this study. The Investigators will limit future access to 
any remaining sample to only those investigators with prior IRB approval for their studies. 
 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety.  
 
The Investigator will retain the data for the entire period of this study and will retain the specified records 
and reports for up to two years after the marketing application is approved for the investigational drug; 

 
Clinical Protocol - Liver 
59 
or, if a marketing application is not submitted or approved for the investigational drug, until two years 
after investigations under the IND have been discontinued and the FDA so notified. The Investigator may 
continue to use and disclose subjects’ de-identified information for the purpose of this study for a 
minimum of seven years after final reporting or publication of the study. If the subject and/or legal 
representative decide to withdraw or be withdrawn from study participation, they may request that the 
study data and samples be destroyed. Subject names or other directly identifiable information will not 
appear on any reports, publications, or other disclosures of clinical study outcomes. 
 
6.5.2 Protection Against Potential Risks of Experimental Intervention 
 
Recipients will receive DCreg infusion and will be carefully monitored by the transplant team in the 7 
West Patient Service Center (7W) or in the Montefiore Clinical and Translational Research Center (MUH-
CTRC).  
 
The 7W unit is a dedicated transplant outpatient care/infusion center is located on the west wing of 
Montefiore Hospital, adjacent to the outpatient clinic. The unit is open 6days a week and is staffed by full 
time experienced transplant nurses. The 7W unit has the capacity to evaluate and manage all complex 
liver transplant recipients. Transplant patients undergo routine laboratory testing; receive oral and IV 
medications including immunosuppressants and chemotherapeutics. In addition, the unit has the 
capability for plasmapheresis or leukapheresis, central venous infusion, and associated monitoring. 
Patients undergoing liver biopsy are routinely monitored here. The staff will be fully trained by study 
investigators prior to the start of the study, and a study physician will oversee all infusions. 
 
The MUH-CTRC is located on the 6th floor of UPMC Montefiore. It provides state-of-the-art infrastructure, 
inpatient and outpatient facilities, specialized equipment, and experienced staff to support researchers 
performing clinical and translational research at the University of Pittsburgh. Pertinent to this trial, the 
MUH-CTRC has three outpatient private rooms, an outpatient suite that includes three beds and 
treatment chairs, six outpatient examination rooms, and a metabolic kitchen. Personnel include 
specialized research nurses, and support staff, all of whom have completed the University of Pittsburgh's 
online training required of individuals engaging in studies with human research participants. As noted 
above, the staff will be fully trained by study investigators prior to the start of the study, and a study 
physician will oversee all infusions. 
 
For the proposed clinical trial, living donors will undergo leukapheresis at the 7 West Patient Service 
Center by the Institute for Transfusion Medicine (ITxM) per standard protocol. The ITxM is staffed by 
highly trained, specialized, laboratory professionals and physicians who are board certified in transfusion 
medicine. We will meet with the staff prior to implementation of this study; however, since no modifications 
to standard leukapheresis procedures will be required, extensive study-specific training is not necessary.  
 
This study employs many standard medical procedures (liver biopsy, blood sampling, infusion, 
EKG/Heart monitoring, administration of approved medications [acetaminophen and diphenhydramine]). 
To minimize risk, all such procedures will be performed by a trained medical professionals at the 
University of Pittsburgh Medical Center. 
 

 
Clinical Protocol - Liver 
60 
To minimize the risks of the survey, subjects do not have to answer any question that makes them feel 
uncomfortable. 
 
As described in Section C2.2, enrollment will initially be paced such that there is sufficient time from one 
patient to the next to identify safety concerns. Moreover, as detailed in Section C6.5, subject safety will 
be closely monitored by an independent DSMB as well as the study physicians. 
 
Minimization of risk assoicated with immunosuppression withdrawal:  
Definition of Allograft Dysfunction and Indication for Allograft Biopsy 
Allograft dysfunction is an unexplained elevation in ALT, AST or Total Bilirubin greater than or equal to 
2.5-fold the upper limit of normal. 
 
When allograft dysfunction occurs, participants will be thoroughly assessed for any concurrent illness or 
alternative diagnosis according to SOC. Liver function tests may be repeated for both verification and re-
assessment. If allograft dysfunction persists for greater than 72 hours and no other etiology can be 
identified, then a for-cause biopsy must be performed. If a for-cause biopsy has been performed and is 
non-diagnostic, further biopsies may be performed at the discretion of the investigator. Mechanistic blood 
samples will be drawn with the initial for-cause biopsy that is performed for each episode. 
 
Presumed Rejection: Every effort will be made to confirm all episodes of suspected rejection with a 
biopsy. However, in cases where this is medically contraindicated, participants may be treated empirically 
based on clinical suspicion. Participants with allograft dysfunction who are treated with an increase in 
immunosuppression without a confirmatory biopsy will be considered to have presumed rejection. 
 
 
In addition to protocol specific training, the Investigator and all sub-investigators, as well as key research 
personnel, listed on University of Pittsburgh Institutional Review Board approved protocol are required to 
complete the human subjects training program developed by the Collaborative Institutional Training 
Institute (CITI).  
 
7. 
COSTS AND PAYMENTS 
 
7.1 COSTS 
 
Subjects will be responsible for the cost of routine clinical services that they would ordinarily receive as 
a transplant recipient or donor. The costs of all research-only services that are provided because of the 
patients’ involvement in this research study will be covered by the study.  
 
7.2 PAYMENTS 
 
Donors will be paid $80.00 for the leukapheresis procedure(s) (up to 2 procedures for up to $160) and 
$20.00 for screening and Day -3 study visits.  All donors will be provided a parking validation ticket for 
each visit.  Donors will be provided a meal for the day of the leukapheresis procedure.  

 
Clinical Protocol - Liver 
61 
 
Recipients will be paid $80.00 for DCreg infusion visit and $20.00 for all other study visits including 
each time the laboratory assessments are during the weaning phase of the trial. Recipients will be 
provided a parking validation ticket for each visit. Recipients will be provided a meal for the day of the 
DCReg infusion.  
 
Subjects will be reimbursed at the end of each study visit regardless of whether the study-related 
procedures are successfully completed. e.g. if the donor comes to Oakland for the leukapheresis visit 
but we determine that the donor cannot undergo leukapheresis, we will still pay the donor $80 + parking 
+ lunch as anticipated. This is because the donor likely cleared their calendar (took a vacation day, 
hired childcare, etc) in anticipation of spending the day in Oakland. 
 
8. 
QUALIFICATIONS AND SOURCE OF SUPPORT 
 
8.1 QUALIFICATIONS OF THE INVESTIGATORS 
 
Angus W. Thomson, PhD, DSc, DSc (Med), FRCPath: Dr. Thomson is a tenured Distinguished 
Professor of Surgery, Professor of Immunology and Microbiology & Molecular Genetics, and Professor 
of Clinical & Translational Science at the University of Pittsburgh School of Medicine. He is Director of 
Transplantation Immunology at the Thomas E. Starzl Transplantation Institute (STI). He has published 
extensively on dendritic cell, T cell and transplantation immunology and has been closely involved in the 
pre-clinical evaluation of several important immunosuppressive agents. He is on the editorial boards of 
leading basic transplantation and immunology journals. He has served as a member of the international 
scientific advisory committee of the Immune Tolerance Network and currently serves on the Steering 
Committee of the NIAID non-human primate (NHP) transplant tolerance cooperative study group. Dr. 
Thomson has published extensively on the immunobiology of rodent, human and NHP regulatory immune 
cells, especially dendritic cells and their role in regulation of organ transplant outcome. He has directed 
and coordinated NIH-funded experimental work on dendritic cells and cell therapy of graft rejection over 
the past 20 years that provides a foundation for the current proposal. His is currently the PI of NIH-funded 
studies evaluating the influence of regulatory immune cell therapy in pre-clinical NHP organ transplant 
models. 
 
Abhinav Humar, MD: Dr. Humar is the Professor of Surgery and Chief, Division of Transplantation 
Surgery. Dr. Humar is the Clinical Director of the Starzl Transplantation Institute. He has been involved 
with kidney transplants since 1999 and has extensive experience with transplants (kidney and liver). He 
also has extensive experience conducting clinical research in organ transplantation. His areas of 
research include partial liver transplants and studies of hepatic regeneration, clinical outcome studies in 
kidney, liver and pancreas transplants, cytomegalovirus (CMV) disease in transplant recipients, 
outcomes in pediatric kidney and liver transplants and small bowel transplantation. His research pursuits 
have resulted in numerous National Institutes of Health (NIH) and multi-year pharmaceutical grants. He 
has more than 200 abstracts accepted at national and international transplant meetings. In addition, he 
has authored or co-authored over 80 manuscripts, 15 book chapters and one book. He oversees all 
clinical and research efforts at the Starzl Tranplantation Institute. 

 
Clinical Protocol - Liver 
62 
 
Anthony Demetris, MD: Dr. Demetris is Professor of Pathology, Starzl Professor of Transplant 
Pathology and Director of the Division of Liver and Transplantation Pathology at the University of 
Pittsburgh and also the Research Histology Shared Resource Laboratory of the University of Pittsburgh. 
His clinical expertise is in native and allograft liver, kidney and heart pathology. In addition, he has 
extensive research experience in immunobiology of tolerance induction and chronic rejection. 
 
Swaytha Ganesh MD: Dr. Ganesh is a Clinical Instructor of Hepatology at the University of Pittsburgh 
School of Medicine. She is a gastroenterology Fellow at the University of Pittsburgh Medical Center. She 
has years of experience in medical instruction and has published numerous abstracts and publications. 
 
Christopher Hughes, MD:  Dr. Christopher Hughes - Associate Professor of Surgery, Surgical Director, 
Liver Transplantation. Dr. Hughes is a solid organ transplant surgeon with extensive clinical and research 
experience. 
 
Fadi G. Lakkis, MD: Dr. Lakkis, a Professor of Surgery, Immunology and Medicine, is the Frank and 
Athena Sarris Chair in Transplantation Biology and the Scientific Director of STI.  He is an internationally-
recognized transplant immunologist. Funded by the NIH, Dr. Lakkis’s laboratory is focused on two 
fundamental areas in transplantation immunology:  (1) allorecognition in the innate immune system, and 
(2) the biology of memory T cells in organ transplantation. Dr. Lakkis is also the PI on a NIH-funded 
human immunology trial investigating in collaboration with Dr. Metes the effects of antibody-induction 
therapy on T and B lymphocyte phenotype and function in renal transplant recipients.  
 
Diana Metes, MD: Dr. Metes is a Professor of Surgery and Immunology at the University of Pittsburgh 
and Director of the Human Immunology Core Lab at the STI. Dr. Metes has broad expertise in human 
immunology, with an emphasis on anti-viral and allo-specific T cell immunity in immunocompromised 
solid organ transplant recipients. Her research, funded by the NIH, has focused on aspects of the innate 
and adaptive immunity to EBV, a latent herpes virus implicated in the immune-pathogenesis of PTLD, 
and on the naïve and memory T cell contribution to human allo-immunity..  
 
Michele Molinari, MD: Dr. Molinari is an Associate Professor of Surgery and the Director of Transplant 
Quality at the Starzl Transplantation Institute. He has extensive experience performing abdominal 
transplants. He also has extensive experience conducting clinical research in organ transplantation. His 
areas of research include liver transplants, cancer and transplantation, and epidemiological studies. He 
has co-authored 49 published articles (original research) in Peer-Reviewed journals, co-authored 42 
abstracts and 18 case reports and review articles. He has greater than 10 years of experience as the 
Program Director of Surgical fellows and has greater than 10 years’ experience as a research mentor 
and thesis advisor to surgical residents and medical students. 
 
Warren Shlomchik, MD: Dr. Shlomchik, Director of Stem Cell Transplantation and Cell Therapies for 
the University of Pittsburgh’s Division of Hematology-Oncology and University of Pittsburgh Cancer 
Institute (UPCI), is a bone marrow transplantation expert and the leader of a clinical trial assessing the 
safety and efficacy of depletion of naïve T cells from human stem cells to prevent GVHD.   
 

 
Clinical Protocol - Liver 
63 
Paul Szabolcs, MD: Dr. Szabolcs is the Chief of the Division of Blood and Marrow Transplantation and 
Cellular Therapies at Children's Hospital of Pittsburgh of UPMC and an NIH-funded Professor of 
Pediatrics at the University of Pittsburgh.  An experienced physician-scientist, Dr. Szabolcs successfully 
filed for an IND and currently has a related R34 from NIAID to plan a clinical trial of combined bone 
marrow and lung transplantation in children with immune deficiency syndromes.  
 
Amit Tevar, MD: Dr. Tevar is Associate Professor of Surgery at the University of Pittsburgh School of 
Medicine and Surgical Director of the Kidney and Kidney-Pancreas Transplant Program at the University 
of Pittsburgh Medical Center, STI. As an experienced transplant surgeon, he is well versed in the 
immunosuppressive requirements and the use of various induction and long-term maintenance therapies 
to optimize graft and patient survival.  
 
Adriana Zeevi, PhD: Dr. Zeevi is a Professor of Pathology, Surgery and Immunology and the Director 
of the Tissue Typing Laboratory that provides 24 hr/ 7 day service to the solid organ and bone marrow 
transplant program UPMC-affiliated hospitals. She provides consultation regarding the sensitization 
status of potential transplant candidates and their status post-transplant. She has over 30 years of 
experience with immune monitoring of solid organ transplant recipients and implementation of various 
assays and novel approaches to evaluate the efficacy of protocols used to desensitize transplant 
candidates and/or to treat antibody mediated rejection in solid organ transplant recipients. 
 
8.2 SOURCES OF SUPPORT 
 
Support for preliminary data collection and planning is currently being provided by: 
Thomas E. Starzl Transplantation Institute at the University of Pittsburgh 
The University of Pittsburgh Clinical and Translational Institute (5 UL1 TR0000005-09) Pilot Program: 
Innovation, Transformation, Translation  
 
Support for the initial 3 years of this project is being provided by UPMC Enterprises, UPMC’s Immune 
Transplant and Therapy Center (ITTCT 88906), LDLT/DC Initiative. 
 
9. 
REFERENCES  
 
1. 
Demetris, A. J., C. Bellamy, S. G. Hubscher, J. O'Leary, P. S. Randhawa, S. Feng, D. Neil, 
R. B. Colvin, G. McCaughan, J. J. Fung, A. Del Bello, F. P. Reinholt, H. Haga, O. Adeyi, A. 
J. Czaja, T. Schiano, M. I. Fiel, M. L. Smith, M. Sebagh, R. Y. Tanigawa, F. Yilmaz, G. 
Alexander, L. Baiocchi, M. Balasubramanian, I. Batal, A. K. Bhan, J. Bucuvalas, C. T. Cerski, 
F. Charlotte, M. E. de Vera, M. ElMonayeri, P. Fontes, E. E. Furth, A. S. Gouw, S. Hafezi-
Bakhtiari, J. Hart, E. Honsova, W. Ismail, T. Itoh, N. C. Jhala, U. Khettry, G. B. Klintmalm, S. 
Knechtle, T. Koshiba, T. Kozlowski, C. R. Lassman, J. Lerut, J. Levitsky, L. Licini, R. Liotta, 
G. Mazariegos, M. I. Minervini, J. Misdraji, T. Mohanakumar, J. Molne, I. Nasser, J. 
Neuberger, M. O'Neil, O. Pappo, L. Petrovic, P. Ruiz, O. Sagol, A. Sanchez Fueyo, E. 
Sasatomi, A. Shaked, M. Shiller, T. Shimizu, B. Sis, A. Sonzogni, H. L. Stevenson, S. N. 
Thung, G. Tisone, A. C. Tsamandas, A. Wernerson, T. Wu, A. Zeevi, and Y. Zen. 2016. 2016 
Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of 

 
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Antibody-Mediated Rejection. American journal of transplantation : official journal of the American 
Society of Transplantation and the American Society of Transplant Surgeons. 
2. 
Al Riyami, D., A. Alam, K. Badovinac, F. Ivis, L. Trpeski, and M. Cantarovich. 2008. 
Decreased survival in liver transplant patients requiring chronic dialysis: a Canadian experience. 
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