Nonhuman Primate Transplant Model Research Articles

Vascularized Thymosternal Composite Tissue Allo- and Xenotransplantation in Nonhuman Primates: Initial Experience

Background:Vascularized composite allotransplantation is constrained by complications associated with standard immunosuppressive strategies. Vascularized thymus and bone marrow have been shown to promote prolonged graft survival in composite organ and soft-tissue vascularized composite allotransplantation models. We report development of a nonhuman primate vascularized thymosternal composite tissue transplant model as a platform to address donor-specific immune tolerance induction strategies.Methods:Vascularized thymosternal allograft (skin, muscle, thymus, sternal bone) was transplanted between MHC-mismatched rhesus monkeys (feasibility studies) and baboons (long-term survival studies), with end-to-side anastomoses of the donor aorta and SVC to the recipient common femoral vessels. A male allograft was transplanted to a female’s lower abdominal wall, and clinically applicable immunosuppression was given. Skin biopsies and immunological assays were completed at regular intervals, and chimerism was quantified using polymerase chain reaction specific for baboon Y chromosome.Results:Four allo- and 2 xenotransplants were performed, demonstrating consistent technical feasibility. In 1 baboon thymosternal allograft recipient treated with anti-CD40–based immunosuppression, loss of peripheral blood microchimerism after day 5 was observed and anticipated graft rejection at 13 days. In the second allograft, when cutaneous erythema and ecchymosis with allograft swelling was treated with anti-thymocyte globulin starting on day 6, microchimerism persisted until immunosuppression was reduced after the first month, and the allograft survived to 87 days, 1 month after cessation of immunosuppression treatment.Conclusions:We established both allo- and xeno- composite vascularized thymosternal transplant preclinical models, which will be useful to investigate the role of primarily vascularized donor bone marrow and thymus.

A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem Cell Gene Editing Strategies for β-Hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

Refining the gold standard

Stem Cells CD34+ cells are the gold-standard target hematopoietic cells for stem cell therapy and transplantation of stem cell–enriched grafts. However, most of the cells within this population do not contribute to engraftment. Radtke et al. used a robust nonhuman primate transplantation model to identify a stem cell–enriched subpopulation of CD34+ cells that was exclusively responsible for engraftment. Cell doses of this subpopulation correlated with neutrophil and platelet recovery and reliably predicted transplant success. Importantly, the authors observed phenotypic and transcriptomic similarities between these cells and human hematopoietic cells with high engraftment and repopulating potential. Sci. Transl. Med. 9 , eaan1145 (2017).

Belatacept-Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells.

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28-CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept- or atacrolimus-based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28+ CD8+ TEMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28+ CD8+ TEMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28- . These data suggest that CD28+ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28+ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation-independent rejection.

Comparative Study of Human and Cynomolgus T-cell Depletion With rabbit anti-thymocyte globulin (rATG) treatment - for dose adjustment in a non-human primate transplantation model

Rabbit-antithymocyte globulin (rATG) is commonly used in solid organ transplantation to prevent and treat allograft rejection. Although rATG is also commonly used in non-human primates, the optimal dose has not been reported. In this study, we evaluated which cumulative dose of rATG was most appropriate for transplantation in a non-human primate (NHP). Cynomolgus monkeys were treated with 5 mg/kg of rATG (Thymoglobulin®, Genzyme Ltd., UK) intravenously twice on day 0 and 2 (a total of 10 mg/kg, n = 2) or 4 times on day 0, 1, 2 and 3 (a total of 20 mg/kg, n = 6). And, we performed an allo-kidney transplantation (KT) in cynomolgus monkeys (n = 4) with a cumulatively 20 mg/kg of rATG induction. Also, their subpopulation of T cells like naïve, central memory and effector memory T cells were compared in the kinetics of human KT patients (n = 7). The kinetics of lymphocytes decreased rapidly at day 1 and then was maintained for 2 weeks in the 20 mg/kg rATG group, while maintenance was not observed in the 10 mg/kg rATG group. During the early period of rATG treatment in NHP KT model, their T cell subpopulation in the 20 mg/kg group showed a similar pattern of change to kidney transplant patients treated with rATG (1.5 mg/kg, 3 days). However, the pattern of B cells was different from those of human KT patients. These data indicate that the lymphocyte-depletion induced by rATG was influenced by the cumulative dose, and that an rATG dose of 20 mg/kg is suitable for induction therapy in renal transplantation in cynomolgus monkeys when compared to human KT.

Interruption of OX40L signaling prevents costimulation blockade-resistant allograft rejection.

The potential of costimulation blockade to serve as a novel transplant immunosuppression strategy has been explored for over 20 years, culminating in the recent clinical approval of belatacept for renal transplant patients. Despite improving long-term graft function and survival compared with calcineurin inhibitors, clinical acceptance of belatacept has been hindered by elevated rates of acute rejection. We examined the signaling pathways required to activate costimulation blockade-resistant alloreactive T cells and identified the OX40/OX40L secondary costimulatory pathway as a promising target. We next sought to improve the clinical efficacy of traditional costimulation blockade using belatacept by coupling it with anti-OX40L. Using a murine transplant model, we demonstrate that combined blockade enhances the suppression of alloreactive T cell proliferation and effector functions including both cytokine release and cytotoxic degranulation. We also show that anti-OX40L may be particularly useful in targeting alloreactive memory T cell responses that are relatively unaffected by traditional costimulation blockade regimens. Finally, we translated this therapy to a clinically relevant nonhuman primate renal transplant model, validating the efficacy of this regimen in a potentially novel steroid- and calcineurin inhibitor-free immunosuppression regimen.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model.

Costimulation blockade (CoB) via belatacept is a lower-morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)-1. LFA-1 exists in two forms: a commonly expressed, low-affinity form and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL-579, each of which targets the high-affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB-resistant rejection.

758. A Nonhuman Primate Transplantation Model to Evaluate Gene Editing Strategies Aimed at Inducing Fetal Hemoglobin Production for the Treatment of Hemoglobinopathies

Fetal hemoglobin (HbF) is the major form of hemoglobin present in newborns but is almost completely replaced by adult hemoglobin after birth, where it constitutes less than 1 percent of total hemoglobin. Hereditary persistence of HbF is linked to mutations at multiple genetic loci that regulate the switch from fetal to adult hemoglobin. The targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcllla-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcllla-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.

(79) - Early CD20+ B Cell Depletion Attenuates Cardiac Allograft Vasculopathy in Anti-CD154 Treated Non-Human Primate Model

Anti-CD154 (αCD154) monotherapy is associated with the development of anti-donor alloantibodies, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. Evidence suggests that peri-transplant B cell depletion may remove an efficient source of donor antigen-specific costimulation, thereby attenuating development of anti-donor alloantibodies and subsequent pathogenic phenomena. Cynomolgus monkeys underwent heterotopic intraabdominal cardiac transplantation, and recipients were treated with either αCD154 (IDEC-131, n=21), αCD20 (Rituximab, n=2), αCD154 + αCD20 (n=7), or αCD154 + αCD20 + rabbit anti-human thymocyte immune globulin (rATG, n=10). Primary graft survival was defined as time to the first rejection episode. All biopsies and explant tissue specimens were assessed for CAV using the 2005 ISHLT revised criteria. Serum BAFF (B-cell activating factor of the tumor necrosis factor family) levels were analyzed by ELISA. Combined αCD154 + αCD20 treatment significantly prolonged graft median survival time (MST >90 days), delayed anti-donor alloantibody production, and attenuated CAV compared to αCD154 monotherapy (MST 28 days, p=0.05). Addition of rATG to αCD154 + αCD20 also prolonged graft MST to >90 days, but it was associated with a 50% incidence of treatment-attributed morbidity. Incidence of severe acute rejection (>1R) and CAV severity within 90 days was reduced in αCD20 treated animals relative to reference groups (p<0.05 for both endpoints). In αCD20 treated recipients, detection of anti-donor IgM and IgG alloantibody and relatively severe CAV were anticipated by appearance of CD20 cells in peripheral blood and was associated with trough αCD154 levels <100 μg/mL. Serum BAFF levels were higher in αCD20 treated animals (d7: 3762±181pg/mL) compared to untreated controls (628±507 pg/mL) and, similarly, in combined therapy (d7: 2687±829 pg/mL) vs. αCD154 controls (371±80 pg/mL). Efficient preemptive CD20+ B cell depletion in combination with selective CD154 inhibition consistently modulates pathogenic alloimmunity and attenuates CAV in this non-human primate heterotopic heart transplantation model; however, the addition of T cell depletion to the combined therapy was associated with significant morbidity in this model.

Nonhuman primate models of transplant tolerance: closer to the holy grail.

Transplantation tolerance, successful acceptance of an organ without the perils of immunosuppression, has been a central goal of transplant research. Many strategies to achieve this tolerance have been examined over the past three decades, culminating in several human trials of transplant tolerance. This progression from the 'benchtop to the clinic' has depended on the successful implementation of these tolerance strategies in nonhuman primates. This review will examine the described methods of transplant tolerance induction in nonhuman primates. Although costimulatory blockade and mixed chimerism have an established record of achieving transplant tolerance in nonhuman primates, some of the most innovative recent techniques of tolerance induction have relied on cellular transfer. This review will fully examine the role of regulatory T-cell transfer and the use of mesenchymal stem/stromal cells to promote tolerance of organ allografts in nonhuman primates. Use of translational nonhuman primate transplant models is a vital intermediate step to advance new approaches of transplant tolerance induction from the lab to the clinic. This review will explore numerous techniques of tolerance induction that have been piloted in primates, including depletional techniques, induction of mixed hematopoietic chimerism, costimulation blockade, and adoptive transfer of tolerogenic cell populations.

Anti-Leukocyte Function-Associated Antigen 1 Therapy in a Nonhuman Primate Renal Transplant Model of Costimulation Blockade-Resistant Rejection.

Costimulation blockade with the fusion protein belatacept provides a desirable side effect profile and improvement in renal function compared with calcineurin inhibition in renal transplantation. This comes at the cost of increased rates of early acute rejection. Blockade of the integrin molecule leukocyte function-associated antigen 1 (LFA-1) has been shown to be an effective adjuvant to costimulation blockade in a rigorous nonhuman primate (NHP) model of islet transplantation; therefore, we sought to test this combination in an NHP renal transplant model. Rhesus macaques received belatacept maintenance therapy with or without the addition of LFA-1 blockade, which was achieved using a murine-derived LFA-1-specific antibody TS1/22. Additional experiments were performed using chimeric rhesus IgG1 (TS1/22R1) or IgG4 (TS1/22R4) variants, each engineered to limit antibody clearance. Despite evidence of proper binding to the target molecule and impaired cellular egress from the intravascular space indicative of a therapeutic effect similar to prior islet studies, LFA-1 blockade failed to significantly prolong graft survival. Furthermore, evidence of impaired protective immunity against cytomegalovirus was observed. These data highlight the difficulties in translating treatment regimens between organ models and suggest that the primarily vascularized renal model is more robust with regard to belatacept-resistant rejection than the islet model.

Regulated apoptosis of genetically modified hematopoietic stem and progenitor cells via an inducible caspase-9 suicide gene in rhesus macaques.

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of "suicide genes" in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the "inducible Caspase-9" (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75% and 94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development.

Orchestration of transplantation tolerance by regulatory dendritic cell therapy or in-situ targeting of dendritic cells.

Extensive research in murine transplant models over the past two decades has convincingly demonstrated the ability of regulatory dendritic cells (DCregs) to promote long-term allograft survival. We review important considerations regarding the source of therapeutic DCregs (donor or recipient) and their mode of action, in-situ targeting of DCregs, and optimal therapeutic regimens to promote DCreg function. Recent studies have defined protocols and mechanisms whereby ex-vivo-generated DCregs of donor or recipient origin subvert allogeneic T-cell responses and promote long-term organ transplant survival. Particular interest has focused on how donor antigen is acquired, processed and presented by autologous dendritic cells, on the stability of DCregs, and on in-situ targeting of dendritic cells to promote their tolerogenic function. New evidence of the therapeutic efficacy of DCregs in a clinically relevant nonhuman primate organ transplant model and production of clinical grade DCregs support early evaluation of DCreg therapy in human graft recipients. We discuss strategies currently used to promote dendritic cell tolerogenicity, including DCreg therapy and in-situ targeting of dendritic cells, with a view to improved understanding of underlying mechanisms and identification of the most promising strategies for therapeutic application.

Pre-Clinical Evaluation of a Novel Domain Antibody Targeting CD154 in a Non-Human Primate Transplant Model.

Interactions between CD40 & CD154 represent a critical pathway for effective T cell activation. Over two decades ago there were tremendous expectations surrounding clinical trials of anti-CD154 antibodies. Unfortunately, outcomes were disappointing as unanticipated thromboembolic events led to cessation of trials. One prevailing hypothesis suggested that anti-CD154 therapy contributed to the development of immune complexes composed of anti-CD154, platelet Fc receptors & soluble CD154. These complexes purportedly led to platelet aggregation & thromboembolism. We have developed a novel CD154 domain antibody (dAb), which lacks Fc binding but retains the ability to block CD40-CD154. Initial screens identified monomeric dAbs against hCD154, which were then formatted as bivalent Fc-silent proteins. Platelet activation studies were performed with soluble CD154 & dAbs targeting CD154 with or without Fc binding capacity. Only reagents with Fc binding activity promoted platelet activation in the presence of soluble CD154. This activation was abolished with the addition of anti-FcγRIIa. Additional in-vitro studies evaluating CD154-dependent B & T cell activation were used to select an optimal clone (BMS-986004). Further evaluation of the novel anti-CD154 dAb was assessed in non-human primates. A single dose of anti-CD154 dAb effectively eliminated the T-dependent Ab response after KLH immunization. Next efficacy was evaluated in a non-human primate renal transplant model. Rhesus macaques received anti-CD154 dAb (20mg/kg iv, qwk to day 70) and underwent a MHC-mismatched renal transplant. BMS 986004 effectively prolonged survival in the majority of animals (>77, >77, >77, 68, 67, >42, 8, & 8 days). 3 of 8 animals were sacrificed after finishing therapy & underwent a full pathologic evaluation, which failed to identify any gross or histologic evidence of thromboembolism. Protective immunity, as assessed by CMV viral reactivation and general clinical condition, was maintained. This report identifies a novel dAb targeting CD154 which effectively prolongs transplant survival yet lacks thromboembolic potential & thus may provide an attractive candidate molecule for clinical development. DISCLOSURES:Price, K.: Employee, Bristol-Myers Squibb. Nadler, S.: Employee, Bristol-Myers Squibb. Adams, A.: Grant/Research Support, Bristol-Myers Squibb.

Cellular Based Immunodepletion and Tomotherapy for Induction of Immunosuppression in a Rhesus Macaque Renal Allotransplant Model.

PURPOSE: To determine the safety and efficacy of inducing an immunosuppressive environment for mixed chimerism in a nonhuman primate renal transplant model. METHODS: Eight rhesus macaques were divided into three treatment groups: Group 1 (n=1) received only anti-thymocyte globulin (ATG)(5 days at 4 mg/kg), Group 2 (n=3) received both ATG and helical tomotherapy based total lymphoid irradiation (TLI) (10 fractions of 1.2 Gy), and Group 3 (n=4) received ATG, TLI and a renal allograft. Animals received maintenance tacrolimus and mycophenolate mofetil. Weekly blood differential counts, serum chemistries, and flow cytometry was performed in all animals. Cytomegalovirus was measured by quantitative PCR. These studies differed from TLI/ATG protocols used in humans, since the latter include anti-viral prophylaxis to prevent CMV reactivation, and vertebral bodies are shielded to prevent severe neutropenia. RESULTS: All animals did well with no fever or major illness. Animal weight temporarily decreased an average of 19.1% following treatment (6.3% Group 1; 14.7% Group 2; 25.7% Group 3), with resolution at a mean of 90 days. Serum chemistries remained normal other than elevated glucose (>200) in 3 animals in groups 2 and 3. ATG immunodepletive therapy alone (Group 1) resulted in lymphocyte depletion that recovered after day 7. Use of ATG and TLI in Groups 2 and 3 demonstrated adequate lymphocyte depletion, lasting beyond 14 days; however, severe neutropenia (ANC<500) was noted in 5 animals between days 9 and 21 in the absence of vertebral body shielding. Two animals in Group 3 developed wound infections (day 7 and 56 respectively), treated successfully with antibiotics. All animals in group 3 without anti-viral prophylaxis developed CMV reactivation requiring antiviral therapy. CONCLUSION: A combination of ATG and helical tomotherapy for TLI can be safely administered to the rhesus macaque, providing effective immunosuppression beyond 2 weeks without toxicity. This is ideal to allow for engraftment of donor HSC and T cell infusion on treatment day 11. CMV reactivation should be treated prophylactically rather than preemptively. Severe neutropenia was well tolerated, however, further adjustments to TLI dose and distribution may result in less neutropenia and less CMV reactivation.

The Effect of CCR5 Blockade On De Novo DSA and Long-Term Graft Survival in Non-Human Primate AMR Model.

Background: Multiple lines of evidence suggest that CCR5 blockade may reduce, delay or even prevent alloantibody production. Previously, we demonstrated that depletion using anti-CD3 immunotoxin (IT) combined with Tacrolimus and Alefacept (AMR regimen) reliably induced early DSA production with antibody-mediated injury in a nonhuman primate kidney transplant model. We hypothesize that short-term CCR5 blockade (Selzentry, maraviroc) will prevent alloantibody formation and substantially promote tolerance in our nonhuman primate model of AMR after anti-CD3 immunotoxin treatment. Methods: All rhesus macaques received the AMR regimen of CD3-IT × 4 days, alefacept × 8 weeks, and tacrolimus maintenance. Five animals were assigned as positive AMR controls, four were treated with additional CCR5 blockade maraviroc (4mg/kg PO daily) for 60 days. Results: Mean graft survival was 91.8±111.2 days (n=4), which was not prolonged compared to AMR control (n=5, 59.4±22.1d). T and B cell repopulation patterns showed no difference between treatment groups. However, B cell isotype switching measured by reduction of IgM+ B cells in peripheral blood was delayed with additional maraviroc treatment.Figure: No Caption available.Two out of 4 maraviroc recipients showed no elevation of DSA. Furthermore, one animal had tacrolimus maintenance discontinued on day 180 and maintained graft function without evidence of subclinical rejection through day 287. Conclusion: Additional CCR5 blockade did not affect peripheral T and B cell numbers but reduced the kinetics of B cell isotype switching (IgM to IgG). Half of the AMR regimen-treated animals responded to additional CCR5 blockade with suppression of DSA, and long-term graft survival was achieved. These results support the translation of selective supplementation with CCR5 blockade for AMR prevention.

Treg Promoting Immunosuppression and Non-Immunogenic Donor Antigen Delivery in a Non-Human Primate Islet Transplant Model.

Treg Promoting Immunosuppression and Non-Immunogenic Donor Antigen Delivery in a Non-Human Primate Islet Transplant Model.

464. Modeling Potential HMGA2-Induced Clonal Expansion in a Non-Human Primate Stem Cell Transplant Model

464. Modeling Potential HMGA2-Induced Clonal Expansion in a Non-Human Primate Stem Cell Transplant Model

Costimulation Blockade Alters Germinal Center Responses and Prevents Antibody-Mediated Rejection

De novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.