Tolerance In Nonhuman Primates Research Articles

449: Cellular Immunity Is Enhanced in a Non-Human Primate Lung Transplantation Model, as Compared to an Analogous Kidney Transplantation Model

We previously reported renal allograft tolerance in non-human primates by inducing mixed-chimerism using a non-myeloablative conditioning regimen. In this study, we applied this same technique to lung transplantation in cynomolgus monkeys.

Comparison of Lung and Kidney Allografts in Induction of Tolerance by a Mixed-Chimerism Approach in Cynomolgus Monkeys

Background We have previously reported the successful induction of renal allograft tolerance in non-human primates using a nonmyeloablative conditioning regimen to produce a mixed-chimeric state in the recipient. In the present study, we applied this same technique to lung allotransplantation in cynomolgus monkeys. Methods Nine pairs of fully major histocompatibility complex (MHC)-mismatched cynomolgus monkeys were used. The conditioning regimen consisted of total body irradiation, thymic irradiation, and antithymocyte globulin. The recipients underwent lung and bone marrow transplantation, followed by anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine. The regimen included anti-CD8 mAb in the last 5 recipients and α 1-antitripsin in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same regimen. Results Transient chimerism developed in all lung recipients, as was previously seen in the kidney recipients. Nonetheless, the lung recipients rejected their allografts significant earlier than the kidney recipients ( P < .01). Conclusions Despite the successful induction of mixed chimerism in recipients of fully MHC-mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. Strategies to overcome this problem include organ-specific modifications of the transplant regimen.

Induction of allograft tolerance in nonhuman primates and humans

Despite remarkable improvement in short-term survival following organ transplantation, long-term results have been less satisfactory, mainly due to chronic rejection or toxicities induced by immunosuppressive drugs. Therefore, induction of specific immunologic tolerance remains an important goal in organ transplantation. Although numerous regimens for the induction of allograft tolerance have been developed in rodents, their application to large animal models has been limited. The mechanisms of action of the approaches that have been successfully applied in monkey models can be divided into three major categories: 1) deletion, 2) co-stimulatory blockade and 3) regulation. Long-term allograft survival has now been achieved in several nonhuman primate models; however, late-onset chronic rejection as well as the toxicity of some of these regimens remain as significant limitations that hamper clinical application.

Results from a Human Renal Allograft Tolerance Trial Evaluating T-Cell Depletion with Alemtuzumab Combined with Deoxyspergualin

Perioperative lymphocyte depletion induces allograft tolerance in some animal models, but in humans has only been shown to reduce immunosuppressive requirements. Without maintenance immunosuppression, depleted human renal allograft recipients experience rejection characterized by infiltration of the allograft with monocytes and macrophages. T-cell depletion combined with a brief course of deoxyspergualin (DSG), a drug with inhibitory effects on monocytes and macrophages, induces tolerance in nonhuman primates. We therefore performed a trial to determine if lymphocyte depletion with alemtuzumab combined with DSG would induce tolerance in humans. Five recipients of live donor kidneys were treated perioperatively with alemtuzumab and DSG and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically, by flow cytometry, and by protocol biopsies analyzed immunohistochemically and with real-time polymerase chain reaction. Results were compared to previously studied patients receiving alemtuzumab alone or standard immunosuppression. Despite profound T-cell depletion and therapeutic DSG dosing, all alemtuzumab/DSG patients developed reversible rejection that was similar in timing, histology, and transcriptional profile to that seen in patients treated with alemtuzumab alone. Chemokine expression was marked prior to and during rejections. We conclude that treatment with alemtuzumab and DSG does not induce tolerance in humans. Chemokine production may not be adequately suppressed using this approach.

IDEC-131 (Anti-CD154), Sirolimus and Donor-Specific Transfusion Facilitate Operational Tolerance in Non-Human Primates

CD154-specific antibody therapy prevents allograft rejection in many experimental transplant models. However, initial clinical transplant trials with anti-CD154 have been disappointing suggesting the need for as of yet undetermined adjuvant therapy. In rodents, donor antigen (e.g., a donor blood transfusion), or mTOR inhibition (e.g., sirolimus), enhances anti-CD154's efficacy. We performed renal transplants in major histocompatibility complex-(MHC) mismatched rhesus monkeys and treated recipients with combinations of the CD154-specific antibody IDEC-131, and/or sirolimus, and/or a pre-transplant donor-specific transfusion (DST). Therapy was withdrawn after 3 months. Triple therapy prevented rejection during therapy in all animals and led to operational tolerance in three of five animals including donor-specific skin graft acceptance in the two animals tested. IDEC-131, sirolimus and DST are highly effective in preventing renal allograft rejection in primates. This apparently clinically applicable regimen is promising for human renal transplant trials.

Nonhuman primates have a high proportion of regulatory T-cells after lymphocyte depletion

Abstract Introduction: Aggressive lymphocyte depletion promotes tolerance in nonhuman primates (NHPs) but not in humans. CD4+ memory-like T-cells impede tolerance in patients treated with lymphocyte depletion, while cells with potential regulatory function (Treg) may promote tolerance. To investigate the differential tolerogenesis in NHPs and humans we questioned whether NHP and human memory and Treg cells responded similarly to depletion. Methods: Three cynomolgus macaques received high-dose rabbit antithymocyte globulin (RATG). Peripheral lymphocytes were analyzed using polychromatic flow cytometry. NHP results were compared with 10 patients depleted with RATG or alemtuzumab. Results: Similar dynamics were observed in CD4+ memory cells, those effecting depletion-refractory rejection. They were relatively resistant to depletion, comprising 17% of lymphocytes in NHPs and 60% in humans, and repopulated briskly. In contrast to humans, Treg (CD4+CD25+ and CD4-CD8-) were resistant to depletion in NHPs. Treg constituted 30% of total lymphocytes at the nadir of depletion in NHPs, but Conclusions: CD4+ memory cells respond similarly to aggressive depletion in NHPs and humans. NHPs manifest a high ratio of Treg to memory T-cells, which may be associated with tolerance. Therefore, post-treatment lymphocyte composition may predict the tolerogenic response to depletion and should be considered when applying preclinical data to clinical trials.

DEVELOPMENT OF A MIXED CHIMERISM-TOLERANCE INDUCING NONMYELOABLATIVE REGIMEN RELEVANT TO CADAVER DONOR TRANSPLANTATION

P1061 Aims: A previously reported nonmyeloablative conditioning protocol (Standard Regimen) that can induce mixed chimerism and renal allograft tolerance in non-human primates, requires a 6-day preparative period before transplant, and is consequently limited to recipients of living donor allografts. In this study, we aimed to modify the regimen for cadaveric donor application. Methods: The standard regimen consists of total body irradiation (TBI), thymic irradiation (TI), antibody treatments and donor bone marrow (DBM), followed by a one-month course of cyclosporine. Various timimg or dosages of TBI, TI and antibodies were studied in six groups of cynomolgus monkeys. Results: Compression of 3 Gy of TBI and 7 Gy of TI into a 24-hour period led to unacceptable toxicity (Regimens A and C). Delayed irradiation and DBM failed to induce chimerism even with additional aCD154 (Regimen B). A single 3 Gy of TBI induced chimerism in 2/3 recipients without TI when combined with aCD154 and ATG (Regimen D). Nevertheless, all recipients treated with Regimen D eventually rejected their allografts. Addition of TI (4Gy) to Regimen D again resulted in toxicity (Regimen E). An antibody combination of ATG, aCD154 and aCD8 in conjunction with reduced dose TBI consistently induced mixed chimerism, less infectious complications, and prolonged graft survival (Regimen F). Conclusions: A single dose of TBI appears to be more effective for inducing chimerism than fractionated dosages. An antibody combination of ATG, aCD154 and aCD8 combined with TBI 2.5 Gy improved consistency of chimerism induction with less risk for infectious complications and appears promissing for induction of tolerance following a less than 24 hours pre-transplant conditioning protocol. The conditioning regimens and mixed chimerismFigureaureteral complication, binfection, clymphoma, dacute rejection, echronic rejection

Metastable tolerance in nonhuman primates and humans.

It is clear that both humans and nonhuman primates can do without immunosuppression for long periods of time before rejecting their allogeneic organ transplants. Immune cell depletion, particularly lymphocyte depletion, is an effective clinical strategy for achieving a tolerant state. Based on nonhuman primate and human studies, evidence suggests that metastable tolerance develops over time in a donor-specific manner and is mediated at least in part by donor antigen-specific regulatory T cells. Suppression of effector T cells by CD8+ and CD4+ T-regulatory cells is mediated by transforming growth factor (TGF)-beta or interleukin 10, and the presence of CD4+ TGF beta(latent) T-cell infiltrates may be a useful diagnostic marker for metastable tolerance. Loss of these cells has been shown to correlate with loss of tolerance. Future efforts to withdraw immunosuppressive drugs and establish a tolerant state will depend importantly on development of such diagnostic immunologic monitoring tools. Some of the reasons that tolerance remains such a challenging goal in clinical transplantation are discussed herein.

RETRACTED: The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation

Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.

Heterologous immunity provides a potent barrier to transplantation tolerance.

Many strategies have been proposed to induce tolerance to transplanted tissue in rodents; however, few if any have shown equal efficacy when tested in nonhuman primate transplant models. We hypothesized that a critical distinction between specific pathogen-free mice and nonhuman primates or human patients is their acquired immune history. Here, we show that a heterologous immune response--specifically, virally induced alloreactive memory--is a potent barrier to tolerance induction. A critical threshold of memory T cells is needed to promote rejection, and CD8(+) "central" memory T cells are primarily responsible. Finally, treatment with deoxyspergualin, an inhibitor of NF-kappa B translocation, together with costimulation blockade, synergistically impairs memory T cell activation and promotes antigen-specific tolerance of memory. These data offer a potential explanation for the difficulty encountered when inducing tolerance in nonhuman primates and human patients and provide insight into the signaling pathways essential for memory T cell activation and function.

Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD154 in nonhuman primate renal transplantation.

Antibodies and fusion proteins specific for CD80, CD86, and CD154 have shown promise as agents capable of inducing donor-specific tolerance in rodents. These agents have also been shown to be synergistic with one another in many settings of counter-adaptive immunity. In the nonhuman primate, monoclonal antibodies specific for CD80 and CD86 have prolonged the time to rejection of renal allografts but have not resulted in tolerance. A monoclonal antibody specific for CD154 has resulted in markedly prolonged survival of kidney, islet, cardiac, and skin allografts, but again most animals have eventually developed rejection after prolonged periods of rejection-free survival off therapy. A combination of monoclonal antibodies specific for CD80, CD86, and CD154 were used in a mismatched nonhuman primate renal-allograft model. Doses used were based on optimized treatment protocols for each agent individually. Treatment of four rhesus macaques with this combination yielded a mean rejection-free survival of 565 days (311-911 days), significantly greater than untreated controls (mean survival=7.0 days, P=0.001) and animals treated with only a combination of anti-CD80 and CD86 (mean survival=191 days, P=0.01). The survival of animals treated with this combination of monoclonal antibodies was not significantly greater than those treated with anti-CD154 alone, but the production of alloantibody was delayed compared with monotherapy anti-CD154. These data suggest that a synergy exists between these agents, particularly with regard to T-dependent B-cell responses, but that they fail to induce durable tolerance in nonhuman primates.

Costimulatory blockade for induction of mixed chimerism and renal allograft tolerance in nonhuman primates

Costimulatory blockade for induction of mixed chimerism and renal allograft tolerance in nonhuman primates

Comparison of horse antithymocyte globulin with other T cell-depleting antibodies for induction of chimerism and renal allograft tolerance in nonhuman primates

Comparison of horse antithymocyte globulin with other T cell-depleting antibodies for induction of chimerism and renal allograft tolerance in nonhuman primates

Tolerability and side effects of anti-CD3-immunotoxin in preclinical testing in kidney and pancreatic islet transplant recipients.

Anti-CD3-immunotoxin (alpha-CD3-IT) promotes allograft tolerance in nonhuman primates owing to efficient depletion of sessile and circulating T cells. Common side effects of vascular leak syndrome, hepatotoxicity, and nephrotoxicity have limited tolerability of other immunotoxins. We report on preclinical studies of alpha-CD3-IT-related side effects. Normal rhesus monkeys received a kidney transplant and alpha-CD3-IT alone (on day -to +2) or in combination with brief peritransplant adjunctive immunosuppressive therapy. Some received donor CD34+ cells. Blood chemistries, complete blood count, weight, liver, and kidney biopsies were examined for immunotoxin-related changes. Five spontaneously diabetic primates also received alpha-CD3-IT, three of whom had a pancreas islet transplant. The main side effect of alpha-CD3-IT, vascular leak syndrome, was entirely prevented by adjunctive immunosuppressive therapy. Renal and liver function tests and biopsies revealed a lack of nephrotoxicity and hepatotoxicity. All had transient weight loss (14+/-5%). Without infusion of donor CD34+ cells, 97% had full weight recovery. Of those given donor CD34+ cells, 50% were euthanized for wasting. Side effects of alpha-CD3-IT are manageable and should not prevent therapeutic application.

Tolerability and Side Effects of Anti-CD3 Immunotoxin (??CD3-IT) in Preclinical Testing in Kidney Allograft Recipients

322 αCD3-IT has shown potential to promote allograft tolerance in nonhuman primates owing to its unusual efficiency in depleting PBL and sessile lymph node (LN) T cells. Common side effects of vascular leak syndrome (VLS) hepatotoxicity and rash have limited host tolerability of most immunotoxins in the past. We postulated that the unique combined properties of αCD3-IT, i.e., stability of the mAb-mutated diphtheria conjugate, high specificity and affinity for the CD3ε receptor and efficient intracellular translocation would limit the systemic side effects that have hindered IT therapy to date. Normal male rhesus macaques underwent kidney transplantation with intrinsic nephrectomy and received either αCD3-IT(n=21) or αCD3 mAb (n=5) at a total dose of 133-150 µg/kg over 3 days, beginning 12-16h pretransplant. Some received additional treatment with 15-deoxspergualin (DSG; 2.5 mg/kg for 4 to 14 days) and methylprednisone(MP for 3 days at a tapering dose from 7 mg/kg). We examined blood chemistries, CBC, plasma cytokines, weight changes, and 24h liver biopsies for IT related side effects. The main side effect of αCD3-IT was VLS, characterized by increased body weight, edema, cytokine release, and decreased serum albumin without proteinuria. DSG and MP entirely prevented the VLS. Liver function tests and CBC values (tabulated below) were similar in recipients given αCD3-IT and αCD3 mAb. Liver biopsies showed an absence of hepatotoxicity after IT with or without DSG/MP. Rash occurred in 20% and resolved in 48h without special treatment. Transient weight loss(14 ± 5%) occurred in all recipients; of IT-treated recipients followed long-term for >3-18 months, 1/19 was euthanized for persistent low weight. Despite a mean 95% and 86% decrease in PBL and LN T cells, respectively, at 1wk post-transplant, and 50% recovery by 2 months, no evidence of increased infection or neoplasia was seen. In summary, side effects of αCD3-IT are manageable and should not prevent therapeutic application. Table

Tolerability and Side Effects of Anti-CD3 Immunotoxin (αCD3-IT) in Preclinical Testing in Kidney Allograft Recipients

322 αCD3-IT has shown potential to promote allograft tolerance in nonhuman primates owing to its unusual efficiency in depleting PBL and sessile lymph node (LN) T cells. Common side effects of vascular leak syndrome (VLS) hepatotoxicity and rash have limited host tolerability of most immunotoxins in the past. We postulated that the unique combined properties of αCD3-IT, i.e., stability of the mAb-mutated diphtheria conjugate, high specificity and affinity for the CD3ε receptor and efficient intracellular translocation would limit the systemic side effects that have hindered IT therapy to date. Normal male rhesus macaques underwent kidney transplantation with intrinsic nephrectomy and received either αCD3-IT (n=21) or αCD3 mAb (n=5) at a total dose of 133-150 μg/kg over 3 days, beginning 12-16h pretransplant. Some received additional treatment with 15-deoxspergualin (DSG; 2.5 mg/kg for 4 to 14 days) and methylprednisone (MP for 3 days at a tapering dose from 7 mg/kg). We examined blood chemistries, CBC, plasma cytokines, weight changes, and 24h liver biopsies for IT related side effects. The main side effect of αCD3-IT was VLS, characterized by increased body weight, edema, cytokine release, and decreased serum albumin without proteinuria. DSG and MP entirely prevented the VLS. Liver function tests and CBC values(tabulated below) were similar in recipients given αCD3-IT andαCD3 mAb. Liver biopsies showed an absence of hepatotoxicity after IT with or without DSG/MP. Rash occurred in 20% and resolved in 48h without special treatment. Transient weight loss (14 ±5%) occurred in all recipients; of IT-treated recipients followed long-term for >3-18 months, 1/19 was euthanized for persistent low weight. Despite a mean 95% and 86% decrease in PBL and LN T cells, respectively, at 1wk post-transplant, and 50% recovery by 2 months, no evidence of increased infection or neoplasia was seen. In summary, side effects of αCD3-IT are manageable and should not prevent therapeutic application. Table