S. Boskovic

Depletion of CD8 memory T cells for induction of tolerance of a previously transplanted kidney allograft.

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti‐CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti‐CD8 monoclonal antibody (cM‐T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide ‘proof of principle’ that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T‐cell function is adequately controlled.

Four Stages and Lack of Stable Accommodation in Chronic Alloantibody-Mediated Renal Allograft Rejection in Cynomolgus Monkeys

The etiology of immunologically mediated chronic renal allograft failure is unclear. One cause is thought to be alloantibodies. Previously in Cynomolgus monkeys, we observed a relationship among donor‐specific alloantibodies (DSA), C4d staining, allograft glomerulopathy, allograft arteriopathy and progressive renal failure. To define the natural history of chronic antibody‐mediated rejection and its effect on renal allograft survival, we now extend this report to include 417 specimens from 143 Cynomolgus monkeys with renal allografts. A subset of animals with long‐term renal allografts made DSA (48%), were C4d positive (29%), developed transplant glomerulopathy (TG) (22%) and chronic allograft arteriopathy (CAA) (19%). These four features were highly correlated and associated with statistically significant shortened allograft survival. Acute cellular rejection, either Banff type 1 or 2, did not correlate with alloantibodies, C4d deposition or TG. However, endarteritis (Banff type 2) correlated with later CAA. Sequential analysis identified four progressive stages of chronic antibody‐mediated rejection: (1) DSA, (2) deposition of C4d, (3) TG and (4) rising creatinine/renal failure. These new findings provide strong evidence that chronic antibody‐mediated rejection develops without enduring stable accommodation, progresses through four defined clinical pathological stages and shortens renal allograft survival.

Host Alloreactive Memory T Cells Influence Tolerance to Kidney Allografts in Nonhuman Primates

Only nonhuman primates with low frequencies of donor-specific memory T cells develop tolerance and accept allogeneic kidney transplants. The Slippery Slope of Transplantation Tolerance Although science is respected as an impartial pursuit, even the most unbiased observer must begin with assumptions to design experiments. But even seemingly reasonable hypotheses don’t always survive experimental scrutiny. Transplantation researchers began with the credible assumption that laboratory mice can serve as a model for human transplantation. Thus, when scientists showed that mice can accept human grafts without life-long immunosuppression, researchers enthusiastically attempted to translate these findings to nonhuman primates and patients—to no avail. Nadazdin et al. now provide a potential explanation for this discrepancy—a pre-existing pool of graft-reactive memory T cells. Memory T cells respond more rapidly and with greater strength than other T cells that have not previously seen a specific antigen. As the name suggests, memory T cells are long-lived in patients and explain in part the success of vaccination in preventing subsequent infections. Laboratory mice lack large numbers of memory T cells because they live in germ-free conditions, unlike people who, despite their best efforts, do not. This lifestyle difference was not thought to be a problem for transplantation studies, because people have not been previously exposed to donor-derived alloantigens and thus were not expected to have a memory response. In an unexpected twist, primates have been shown to have a relatively high frequency of alloreactive memory T cells before transplantation, perhaps as a result of cross-reactivity from previous infections. Nadazdin et al. now investigate the effects of these preexisting alloreactive memory T cells on transplant tolerance in nonhuman primates. The authors found that transplanted organs are rejected from monkeys with high numbers of preexisting alloreactive memory T cells, but survive long-term in monkeys with low numbers of these cells. Indeed, the animals with low numbers of alloreactive memory cells were rendered tolerant to the transplanted kidney, which was not rejected despite a lack of continued immunosuppression. This tolerance was allospecific, because both tolerant and rejecting monkeys had similar levels of homeostatic memory T cell expansion, but only rejecting monkeys displayed expanded levels of donor-reactive memory T cells after transplantation. These findings suggest two approaches to improving tolerance induction in transplant patients: One could either pair grafts with patients who have low numbers of donor-specific memory T cells or devise a way to eliminate these memory T cells from patients before transplantation. In this case, questioning assumptions did not validate the assumption, but instead yielded new information that may help researchers overcome barriers to transplant tolerance. Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients.

Chronic Antibody Mediated Rejection of Renal Allografts: Pathological, Serological and Immunologic Features in Nonhuman Primates

The pathogenesis of late renal allograft loss is heterogeneous and difficult to diagnose. We have analyzed renal allografts in nonhuman primates to determine the relationship between alloantibodies and the graft pathology of late graft loss. Seventeen Cynomolgus monkeys were chosen from among those on several protocols for renal allotransplantation with mixed chimerism induction so that animals with and without alloantibodies were included. All animals received transient CD154 blockade and short‐term cyclosporine treatment until day 28. Serial blood samples were tested for alloantibodies. Protocol biopsies and autopsy kidneys were scored for pathology and C4d deposition. Group 1, defined by complete lack of C4d deposition (24 tissue samples; 8 recipients), had no detectable alloantibodies (33 serum samples; 1–7 samples per recipient) and no evidence of chronic rejection. Three survived greater than 2 years with normal function and histology. Group 2, defined as having C4d deposition in peritubular capillaries, all made alloantibodies (100%), and most grafts later showed chronic allograft glomerulopathy (89%), and/or arteriopathy (89%). All grafts in Group 2 failed (3–27 months). Pathologic lesions of typical of chronic rejection in humans develop in monkeys, correlate with antecedent alloantibodies/C4d deposition and predict chronic rejection rather than durable accommodation.

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such “delayed tolerance” can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor‐specific memory T‐cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long‐term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

Phenotype, Distribution and Alloreactive Properties of Memory T Cells from Cynomolgus Monkeys

The high frequency of memory T cells present in primates is thought to represent a major barrier to tolerance induction in transplantation. Therefore, it is crucial to characterize these memory T cells and determine their functional properties. High numbers of memory T cells were detected in peripheral blood and all lymphoid tissues except lymph nodes, which were essentially the site of naïve T cells. The majority of CD8+ memory T cells were effector memory cells located in the blood and bone marrow while most CD4+ memory T cells were central memory cells present in the spleen. Next, memory T cells from over 100 monkeys were tested for their response to alloantigens by ELISPOT. Memory alloreactivity mediated via direct but not indirect allorecognition was detected in all animals. The frequency of allospecific memory T cells varied dramatically depending upon the nature of the responder/stimulator monkey combination tested. MHC gene matching was generally associated with a low‐memory alloreactivity. Nevertheless, low anamnestic alloresponses were also found in a significant number of fully MHC‐mismatched monkey combinations. These results show that selected donor/recipient combinations displaying a low memory alloresponsiveness can be found. These combinations may be more favorable for transplant tolerance induction.

Evaluation of recombinant human soluble dimeric tumor necrosis factor receptor for prevention of OKT3-associated acute clinical syndrome

Tumor necrosis factor alpha (TNFa) has been shown to be the primary cytokine responsible for the OKT3-induced acute clinical syndrome (OKT3-ACS). Recombinant human soluble tumor necrosis factor receptor (TNFR:Fc) is a dimer of the p80 TNF receptor, which binds both TNFa and lymphotoxin (LT). Renal allograft recipients undergoing OKT3 therapy for steroid-resistant rejection were randomized to receive OKT3 alone or in combination with TNFR:Fc to determine its safety and efficacy in decreasing the severity of OKT3-ACS and in restoring renal function. Six of 12 patients were given TNFR:Fc prior to each of the first two injections of OKT3. All patients were monitored for manifestations of OKT3-ACS and changes in renal function. In addition, serial serum samples were assayed for TNFa and TNFR:Fc levels (ELISA) and TNFa bioactivity (L929). No adverse side effects were identified in patients receiving TNFR:Fc. Patients treated with TNFR:Fc had significantly fewer symptoms by day 2 of OKT3, and had a lower overall incidence of chills and arthralgias. Renal dysfunction reversed within 24 hr in the TNFR:Fc-treated group in contrast to the 48-72-hr delay in the control group. Antigenic TNFa levels increased in the control group from < 10 pg/ml pre OKT3 to a mean peak level of 30 +/- 13 pg/ml on day 1 and decreased to pretreatment levels by day 2. TNFR:Fc-treated patients had a mean peak TNFa level of 235 +/- 135 pg/ml, suggesting a carrier effect of TNFR:Fc. In contrast, bioactivity was barely detectable (mean 20 +/- 14 pg/ml) in the day 1 samples from TNFR:Fc-treated patients, whereas significant bioactivity (peak mean 60 +/- 35 pg/ml) was detected in sera from control patients. TNF receptor levels reached 600 ng/ml in treated patients and remained elevated for up to 18 days confirming the long half-life of TNFR:Fc. This phase 1 trial demonstrates that TNFR:Fc is well tolerated and may limit the severity of OKT3-ACS. The most significant observation was a more rapid improvement in renal function in the TNFR:Fc-treated patients. The absence of TNFa bioactivity indicates that TNFR:Fc functions as a TNF antagonist. Further evaluation of higher doses of TNFR:Fc in OKT3-treated patients is currently in progress.

Long-term islet allograft function in the absence of chronic immunosuppression : A case report of a nonhuman primate previously made tolerant to a renal allograft from the same donor

UNLABELLED Development of mixed chimerism by donor bone marrow transplantation (DBMT) has led to long-term tolerance of solid organ allografts in nonhuman primates. As an initial attempt to extend this approach to cellular transplant, islet transplant from the same donor was attempted in the recipient previously made tolerant to a kidney allograft. METHODS After the conditioning with ATG, total body irradiation, thymic irradiation, and splenectomy, DBMT was performed followed by 4 weeks of cyclosporine. Kidney transplantation and native nephrectomies were subsequently performed on day 89. After 2.8 years of DBMT, diabetes was induced by streptozocin (STZ) and islets from bone marrow and kidney donor were transplanted without immunosuppression. RESULTS After DBMT, the recipient developed chimerism and no evidence of kidney rejection for more than 1000 days. STZ induced diabetes was reversed after the islet transplantation. Islet biopsies demonstrated insulin staining without rejection. Although the recipient became diabetic 300 days after islet transplantation, viable transplanted islets were found in the liver and under the kidney capsule without any evidence of rejection. CONCLUSION Tolerance with a nonmyeloablative conditioning can allow successful pancreatic islet transplantation without immunosuppression. Because no histological evidence of rejection was identified, recurrent diabetes is presumed to be inadequate islet mass.

Tolerance of Lung Allografts Achieved in Nonhuman Primates via Mixed Hematopoietic Chimerism

While the induction of transient mixed chimerism has tolerized MHC‐mismatched renal grafts in nonhuman primates and patients, this approach has not been successful for more immunogenic organs. Here, we describe a modified delayed‐tolerance‐induction protocol resulting in three out of four monkeys achieving long‐term lung allograft survival without ongoing immunosuppression. Two of the tolerant monkeys displayed stable mixed lymphoid chimerism, and the other showed transient chimerism. Serial biopsies and post‐mortem specimens from the tolerant monkeys revealed no signs of chronic rejection. The tolerant recipients also exhibited T cell unresponsiveness and a lack of alloantibody. This is the first report of durable mixed chimerism and successful tolerance induction of MHC‐mismatched lungs in primates.

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 alpha 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.