Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Innate immunity and resistance to tolerogenesis in allotransplantation.

The development of immunosuppressive drugs to control adaptive immune responses has led to the success of transplantation as a therapy for end-stage organ failure. However, these agents are largely ineffective in suppressing components of the innate immune system. This distinction has gained in clinical significance as mounting evidence now indicates that innate immune responses play important roles in the acute and chronic rejection of whole organ allografts. For instance, whereas clinical interest in natural killer (NK) cells was once largely confined to the field of bone marrow transplantation, recent findings suggest that these cells can also participate in the acute rejection of cardiac allografts and prevent tolerance induction. Stimulation of Toll-like receptors (TLRs), another important component of innate immunity, by endogenous ligands released in response to ischemia/reperfusion is now known to cause an inflammatory milieu favorable to graft rejection and abrogation of tolerance. Emerging data suggest that activation of complement is linked to acute rejection and interferes with tolerance. In summary, the conventional wisdom that the innate immune system is of little importance in whole organ transplantation is no longer tenable. The addition of strategies that target TLRs, NK cells, complement, and other components of the innate immune system will be necessary to eventually achieve long-term tolerance to human allograft recipients.

Differential effects of denileukin diftitox IL-2 immunotoxin on NK and regulatory T cells in nonhuman primates.

Denileukin diftitox (DD), a fusion protein comprising IL-2 and diphtheria toxin, was initially expected to enhance antitumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high-affinity IL-2R (α-ß-γ trimers). Although DD was shown to deplete some Tregs in primates, its effects on NK cells (CD16(+)CD8(+)NKG2A(+)CD3(-)), which constitutively express the intermediate-affinity IL-2R (ß-γ dimers) and play a critical role in antitumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected i.v. with two doses of DD (8 or 18 µg/kg). This treatment resulted in a rapid, but short-term, reduction in detectable peripheral blood resting Tregs (CD4(+)CD45RA(+)Foxp3(+)) and a transient increase in the number of activated Tregs (CD4(+)CD45RA(-)Foxp3(high)), followed by their partial depletion (50-60%). In contrast, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells compared with Tregs. Coadministration of DD with IL-15, which binds to IL-2Rß-γ, abrogated DD-induced NK cell deletion in vitro and in vivo, whereas it did not affect Treg elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon that might impair its antitumoral properties. However, coadministration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells, while allowing the depletion of immunosuppressive Tregs in cancer patients.

Effects of an agonist interleukin-2/Fc fusion protein, a mutant antagonist interleukin-15/Fc fusion protein, and sirolimus on cardiac allograft survival in non-human primates.

BACKGROUND: To tilt the immunologic balance toward tolerance and away from rejection, non-human primate recipients of cardiac allografts were treated with interleukin (IL)-2/Fc, mutant (m) antagonist type mIL-15/Fc, and sirolimus. METHODS: Heterotopic heart transplants were performed on 8 fully mismatched cynomolgus macaques. An untreated control recipient rejected its graft by post-operative Day 6. The remaining 7 animals received oral or intramuscular immunosuppression with sirolimus. A recipient treated with sirolimus alone rejected at the end of 28 days of immunosuppression. The remaining 6 monkeys also received IL-2/Fc and mIL-15/Fc intramuscularly until 28 days after transplant. One animal received a second 28-day course of fusion protein starting at Day 50. In these 6 animals, sirolimus was continued for 28 days (n = 4) or until protein levels were low (n = 2). RESULTS: In the 4 monkeys treated with a 28-day course of sirolimus and fusion proteins, mean graft survival was 51.5 days (range, 28-76 days). The animal receiving a second course of fusion protein rejected its graft on Day 177, despite detectable levels of the fusion proteins and sirolimus. The central memory, effector memory, and naïve CD4(+) and CD8(+) T-cell populations in the peripheral blood did not change significantly during fusion protein administration. A 2.5-fold expansion in CD4(+)CD25(+) lymphocytes occurred in recipients treated with fusion proteins and sirolimus that was not observed in the recipient treated with sirolimus alone. CONCLUSIONS: Although IL-2/Fc, mIL-15/Fc, and sirolimus administered in this manner permitted modest prolongation of graft survival and expansion of CD4(+)CD25(+) T cells, tolerance was not achieved.

Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in nonhuman primates.

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.

Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates.

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.

Stem cell mobilization and collection for induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys.

BACKGROUND: We have previously observed that donor bone marrow hematopoietic stem cells successfully induce transient mixed chimerism and renal allograft tolerance following non-myeloablative conditioning of the recipient. Stem cells isolated from the peripheral blood (PBSC) may provide similar benefits. We sought to determine the most effective method of mobilizing PBSC for this approach and the effects of differing conditioning regimens on their engraftment. METHODS: A standard dose (10 µg/kg) or high dose (100 µg/kg) of granulocyte colony-stimulating factor (GCSF) with or without stem cell factor (SCF) was administered to the donor, and PBSC were collected by leukapheresis. Cynomolgus monkey recipients underwent a nonmyeloablative conditioning regimen (total body irradiation, thymic irradiation, and ATG) with splenectomy (splenectomy group) or a short course of anti-CD154 antibody (aCD154) (aCD154 group). Recipients then received combined kidney and PBSC transplantation and a 1-mo post-transplant course of cyclosporine. RESULTS: Treatments with either two cytokines (GCSF+SCF) or high dose GCSF provided significantly more hematopoietic progenitor cells than standard dose GCSF alone. Recipients in the aCD154 group developed significantly higher myeloid and lymphoid chimerism (P < 0.0001 and P = 0.0002, respectively) than those in the splenectomy group. Longer term renal allograft survival without immunosuppression was also observed in the aCD154 group, while two of three recipients in the splenectomy group rejected their allografts soon after discontinuation of immunosuppression. CONCLUSIONS: Protocols including administration of two cytokines (GCSF + SCF) or high dose GCSF alone significantly mobilized more PBSC than standard dose GCSF alone. The recipients of PBSC consistently developed excellent chimerism and survived long-term without immunosuppression, when treated with CD154 blockade.

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.

Limited efficacy and unacceptable toxicity of cyclophosphamide for the induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys.

To induce mixed chimerism and renal allograft tolerance in cynomolgus monkeys, cyclophosphamide (CP) and total body irradiation (TBI) were compared as part of a nonmyeloablative conditioning regimen. CP induced dose-dependent neutropenia and lymphopenia, but hematopoietic recovery was more rapid than that observed in the TBI group. Absolute B cell counts after CP were significantly higher (P<0.01) than those in the TBI group. With CP, a total dose of 200 mg/kg with CD154 blockade regularly induced multilineage chimerism. Nevertheless, the recipients failed to achieve long-term survival because of rejection (3 of 5), posttransplantation B cell lymphoma (1 of 5), and toxicities of CP (1 of 5). As previously reported, 3 Gy of TBI with either splenectomy or CD154 blockade induced mixed chimerism and renal allograft tolerance, with significantly less morbidity and mortality than that produced by CP. Thus, TBI is more effective and less toxic than CP as part of a nonmyeloablative regimen for the induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys.

Monitoring antidonor alloantibodies as a predictive assay for renal allograft tolerance/long-term observations in nonhuman primates.

BACKGROUND: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients. METHODS: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4). RESULTS: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR. CONCLUSION: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.

CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates.

Costimulatory blockade with anti-CD154 monoclonal antibody (aCD154) prolongs allograft survival in nonhuman primates, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding CD154 blockade to a chimerism inducing nonmyeloablative regimen in primates. We observed a significant improvement of donor bone marrow (DBM) engraftment, which has been associated with a lower incidence of acute rejection and long-term survival of renal allografts without the need for previously required splenectomy. Among the long-term survivors, four never showed evidence of rejection, with the longest survival exceeding 1700 days following discontinuation of immunosuppression. Nevertheless, late chronic rejection was observed in three of eight recipients, indicating the necessity of further modifications of the regimen. Control recipients receiving no DBM or donor splenocytes in place of DBM rejected their allografts. Thus, DBM engraftment with, at least, transient mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Modification of the original mixed chimerism approach, by the addition of costimulatory blockade, has been shown to enhance mixed chimerism and induce renal allograft tolerance with less morbidity in nonhuman primates.

Thrombophilia associated with anti-CD154 monoclonal antibody treatment and its prophylaxis in nonhuman primates.

BACKGROUND: The authors previously reported thromboembolic complications associated with anti-CD154 monoclonal antibody (mAb) treatment in nonhuman primates. The underlying mechanisms of this complication and its management have not been established. METHODS: Eighty cynomolgus monkey renal allograft recipients treated with anti-CD154 mAb were studied for the incidence of thrombosis and its prophylaxis. RESULTS: Without anticoagulation prophylaxis, thromboembolic complications were seen in 5 of 11 recipients. With addition of perioperative heparin, the incidence was decreased to 2 of 10. No further improvement was observed by adding intraoperative prostaglandin (PG) E1. However, addition of ketorolac tromethamine to PGE1 and heparin decreased the incidence of thrombosis (one of eight). Most recently, the authors have found that ketorolac administration alone resulted in no thrombosis in 25 consecutive recipients. CONCLUSIONS: Ketorolac is remarkably effective in preventing thromboembolism associated with anti-CD154 mAb treatment, suggesting the mechanism underlying this complication may be related to platelet activation leading to enhanced aggregation.

Effect of mixed hematopoietic chimerism on cardiac allograft survival in cynomolgus monkeys.

BACKGROUND: We have previously reported the successful induction of mixed chimerism and long-term acceptance of renal allografts in MHC-mismatched nonhuman primates after nonmyeloablative conditioning and donor bone marrow transplantation. In this study, we extended our regimen to cardiac allotransplantation and compared the immunological responses of heart and kidney allograft recipients. METHODS: Five cynomolgus monkeys were conditioned with low-dose total body irradiation (1.5 Gy on days -6 and -5), supplemental thymic irradiation (7 Gy on day -1), antithymocyte globulin (50 mg/kg on days -2, -1, and 0), splenectomy (day 0), donor bone marrow transplantation (day 0), and a 4-week posttransplant course of cyclosporine. Heart allografts from MHC-mismatched donors were transplanted heterotopically on day 0. RESULTS: Two monkeys failed to develop multilineage chimerism and rejected their allografts soon after cyclosporine was stopped (postoperative days [PODs] 43 and 56). Three monkeys developed multilineage chimerism, which persisted 20 to 43 days posttransplant by flow cytometric analysis and to POD 124 by polymerase chain reaction analysis. Allograft survival in these recipients was prolonged to 138, 428, and 509 days, and in vitro mixed leukocyte reaction and cell-mediated lympholysis (CML) assays demonstrated donor-specific hyporesponsiveness. However, in contrast to kidney allograft recipients, long-term heart allograft recipients eventually developed humoral and cellular immunity against the donor and rejected the grafts. At the time of rejection, 1.3% to 9.5% of donor coronary arteries exhibited intimal proliferation. CONCLUSIONS: The induction of transient mixed hematopoietic chimerism leads to long-term heart allograft survival in MHC disparate monkeys without chronic immunosuppression. However, unlike kidney allografts, full tolerance to cardiac allografts was not achieved. Organ-specific modifications of the preparative regimen may be necessary to prevent the chronic cellular and humoral immune responses elicited by cardiac allografts.