The effect of thymectomy on tolerance induction and cardiac allograft vasculopathy in a miniature swine heart/kidney transplantation model.

BACKGROUND: We have previously demonstrated that MHC class I disparate hearts transplanted into miniature swine treated with a short course of cyclosporine developed florid cardiac allograft vasculopathy (CAV) and were rejected within 55 days. However, when a donor-specific kidney is cotransplanted with the heart allograft, recipients become tolerant to donor antigen and accept both allografts long-term without the development of CAV. In the present study, we have investigated the role of the host thymus in the induction of tolerance and prevention of CAV in heart/kidney recipients. METHODS: Total thymectomies were performed in six animals (postoperative day [POD]-21), which on day 0 received either an isolated MHC class I disparate heart allograft (n=3) or combined class I disparate heart and kidney allografts (n=3), followed in both cases by a 12-day course of cyclosporine (POD 0-11). Graft survival and the development of CAV in these thymectomized recipients were compared to the same parameters in non-thymectomized, cyclosporine-treated recipients bearing either class I disparate heart allografts (n=5) or heart and kidney allografts (n=4). RESULTS: In the group of animals bearing isolated class I disparate heart allografts, the thymectomized recipients rejected their allografts earlier (POD 8, 22, 27) than the non-thymectomized recipients (POD 33,35,45,47,55). The donor hearts in both the thymectomized and non-thymectomized animals developed florid CAV. In the group of animals bearing combined class I disparate heart and kidney allografts, the nonthymectomized recipients accepted both donor organs long term with no evidence of CAV. In contrast, none of the thymectomized heart/kidney recipients survived >100 days, and they all developed the intimal proliferation of CAV. CONCLUSION: Thymic-dependent mechanisms are necessary for the induction of acquired tolerance and prevention of CAV in porcine heart/kidney recipients.     

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates

BACKGROUND: Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. METHOD: Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). RESULTS: In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. <5 days). CONCLUSION: Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.     

Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance

BACKGROUND: Multilineage chimerism and long-term acceptance of renal allografts has been produced in non-human primates conditioned with a nonmyeloablative regimen. Our study was undertaken to evaluate the immunological and pathological status of long-term survivors and to define the role of splenectomy and of the primarily vascularized kidney in the regimen. METHOD: Monkeys were treated with the basic regimen, including: total body irradiation, thymic irradiation, antithymocyte globulin, donor bone marrow transplantation, and a 4-week course of cyclosporine after which no further immunosuppression was given. They were divided into four groups according to the timing of kidney transplantation (KTx) and splenectomy as follows; group A (n=13): KTx and splenectomy on the day of donor bone marrow transplantation (day 0); group B (n=3): KTx on day 0 without splenectomy; group C (n=7): splenectomy on day 0 but delayed KTx until 3 to 16 weeks post-donor bone marrow transplantation; group D (n=3): both splenectomy and KTx delayed until day 120 post-donor bone marrow transplantation. RESULTS: In group A, 11 of 13 monkeys developed chimerism and 9 monkeys achieved long-term survival of 4 to 70 months without evidence of chronic vascular rejection. Alloantibodies were detected in only one long-term survivor. In contrast, all three monkeys in group B developed alloantibodies and rejected their allografts. In group C, long-term survival without alloantibody production was observed in two of three monkeys that had developed chimerism. In group D, all three recipients were sensitized and rejected the kidney allografts rapidly after transplantation. CONCLUSIONS: 1) Production of anti-donor antibody was prevented in most recipients that developed mixed chimerism in the regimens with splenectomy at the time of donor bone marrow transplantation. 2) If splenectomy is not included in the initial conditioning regimen, induction of B cell tolerance is less likely and the result is late onset of alloantibody production and allograft rejection. 3) Immediate transplantation of the kidney at the time of recipient conditioning is not essential for induction of donor specific hyporesponsiveness by bone marrow transplantation.     

Development and maintenance of donor-specific chimerism in semi-allogenic and fully major histocompatibility complex mismatched facial allograft transplants

BACKGROUND: The clinical application of composite tissue allograft transplants opened the discussion on the restoration of facial deformities by allotransplantation. We introduce a hemifacial allograft transplant model to investigate the rationale for the development of operational tolerance across a major histocompatibility complex (MHC) barrier. MATERIAL AND METHODS: Thirty rats were studied in five groups of six animals each. The composite hemiface isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1(1+n) donors (group 2) and fully allogenic transplantations from ACI (RT1a) donors (group 3) to LEW (RT1(1)) recipients. In the allograft treatment groups, recipients of LBN (group 4) and ACI donors (group 5) were treated with cyclosporine A monotherapy (16 mg/kg/day, tapered to 2 mg/kg/day). Face allografts were evaluated clinically and histologically. Donor-specific chimerism for MHC class I RT1n and RT1a antigens was assessed by flow cytometry. Mixed lymphocyte reaction for donor-specific tolerance in vitro was tested at day 160 posttransplant. RESULTS: Isograft controls survived indefinitely. All nontreated allografts rejected within 5 to 8 days posttransplant. Long-term survival was achieved in 100% of LBN (up to 400 days) and ACI (up to 330 days) recipients. At day 160, posttransplant donor-specific chimerism was present in recipients of LBN (10.14% CD4/RT1n, 6.38% CD8/RT1n, 10.02% CD45RA/RT1n) and ACI (17.54% CD4/RT1a, 9.28% CD8/RT1a) transplants, and mixed lymphocyte reaction confirmed tolerance in recipients of LBN transplants and moderate reactivity in recipients of ACI allografts. CONCLUSION: Operational tolerance was induced in hemiface allograft transplants across an MHC barrier under cyclosporine A monotherapy protocol. It was associated directly with the presence of multilineage donor-specific chimerism.     

Demonstration of multilineage chimerism in a nonhuman primate concordant xenograft model

Abstract: Prior studies from our laboratory have demonstrated that a nonmyeloablative conditioning regimen can induce transient mixed chimerism and renal allograft tolerance between MHC disparate cynomolgus monkeys. We have also shown that this preparative regimen can be extended to a concordant baboon to cynomolgus xenograft model by adding, to the post transplant protocol, therapy designed to prevent antibody production. Here we examine the use of brequinar (BQR) for this purpose and the efficacy of two new reagents developed to demonstrate the establishment of chimerism in the xenograft recipients. The cynomolgus recipients were conditioned with WBI (300 cGy), TI (700 cGy), ATG, cyclosporine, and brequinar sodium. To detect engraftment of the donor marrow, we prepared a polyclonal cynomolgus anti-baboon antibody (CABA) and a monoclonal antibody (215.1), which distinguish baboon and cynomolgus lymphocytes and granulocytes. We employed flow cytometry analysis to detect multilineage chimerism in the xenograft recipients. Five of the six recipients monitored using our new reagents (CABA and 215.1) developed detectable chimerism and only one of these animals lost its kidney to rejection. However, other complications have not permitted assessment of long-term outcome. The features of the multilineage chimerism included the detection of donor granulocytes (1.8–77.4%) and lymphocytes (2.4–22.2%) for 9 to 37 days. Our new reagents permit the detection of multilineage mixed chimerism, which may be a predictor of xenograft tolerance. We also conclude that brequinar may be effective in preventing antibody formation, but because of its toxicity, it is probably not the drug of choice for extension of the mixed chimerism protocol to concordant xenografts.     

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.     

Permanent rat cardiac allograft survival induced by ultraviolet B-irradiated donor lymphocytes and peritransplant cyclosporine

This study examines the effect of pretreatment with 10(8) ultraviolet B-irradiated donor leukocytes (UV-DL) with or without peritransplant cyclosporine (CyA) treatment (20 mg/kg on days 0, +1, and +2 relative to transplantation) on rat cardiac allograft survival across major histocompatibility loci. A single UV-DL pretreatment on day -3 or -7 (before transplantation) significantly prolonged survival of heart allografts from Wistar-Furth rats (W/F) in Lewis recipients from 6.8 +/- 0.8 days to 18.4 +/- 2.1 and 17.6 +/- 1.5 days (p less than 0.001), respectively. Multiple UV-DL infusions on days -14 and -7 increased the mean survival time to 20.0 +/- 0.9 days (p less than 0.001). Similarly, UV-DL infusion on day -3 or -7 significantly prolonged the mean survival time of heart allografts from ACI rats in Lewis rats. A single or multiple UV-DL infusions combined with peritransplant CyA led specifically to permanent W/F cardiac allograft survival (more than 200 days) in all recipients. Similarly, UV-DL infusion combined with peritransplant CyA led to indefinite survival of ACI cardiac allografts in two thirds of Lewis recipients. Adoptive transfer of splenocytes from long-term recipients of cardiac allografts, which specifically prolonged donor test grafts in syngeneic hosts, suggests that unresponsiveness to cardiac allografts is, in part, dependent on suppressor cells. This study emphasizes the importance of UV irradiation of DLs in the modulation of alloreactivity and the induction of donor-specific unresponsiveness in adult animals.     

Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine

BACKGROUND: Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. METHODS: Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day coprse of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. RESULTS: Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6-9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. CONCLUSIONS: Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.     

Induction of tolerance to heart transplants by simultaneous cotransplantation of donor kidneys may depend on a radiation-sensitive renal-cell population

BACKGROUND: To determine the mechanism by which cotransplantation of a donor kidney and heart allograft induces tolerance to both organs in miniature swine, we examined the renal elements responsible for tolerance induction. METHODS: Recipients received 12 days of cyclosporine, and transplants were performed across a major histocompatibility complex (MHC) class I mismatch. Group 1 animals received heart transplants (n=5); group 2 animals received heart and kidney allografts with no other manipulation (n=4); group 3 animals received heart transplants and donor-specific renal parenchymal cells (n=4); group 4 animals received heart and kidney allografts from lethally irradiated donors (n=7); group 5 animals received irradiated hearts and nonirradiated kidneys (n=2); group 6 animals received nonirradiated hearts and peripheral blood leukocytes from swine MHC matched to recipients and becoming tolerant to donor antigen (n=2); group 7 animals received nonirradiated hearts and donor-specific peripheral blood monocyte cells (PBMC) (n=2). RESULTS: Animals in group 1 developed vasculopathy and fulminant rejection by day 55. Animals in group 2 never developed vascular lesions. Parenchymal kidney cell infusion (group 3) did not prolong cardiac survival. Animals in group 4 developed arteriopathy by postoperative day (POD) 28. Group 5 recipients accepted allografts without vascular lesions. Adoptive transfer of leukocytes from tolerant swine (group 6) prolonged cardiac graft survival as much as 123 days, whereas donor PBMC infusion (group 7) did not affect cardiac survival or development of arteriopathy. CONCLUSIONS: Radiosensitive elements in kidney allograft may be responsible for tolerance induction and prevention of chronic vascular lesions in recipients of simultaneous heart and kidney allografts.     

Donor-specific tolerance in fully major histocompatibility major histocompatibility complex-mismatched limb allograft transplants under an anti-alphabeta T-cell receptor monoclonal antibody and cyclosporine A protocol

BACKGROUND: Recent studies have demonstrated that treatment with alphabeta-T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined alphabeta-TCR-CsA protocol. METHODS: The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of alphabeta-TCR and CsA or a combination of alphabeta-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. RESULTS: Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-TCR-CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (AxC Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. CONCLUSIONS: Combined therapy of alphabeta-TCR-CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.     

Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation

BACKGROUND: Rodents and dogs conditioned with total-lymphoid irradiation (TLI), with or without antithymocyte globulin (ATG), have been shown to develop mixed chimerism and immune tolerance without graft-versus-host disease (GVHD) after the infusion of major histocompatability complex (MHC)-mismatched donor bone marrow cells given alone or in combination with an organ allograft. METHODS: Four human leukocyte antigen (HLA)-mismatched recipients of living donor kidney transplants were conditioned with TLI and ATG posttransplantation and infused with cyropreserved donor granulocyte colony-stimulating factor (G-CSF) "mobilized" hematopoietic progenitor (CD34+) cells (3-5x10(6) cells/kg) thereafter. Maintenance prednisone and cyclosporine dosages were tapered, and recipients were monitored for chimerism, GVHD, graft function, T-cell subsets in the blood, and antidonor reactivity in the mixed leukocyte reaction (MLR). RESULTS: Three of the four patients achieved multilineage macrochimerism, with up to 16% of donor-type cells among blood mononuclear cells without evidence of GVHD. Prolonged depletion of CD4+ T cells was observed in all four patients. Rejection episodes were not observed in the three macrochimeric recipients, and immunosuppressive drugs were withdrawn in the first patient by 12 months. Prednisone was withdrawn from a second patient at 9 months, and cyclosporine was tapered thereafter. CONCLUSIONS: Multilineage macrochimerism can be achieved without GVHD in HLA-mismatched recipients of combined kidney and hematopoietic progenitor transplants. Conditioning of the host with posttransplant TLI and ATG was nonmyeloablative and was not associated with severe infections. Recipients continue to be studied for the development of immune tolerance.     

A clinically relevant CTLA4-Ig-based regimen induces chimerism and tolerance to heart grafts

BACKGROUND: We determined whether a nontoxic CTLA4-Ig-based conditioning regimen effected mixed chimerism and donor-specific tolerance when heart and bone marrow were transplanted simultaneously. METHODS: Fully mismatched rat strain combinations were used. Recipients received total-body irradiation (300 centigrays), bone marrow (10(8) cells), and cardiac transplants from the donor on day 0. Subsequently, recipient animals received CTLA4-Ig (2 mg/kg, every other day, x 5 doses), tacrolimus (1 mg/kg/day; days 0 to 9), and one dose (10 mg) of antilymphocyte serum on day 10. RESULTS: All bone marrow recipients (n = 7) developed mixed chimerism (mean = 25% +/- 9% at 1 year) and accepted cardiac allografts permanently (> 375 +/- 32 days). Recipients that received conditioning regimen but no bone marrow (n = 5) rejected donor hearts within 51 +/- 13 days (p < 0.01). Recipients that accepted heart grafts also permanently accepted (> 180 days) donor-specific skin grafts, but rapidly rejected (< 10 days) third-party skin grafts. CONCLUSIONS: A nontoxic CTLA4-Ig-based conditioning regimen effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously. This regimen may have clinical application.     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.     

Induction of Cardiac Allograft Tolerance Across a Full MHC Barrier in Miniature Swine by Donor Kidney Cotransplantation

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12‐day course of high‐dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC‐mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor‐specific responsiveness in cell‐mediated lympholysis and mixed‐lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third‐party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor‐specific unresponsiveness in vitro. While kidney‐induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance‐resistant organs.     

Intrathymic injection of donor alloantigens induces donor-specific vascularized allograft tolerance without immunosuppression.

The induction of donor-specific tolerance could prevent the side effects of immunosuppression while improving allograft survival. Male adult Buffalo (RT1b) rats underwent an intrathymic (IT), portal venous (PV), intrasplenic (IS), or subcutaneous (SQ) injection of 25 x 10(6) major histocompatibility complex (MHC) mismatched Lewis (RT1(1)), UV-B-irradiated Lewis (RT1(1)), ACI (RT1a), or syngeneic Buffalo (RT1b) splenocytes. At the completion of the donor alloantigen injection, 1 mL rabbit anti-rat lymphocyte serum (ALS) was administered intraperitoneally to the Buffalo recipients, and 21 days later a heterotopic Lewis or ACI heart was transplanted. Intrathymic injection of donor alloantigen induced a donor-specific tolerance that allowed the cardiac allograft to survive indefinitely (mean survival time [MST] > 140.7 days) in 84% of the recipients without further immunosuppression, whereas groups receiving antigen injections at other sites (PV, IS, and SQ) plus ALS rejected cardiac allografts in normal fashion (MST approximately 8.0 days). Buffalo recipient rats with long-term surviving Lewis cardiac allografts after Lewis IT injection and ALS subsequently rejected a heterotopic third-party ACI cardiac allograft in normal fashion (MST approximately 7 days), whereas a second Lewis cardiac allograft was not rejected (MST > 116 days). Microchimerism is unlikely because Lewis allograft survival was also prolonged (MST > 38.7 days) in rats receiving UV-B-irradiated splenocytes IT, which cannot proliferate. Survival of Lewis renal allografts was also prolonged, but not indefinitely, in Buffalo recipients possessing a long-term surviving Lewis cardiac allograft (MST approximately 17.6 days versus 7 days for control). This model emphasizes the potential role of exposure of immature thymocytes to foreign donor alloantigens during maturation in the thymic environment for the development of unresponsiveness to an MHC-mismatched donor-specific vascularized allograft.     

The kinetics of tolerance induction by nondepleting anti-CD4 monoclonal antibody (RIB 5/2) plus intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. The monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating the recipient T cells. We have successfully induced tolerance to rat heart allografts by recipient pretreatment with a single dose of RIB 5/2 plus intravenous administration of donor splenocytes. In this study, we explored whether this potent regimen could induce tolerance to the more resistant kidney and skin allografts. Furthermore, we examined the kinetics and requirements for tolerance to be met by a single dose of RIB 5/2 plus i.v. alloantigen. METHODS: The efficacy of a single i.p. dose of 20 mg/kg RIB 5/2 plus i.v. donor antigen (25x10(6) splenocyte) pretreatment 0, 21, or 40 days before receipt of an MHC-mismatched Lewis (RT1l) to Buffalo (RT1b) rat cardiac, renal, or skin allograft was studied. Another group of Buffalo recipients treated with RIB 5/2 plus an i.v. alloantigen +/-thymectomy received kidney transplants after 40 days. Attempts to prevent tolerance used interleukin-2 or prior sensitization. Mixed lymphocyte cultures, cytotoxic assays, and precursor frequencies of helper and cytotoxic cells, by limiting dilution analysis, serially measured in vitro cell-mediated immunity. RESULTS: RIB 5/2 administration combined with i.v. alloantigen 21 days before induced tolerance to heart and kidney allografts but did not prolong skin graft survival. In contrast, kidney allografts delayed for 40 days after pretreatment were acutely rejected and survival was not affected by the thymectomy. MLC, CTL, and pTH, and pCTL precursor frequencies from recipients of long-term grafts were specifically suppressed to donor, but not third party, alloantigen. CONCLUSION: A single dose of the nondepleting anti-CD4 monoclonal antibody, RIB 5/2, plus i.v. alloantigen is a potent inducer of tolerance to heart and kidney, but not skin, allografts. The RIB 5/2-induced donor unresponsiveness to a delayed kidney or cardiac allograft is time dependent but can be prolonged if specific alloantigen is present. Suppression of cell-mediated allo-immune responsiveness correlates with allograft acceptance.     

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine

A 12‐day course of high‐dose tacrolimus induces tolerance of major histocompatibility complex–mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart–lung cotransplantation using the same induction protocol. Hearts (n = 3), heart–kidneys (n = 3), lungs (n = 6), and hearts–lungs (n = 3) were transplanted into fully major histocompatibility complex–mismatched recipients treated with high‐dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart–kidney recipients and five of six lung recipients demonstrated long‐term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor‐specific unresponsiveness by cell‐mediated lympholysis/mixed‐lymphocyte reaction. In contrast, heart–lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.     

Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade

BACKGROUND: Clinical composite-tissue (hand) transplantation between genetically disparate individuals currently requires potent, nonspecific immunosuppressive agents that are neither completely successful in preventing acute episodes of rejection nor free from complications. The reliance on long-term immunosuppression has prompted this study to achieve donor-specific transplantation tolerance in adult recipients using a nontoxic, nonmyeloablative protocol. METHODS: Fully mismatched, 4- to 6-week-old ACI (RT1Aa) and Wistar Furth (WF) rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig at 2 mg/kg per day (alternate days) in combination with tacrolimus at 1 mg/kg per day (daily) from day 0 through day +10, antilymphocyte serum at 10 mg at day +10 (single dose), and total-body irradiation t 300 cGy (day 0) before bone-marrow transplantation (BMT) (day 0) with 100 x 10(6) T-cell-depleted bone marrow cells. Hindlimb transplants were performed 4 weeks postBMT. Multilineage donor hematopoiesis was determined pre- and posttransplant using flow cytometry. In vitro T-cell responses were evaluated by mixed lymphocyte reactivity assays. RESULTS: CD28 blockade in a transplant model of mixed chimerism effectively aborts T-cell clonal expansion in vitro and in vivo, inhibits the development of acute and chronic rejection of vascularized hindlimb allografts in rats (ACI limbs to ACI-->WF chimeras, n=5; WF limbs to ACI-->WF chimeras, n=4), and subsequently leads to long-term survival of allogeneic skin grafts (n=9). Third-party (F344, n=4) transplants were uniformly rejected within 14 days posttransplant. Multilineage donor hematopoiesis was demonstrated pre- and posttransplant. Donor chimerism, present postBMT, increased throughout the study (pretransplant range 2-28%, mean 17%; posttransplant range 5-49%, mean 34%). Transplant recipients maintained full reactivity to respond to third-party antigens without harmful manifestations of graft-versus-host disease. CONCLUSIONS: Although efforts have been made to induce tolerance to composite tissue allografts in adult recipients, thus far, none have succeeded without toxic, myeloablative host preconditioning. Our demonstration that tolerance can be achieved with minimal preconditioning provides a rationale for application to large animals and humans and suggests that although composite tissue allografts may have a significant skin component (and are therefore felt to be highly antigenic), protocols used to induce tolerance to organ transplants may be equally applicable to composite-tissue allotransplantation.     

CTLA4-Ig-based conditioning regimen to induce tolerance to cardiac allografts

BACKGROUND: Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. METHODS: Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. RESULTS: Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. CONCLUSIONS: The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.     

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. I. Prolongation of rat cardiac and renal allograft survival by the PG27 extract and immunosuppressive synergy in combination therapy with cyclosporine

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii. We tested PG27 in rat cardiac and renal allotransplantation, and we examined the immunosuppressive interaction with cyclosporine (CsA). METHODS: Brown Norway (BN) rat heart or kidney allografts were transplanted into the abdomen of Lewis rats, which were treated by the intraperitoneal or oral route with PG27, CsA, or both. RESULTS: PG27 administered intraperitoneally to Lewis recipients for 16 days at 10-30 mg/kg/day significantly increased the median survival time of BN heart allografts from 7 to 18-22 days. Oral administration was effective, with cardiac allograft survival prolonged to > 100 days with 52 days of treatment. PG27 at 20-30 mg/kg/day significantly extended the median survival time of BN kidney allograft recipients from 9 to 36.5-77 days, and 30 mg/kg/day for 52 days extended survival beyond 200 days. PG27 combined with CsA significantly enhanced heart and kidney allograft survival, even at doses of CsA ineffective when administered alone. The addition of 5 or 10 mg/kg/day PG27 reduced by 50-75% the CsA dose needed for 100% kidney allograft survival. The combination index was less than 1.0, indicating synergy of PG27 with CsA in prolonging cardiac and renal allograft survival. Furthermore, the PG27/CsA combination exerted a positive influence on renal allograft function. PG490 (triptolide, a constituent of PG27) and PG490-88 (a semisynthetic derivative of PG490) suppressed rejection of cardiac and renal allografts. CONCLUSIONS: The PG27 herbal extract demonstrated immunosuppressive activity by prolonging heart and kidney allograft survival, displaying synergy in the immunosuppressive interaction with CsA, and improving renal allograft function in combination with CsA. PG490 and PG490-88 compounds were also effective.