Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.     

Donor-specific renal,but not cardiac,allograft tolerance promotes engraftment of the normally rejected rat skin graft

This study examined whether a heart or kidney graft could provide protection for the more resistant skin graft. Buffalo rat recipients were given a single dose of RIB 5/2 (non-depleting anti-CD4 mAb) plus i.v. Lewis splenocytes 21 days before being given Lewis heart or kidney grafts. Lewis skin was grafted either simultaneously with, or after, long-term (>50 days) Lewis heart or kidney allograft acceptance. Immune responsiveness was analyzed by in vitro mixed lymphocyte culture (MLC), cytotoxic T lymphocytes (CTLs), and limiting dilution analysis (LDA). While i.v. alloantigen plus RIB 5/2 resulted in long-term acceptance of heart and kidney, survival of skin grafts alone was not prolonged. However, simultaneous transplantation with kidney, but not heart, resulted in long-term skin graft acceptance, while skin grafts subsequently grafted to recipients tolerant to kidney or heart were not accepted. In vitro analysis revealed a down-regulation of proliferation, cytotoxicity, and precursor T-helper cells (pThs)/precursor cytotoxic T lymphocytes (pCTLs) in Buffalo recipients accepting Lewis kidney and skin allografts. While RIB 5/2 plus Lewis splenocytes do not prolong the survival of skin grafts, Lewis skin grafted simultaneously with a kidney, but not heart, is accepted indefinitely and provides donor-specific protection for a subsequent skin graft.     

Effect of nondepleting anti-CD4 monoclonal antibody (Rib 5/2) plus donor antigen pretreatment in peripheral nerve allotransplantation

Peripheral nerve allotransplantation allows the reconstruction of injuries with long nerve gaps that are otherwise unsalvageable. In this study, the efficacy of anti-CD4 monoclonal antibody (mAb) combined with donor antigen pretreatment in prolonging the survival of short peripheral nerve allografts was investigated in a rodent model. Such an approach could potentially avoid the need for systemic immunosuppression and its concomitant morbidities. Buffalo rats received either nerve isografts or nerve allografts from Lewis rats. Untreated isograft and allograft groups were used as controls. Allograft recipients received either a single dose of RIB 5/2, a nondepleting anti-CD4 mAb, a single dose of Lewis splenocytes, or both antigen and RIB 5/2, 7 days prior to transplantation. Flow cytometric analysis verified that the T-lymphocyte population was maintained, while CD4 expression was downregulated by RIB 5/2. Histologic evaluation demonstrated better regeneration in the allograft recipients receiving both donor antigen and antibody, compared to recipients of untreated allografts or treatment with antigen or antibody alone.     

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.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.     

MHC class II presenting cells are necessary for the induction of intrathymic tolerance.

OBJECTIVE: This study determined the form of cellular donor MHC alloantigen necessary for the induction of intrathymic tolerance. BACKGROUND: The authors have achieved indefinite donor-specific tolerance, to a fully MHC-disparate rat heterotopic cardiac allograft, after the pretransplant intrathymic injection of unfractionated donor splenocytes and a single injection of rabbit anti-rat lymphocyte serum (ALS), without subsequent immunosuppression. METHODS: Male 4-12-week-old Buffalo (RT1b) rats underwent an intrathymic injection of either fractionated Lewis (RT1(1)) red blood cells (purified by Ficoll gradient) or T lymphocytes (purified by nylon wool column and plastic adherence), both of which express only MHC class I alloantigens, or B lymphocytes, macrophages, and dendritic cells (purified by plastic adherence) which express both MHC class I and class II alloantigens. At the completion of alloantigen injection the Buffalo recipient rats were given 1 ml of ALS intraperitoneally. Twenty-one days later a heterotopic Lewis heart was transplanted. RESULTS: The intrathymic injection of the fractions of Lewis MHC class I and class II expressing B lymphocytes, macrophages, and dendritic cells induced a donor-specific tolerance that resulted in indefinite Lewis cardiac allograft survival (MST > 125 days) in all recipients without further immunosuppression, whereas groups receiving MHC class I expressing red blood cell or T lymphocyte injections plus ALS rejected Lewis cardiac allografts with a MST of 7.3 and 16.5 days, respectively, thus indicating that the MHC class II expressing cell is necessary for the induction of intrathymic tolerance. Buffalo recipients with a long-term surviving Lewis cardiac allograft, after Lewis MHC class II expressing cells were still able to reject a third-party heterotopic ACI (RT1a) cardiac allograft in normal time (MST = 7.0 days), but did not reject a second Lewis cardiac allograft (MST > 100 days). Additionally, the intrathymic injection of MHC class II expressing cells resulted in decreased interleukin-2 (IL-2) production and an 80% decrease in in vitro donor-specific cell mediated cytotoxicity, whereas the cytolytic response to a third party was unaltered. CONCLUSION: Donor MHC class II, and not class I, expressing cells are the cells in donor splenocytes, injected intrathymically, responsible for the development of donor-specific allograft tolerance.     

Portal venous ultraviolet B-irradiated donor alloantigen prevents rejection in circumferential rat tracheal allografts.

BACKGROUND: Before tracheal transplantation can be considered as a method of reconstruction in patients with extensive circumferential tracheal defects, we must achieve a state of nontoxic, donor-specific tolerance so that the risks of such a transplant do not outweigh the benefits. OBJECTIVE: Our objective was to determine whether a single intraportal injection of modified donor alloantigen achieves donor-specific immunosuppression for major histocompatibility complex-mismatched rat tracheal allografts. STUDY DESIGN: Buffalo (recipient) rats were pretreated with either a single portal-vein administration of ultraviolet B (UVB)-irradiated donor splenocytes (n = 4) or an intraportal inoculation of nonirradiated donor splenocytes (n = 4). Major histocompatibility complex-mismatched Lewis (donor) tracheal allograft segments were then grafted into treatment groups 7 days after donor-cell pretreatment. Tracheal rejection was assessed by histologic analysis, mucosal cilia motility, and in vitro immunologic assessment. RESULTS: The UVB-treated group demonstrated no acute or chronic rejection as well as complete functional recovery. In vitro immunologic assessment demonstrated a donor-specific hyporesponsiveness and donor allospecificity. Untreated animals and those receiving nonirradiated donor splenocytes showed acute rejection of their tracheal allografts. CONCLUSION: Recipient pretreatment with intraportally administered UVB-irradiated donor splenocytes prevents rejection of circumferential rat tracheal allograft segments by inducing a donor-specific immune hyporesponsiveness.     

Variable allograft responses to pretreatment with donor splenocytes treated with mitomycin C in the rat

In an attempt to investigate the nonspecificity of the effect of administration of donor splenocytes treated with mitomycin C (MMC) 7 days before transplantation in inducing immunological unresponsiveness, the survival rates of liver, heart, small bowel, and skin allografts were compared in the RT1-incompatible ACI(RT1a) to LEW(RT1l) rat combination. ACI donor splenocytes (3 x 10(6)) treated with MMC were administered i.p. or i.v. via the penile vein 7 days before transplantation. Both routes of administration prolonged the survival of hepatic allografts (greater than 87.2 +/- 22.2 days and greater than 78.9 +/- 28.2 days, respectively), compared with controls (10.6 +/- 0.5 days). Cardiac allograft survival in untreated controls was 6.0 +/- 0.4 days. A single i.v. injection of 3 x 10(6) donor splenocytes resulted in significantly prolonged survival (10.0 +/- 4.3 days), whereas i.p. injection showed rejection at a mean of 6.0 +/- 1.2 days. A single i.p. injection of 3 x 10(6) splenocytes did not increase survival of small bowel allografts (10.3 +/- 4.8 days), compared with controls (8.8 +/- 1.8 days). On the other hand, a single i.v. injection of donor splenocytes prior to transplantation significantly prolonged survival of small bowel allografts (13.4 +/- 3.5 days). No signs of graft-versus-host reaction (GVHR) were observed during these experiments. Neither a single i.p. nor a single i.v. injection of donor splenocytes resulted in prolonged survival of ACI-to-LEW skin allografts (6.3 +/- 0.8 days and 6.6 +/- 0.9 days, respectively), compared with controls (5.7 +/- 0.5 days). Interestingly, we confirmed the relative ease with which survival of hepatic allografts can be prolonged in the rat by donor antigen treatment alone, even in a strongly rejecting RT1-incompatible rat strain combination, in contrast to other organ allografts such as heart, small intestine, and skin. The discrepancy in survival of different organ allografts following pretreatment with donor splenocytes treated with MMC may be explained by a difference in the immunogenicity of the organs transplanted or other factors.     

Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb.

BACKGROUND: We previously reported the association among donor leukocyte chimerism, apoptosis of presumedly IL-2-deficient graft-infiltrating host cells, and the spontaneous donor-specific tolerance induced by liver but not heart allografts in mice. Survival of the rejection-prone heart allografts in the same strain combination is modestly prolonged by the pretransplant infusion of immature, costimulatory molecule-(CM) deficient donor dendritic cells (DC), an effect that is markedly potentiated by concomitant CM blockade with anti-CD40L (CD154) monoclonal antibody (mAb). We investigated whether the long survival of the heart allografts in the pretreated mice was associated with donor leukocyte chimerism and apoptosis of graft-infiltrating cells, if these end points were similar to those in the spontaneously tolerant liver transplant model, and whether the pretreatment effect was dependent on sustained inhibition of CM expression of the infused immature donor DC. In addition, apoptosis was assessed in the host spleen and lymph nodes, a critical determination not reported in previous studies of either spontaneous or "treatment-aided" organ tolerance models. METHODS: Seven days before transplantation of hearts from B10 (H-2b) donors, 2x10(6) donor-derived immature DC were infused i.v. into C3H (H-2k) recipient mice with or without a concomitant i.p. injection of anti-CD40L mAb. Donor cells were detected posttransplantation by immunohistochemical staining for major histocompatibility complex class II (I-Ab) in the cells of recipient lymphoid tissue. CM expression was determined by two-color labeling. Host responses to donor alloantigen were quantified by mixed leukocyte reaction, and cytotoxic T lymphocyte (CTL) assays. Apoptotic death in graft-infiltrating cells and in areas of T-dependent lymphoid tissue was visualized by terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick-end labeling and quantitative spectrofluorometry. Interleukin-2 production and localization were estimated by immunohistochemistry. RESULTS: Compared with control heart transplantation or heart transplantation after only DC administration, concomitant pretreatment with immature donor DC and anti-CD40L mAb caused sustained elevation of donor (I-Ab+) cells (microchimerism) in the spleen including T cell areas. More than 80% of the I-Ab+ cells in combined treatment animals also were CD86+, reflecting failure of the mAb to inhibit CD40/ CD80/CD86 up-regulation on immature DC in vitro after their interaction with host T cells. Donor-specific CTL activity in graft-infiltrating cells and spleen cell populations of these animals was present on day 8, but decreased strikingly to normal control levels by day 14. The decrease was associated with enhanced apoptosis of graft-infiltrating cells and of cells in the spleen where interleukin-2 production was inhibited. The highest levels of splenic microchimerism were found in mice with long surviving grafts (>100 days). In contrast, CTL activity was persistently elevated in control heart graft recipients with comparatively low levels of apoptotic activity and high levels of interleukin-2. CONCLUSION: The donor-specific acceptance of rejection-prone heart allografts by recipients pretreated with immature donor DC and anti-CD40L mAb is not dependent on sustained inhibition of donor DC CM (CD86) expression. Instead, the pretreatment facilitates a tolerogenic cascade similar to that in spontaneously tolerant liver recipients that involves: (1) chimerism-driven immune activation, succeeded by deletion of host immune responder cells by apoptosis in the spleen and allograft that is linked to interleukin-2 deficiency in both locations and (2) persistence of comparatively large numbers of donor-derived leukocytes. These tolerogenic mechanisms are thought to be generic, explaining the tolerance induced by allografts spontaneously, or with the aid of various kinds of immunosuppression.     

Evidence for epitope spreading and active suppression in skin graft tolerance after donor-specific transfusion.

BACKGROUND: To clarify the controversial results in the literature regarding the role of donor-specific transfusion (DST) on allograft survival, we have examined the influence of the following on DST-induced allograft survival in a 2C transgenic mouse model: varying the time between DST and transplantation; the role of MHC disparities between donor and recipient; whether tolerance induced by DST spreads to skin allografts expressing other alloantigens; and whether cyclosporine (CsA) treatment could further modulate skin allograft tolerance after DST. METHODS AND RESULTS: The studies were performed in both 2C anti-Ld (MHC class I) transgenic and normal (nontransgenic) mice. Our data demonstrate that a single infusion of Ld-mismatched lymphocytes 7 days before transplantation leads to permanent acceptance of donor-specific skin allografts in both transgenic (58/58) and nontransgenic (8/8) mice in the absence of any other nonspecific immunosuppressive treatment. Pretransplantation DST from donors mismatched for more than one MHC antigen (Ag) has no beneficial effect on subsequent donor skin allograft survival. However, Ld plus multiple minor histocompatibility (mH) Ag-mismatched DST induced permanent acceptance of donor-specific skin allografts. Tolerance induced by one-locus Ld-mismatched DST spreads to skin allografts expressing either two-locus Ld or one-locus Ld plus multiple mH Ags. Administration of CsA after DST diminished skin allograft survival, rather than enhancing it, suggesting that tolerance in this model system is established by an active immunological process sensitive to CsA. CONCLUSIONS: (1) Pretransplantation infusion of Ld-mismatched lymphocytes in the presence or absence of multiple mH mismatches induces permanent survival of donor-specific skin allografts. (2) CsA abrogates DST-induced transplantation tolerance.     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

Donor brain death significantly interferes with tolerance induction protocols

Studies in rodents showed that antibodies are able to induce tolerance of allografts. As clinical results are unsatisfactory and deceased donors are still the main source of organ transplants, we investigated whether donor brain-death impacts on tolerance induction after experimental kidney transplantation. Anti-CD4 monoclonal antibodies (RIB 5/2; 2.5 mg/kg × 5 days) treated and untreated recipients of brain-dead donor grafts were compared with RIB 5/2 treated and untreated recipients of living donor grafts (F344-to-Lewis). All recipients received low-dose CsA (1.5 mg/kg × 10 days). Kidneys were recovered 4, 16 and 40 weeks after transplantation and examined by morphology, immunohistology and flow cytometry. Renal function was monitored monthly. RIB 5/2 treatment significantly decreased proteinuria in recipients of living donor allografts when compared with living donor controls. After 40 weeks, inflammatory cell infiltration and MHC class II expression were reduced while morphologic alterations were minimal. In contrast, treatment of brain-dead graft recipients had no impact on graft function. Structural changes and graft infiltration were comparable to brain-dead donor controls at all time points. RIB 5/2 treatment significantly improved graft function in recipients of living donor grafts; however, it was not effective in recipients of brain-dead donor organs.     

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.     

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.     

Rapamycin, a potent immunosuppressive drug for vascularized heart, kidney, and small bowel transplantation in the rat

The effectiveness of rapamycin (RAPA) was examined for heart, kidney, and small bowel allografts in rats. Untreated or vehicle only-infused Wistar Furth (RT1u) recipients rejected Buffalo (RT1b) heart allografts within a mean survival time (MST) of 6.5 +/- 0.5 and 6.3 +/- 0.5 days, respectively. In contrast, a 14-day continuous intravenous (i.v.) infusion by an osmotic pump of 0.08 mg/kg/day RAPA to WFu recipients prolonged BUF heart allograft survival to an MST of 34.4 +/- 12.1 days (P = 0.0001). There was a graded dose-response to 0.16 mg/kg (39.0 +/- 8.7 days; P = 0.0001), 0.32 mg/kg (55.7 +/- 3.3 days; P = 0.0001) and 0.8 mg/kg (48.0 +/- 3.6; P = 0.0001). Furthermore, intraarterial/intragraft but not i.v. infusion of 0.02 mg/kg/day prolonged BUF heart allografts--namely, an MST of 14.6 +/- 1.4 days versus 8.6 +/- 2.6 days (P = 0.0001), respectively. Local delivery doses of RAPA were about as effective as the same dose delivered i.v.: 0.08 mg/kg MST 37.0 +/- 18.3 days (P = 0.0001); 0.32 mg/kg, 40.0 +/- 3.9 days (P = 0.0001); and 0.8 mg/kg, 54.8 +/- 8.2 days (P = 0.0001). Systemic i.v. RAPA therapy with 0.08 or 0.8 mg/kg/day prolonged the survival of BUF kidney grafts in WFu recipients--namely, an MST of 52.7 +/- 42.7 (NS) and 90.2 +/- 62.4 (P = 0.001) days, respectively, versus an MST of 11.6 +/- 1.5 days in control WFu recipients only infused with vehicle. While normal WFu rats reject heterotopic BUF small bowel allografts within an MST of 10.0 days, a 14-day course of i.v. RAPA treatment significantly (P = 0.0001) prolonged small bowel allograft survival to an MST of 26.8 +/- 3.7 days.     

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.     

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 transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody.

The T-cell-mediated immune response usually results in the rapid destruction of organ allografts transplanted between murine strains incompatible for major and minor histocompatibility antigens. This response may be modified by pretreatment with either donor-specific antigen or anti-CD4 monoclonal antibody. Previous work by others has shown that combined treatment of mice with soluble protein antigens and anti-CD4 monoclonal antibody can produce antigen-specific B cell unresponsiveness that continues long after the nonspecific immunosuppressive effect of the mAb treatment has resolved. Following this principle we have shown that adult C3H/He mice can be made specifically unresponsive to vascularized C57BL/10 cardiac allografts by pretreating the recipient with donor alloantigen under the cover of a brief course of mAb against CD4. A full-dose response analysis shows that the dose of mAb is critically important for the successful induction of tolerance. Tolerance induction using this protocol is dependent on treatment with donor major histocompatibility complex antigens and occurs in the presence of marked depletion but not complete elimination of the CD4+ T cell subset. The unresponsiveness to alloantigen is antigen specific, as determined by the ineffectiveness of third-party (C57BL/10) alloantigen when combined with anti-CD4 mAb to induce long-term survival of BALB/c allografts in C3H/He recipients. The tolerant state is specific and effective in the long-term as indicated by the specific acceptance of C57BL/10 skin grafts in recipients with surviving C57BL/10 cardiac allografts. This study provides a simple method for the successful induction of specific transplantation tolerance in the adult across a full H-2 major and minor antigen mismatch strain combination. The results illustrate the important role of the CD4 molecule in the T cell response to alloantigen in vivo and suggest possibilities for the therapeutic manipulation of complex immune reactions.