Development of a combined cardiac and aortic transplant model to investigate the development of transplant arteriosclerosis in the mouse.

BACKGROUND: The degree of transplant arteriosclerosis in murine cardiac allografts is difficult to assess. Aortic allografts represent an alternative model for evaluating the impact of novel transplant strategies on transplant arteriosclerosis in which the vascular changes can be quantified easily. However, it remains controversial as to whether vascular lesions seen in this model are equivalent to those that develop in solid-organ transplants. The aim of this study was to develop a model of combined cardiac and aortic transplantation to allow more precise quantification of transplant arteriosclerosis and to establish a correlation between the lesions that develop in the 2 types of graft. METHODS: CBA (H2(k)) recipients received a C57BL/10 (H2(b)) cervical cardiac allograft on Day 0 and a C57BL/10 (H2(b)) abdominal aortic allograft on Day 1. Recipients were treated with anti-CD154 mAb (MR1) on Days 0, 2, and 4. We performed histology and morphometric measurements for both grafts 30 days after transplantation. RESULTS: We observed significant intimal proliferation in both the cervical cardiac and abdominal aortic allografts from recipients treated with anti-CD154 mAb (heart, 64% +/- 9%; aorta, 67% +/- 8%; n = 5). Abdominal aortic grafts transplanted alone into anti-CD154-treated recipients developed a degree of transplant arteriosclerosis equivalent to that seen in the aortic grafts of the combined group (aorta alone, 68% +/- 9%, vs aorta + heart, 67% +/- 8%; n = 5). CONCLUSIONS: This combined cardiac and aortic transplant model permitted quantitative assessment of transplant arteriosclerosis while monitoring graft survival by cardiac palpation. Furthermore, development of transplant arteriosclerosis was equivalent in abdominal aortic allografts either in the presence or absence of an additional solid- organ transplant.     

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade

BACKGROUND: The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade. METHODS: BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. RESULTS: Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. CONCLUSIONS: Our data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells

BACKGROUND: Treatment with a donor-specific transfusion (DST) and a brief course of anti-mouse CD154 (anti-CD40-ligand) monoclonal antibody (mAb) prolongs the survival of both allografts and rat xenografts in mice. The mechanism by which allograft survival is prolonged is incompletely understood, but depends in part on the presence of CD4+ cells and the deletion of alloreactive CD8+ T cells. Less is known about the mechanism by which this protocol prolongs xenograft survival. METHODS: We measured rat islet and skin xenograft survival in euthymic and thymectomized mice treated with combinations of DST, anti-CD154 mAb, anti-CD4 mAb, and anti-CD8 mAb. Recipients included C57BL/6, C57BL/6-scid, C57BL/6-CD4null, and C57BL/6-CD8null mice. RESULTS: Pretreatment with a depleting anti-CD4 mAb markedly prolonged the survival of both skin and islet xenografts in mice given DST plus anti-CD154 mAb. Comparable prolongation of xenograft survival was obtained in C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb. In contrast, anti-CD8 mAb did not prolong the survival of either islet or skin xenografts in mice treated with DST and anti-CD154 mAb. Thymectomy did not influence xenograft survival in any treatment group. Adoptive transfer of splenocytes from C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb and bearing long-term skin xenografts revealed the presence of residual xenoreactive cells. CONCLUSIONS: These data suggest that treatment with DST and anti-CD154 mAb induces a state of "functional" transplantation tolerance. They also support the hypothesis that both the induction and maintenance of graft survival based on this protocol depend on different cellular mechanisms in allogeneic and xenogeneic model systems.     

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.     

Regulation of transplant arteriosclerosis by CD25+CD4+ T cells generated to alloantigen in vivo

BACKGROUND: CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis. METHODS: CD25+CD4+ T cells from CBA mice that were pretreated with C57BL/6 (B.6) blood (donor-specific transfusion, DST) and nondepleting anti-CD4 Ab (YTS 177) were cotransferred with na?ve CBA CD25-CD4+"effector" T cells into CBA-rag-/- mice. These animals received aorta transplants from B.6 CD31-/- donors. CBA wild-type recipients of B.6 aorta grafts were pretreated with 177/DST directly. Some animals received 6x10(5) CD25+CD4+ T cells from pretreated mice to augment regulation on day -1. Grafts were harvested on day 30. RESULTS: Luminal occlusion of the graft caused by neointima formation was 29.3+/-19.4% (n=5) after transfer of effector T cells only. Co-transfer of CD25+CD4+ regulators reduced occlusion significantly (2.4+/-3.3%, n=3; P=0.009). This effect was partially abrogated in the presence of a CTLA4 blocking Ab (11.1+/-4.7%, n=4; P=0.008). Pretreating immunocompetent CBA recipients of B.6 aortic allografts with 177/DST did not reduce transplant arteriosclerosis significantly (43.0+/-15.7%, n=5 vs. 56.6+/-16.8%, n=5; 177/DST vs. controls; P=0.22). However, when pretreated primary CBA recipients received an additional transfer of 6 x 10(5) CD25+CD4+ T cells procured from other mice pretreated with 177/DST before transplantation, luminal occlusion of the graft was markedly reduced (33.0+/-7.6%, n=5; P=0.002). CONCLUSION: Regulatory T cells generated in vivo to alloantigen can prevent CD25-CD4+ T-cell-mediated transplant arteriosclerosis. In immunocompetent recipients, these cells have potential to be used as cellular immunotherapy to control transplant arteriosclerosis.     

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.     

The complement dependent cytotoxicity (CDC) immune effector mechanism contributes to anti-CD154 induced immunosuppression

BACKGROUND: In many situations, anti-CD154 (CD40 ligand) monoclonal antibody (mAb) treatment is very potent in producing allograft tolerance. In accordance to our previously reported results, combined donor specific transfusion (DST)3 plus anti-CD154 mAb (MR1) treatment enables the permanent engraftment of DBA/2 (H-2(d)) islets into B6AF1 (H-2(b/kd)) recipients in all cases. It has been widely assumed that the MR1 anti-154 is a noncytolytic neutralizing mAb, and it exerts immune suppressive effects by blockade of CD40/CD154 signal pathway. In this study, we sought to test the role of complement dependent cytotoxicity (CDC) immune effector mechanism in MR1 anti-CD154 induced immunosuppression. METHODS: We have evaluated the contributions of CDC in the context of the potent tolerizing effects of DST plus anti-CD154 mAb treatment regiment in recipients of islet allografts. We have used CD40 knockout (KO) mice and complement C5 deficient mice DBA/2 as islet allograft recipients as well as cobra venom factor (CVF), a complement blocker, treatment. RESULTS: The absence of direct and indirect CD40/CD154 pathway signals does not prevent islet allograft acute rejection. Interestingly, MR1 anti-CD154 induces islet allograft tolerance in the absence of CD40/CD154 pathway. In a wild-type major histocompatibility complex (MHC) mismatched strain combination, DST results in accelerated islet allograft rejection. Combination of DST and MR1 anti-CD154 treatment prevents presensitization and permits permanent engraftment. However, administration of CVF abolishes the tolerance induction. Moreover, DST plus MR1 anti-CD154 regiment, a potent tolerizing therapy, does not prevent acute islet allograft rejection when complement C5 deficient DBA/2 mice are used as recipients. Thus, the mechanisms of the tolerizing effects by MR1 anti-CD154 are not limited to blockade of CD40/CD154 signals. The CDC immune effector mechanism contributes to MR1 anti-CD154 induced immunosuppression.     

Anti-CD40L monoclonal antibody treatment induces long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts

The CD40/CD40L costimulatory pathway plays a crucial role in allograft rejection. The purpose of this study was to determine the effectiveness of anti-CD40L monoclonal antibody (mAb) treatment as a method to induce long-term, tissue-specific, immunologic hyporesponsiveness to peripheral nerve allografts. Sciatic nerve allografts were performed from BALB/c donor mice into C57BL/6 recipients. Anti-CD40L mAb (1 mg) was administered intraperitoneally to recipient mice on postoperative days 0, 1, and 2. After a 14-, 28-, or 60-day recovery period, the mice were rechallenged with either a BALB/c cardiac or peripheral nerve allograft. Rejection was assessed by measuring the production of interferon gamma (IFN-gamma), interleukin (IL)-2, -4, and -5, and alloantibodies immunoglobulin (Ig) M and IgG. IFN-gamma, IL-2, IL-4, IL-5, IgM, and IgG responses were much lower in the anti-CD40L mAb group compared with controls. Nerve allograft and nerve isograft rechallenge 60 days following the original nerve allotransplantation produced low cytokine responses, whereas cardiac allograft rechallenge produced high cytokine production, indicative of acute rejection. Short-term anti-CD40L treatment may cause long-term, tissue-specific, immunologic hyporesponsiveness. This may allow time for native axons to traverse the transplanted nerve allograft and replace the graft with autogenous peripheral nerve tissue.     

Alloreactive (CD4-Independent) CD8+ T Cells Jeopardize Long-Term Survival of Intrahepatic Islet Allografts

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8⁺ T cells that are independent of CD4⁺ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8⁺ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60–90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8⁺ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allo specific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.     

Marked inhibition of transplant vascular sclerosis by in vivo-mobilized donor dendritic cells and anti-CD154 mAb

BACKGROUND: Combination of donor dendritic cells (DC) and anti-CD40 Ligand (L) (CD154) monoclonal antibody (mAb) markedly prolongs heart or skin allograft survival, but the influence of this strategy in models of chronic rejection is unknown. Our aim was to ascertain the influence of in vivo-mobilized immature donor DC plus anti-CD40L mAb on vascular sclerosis in functional murine aortic allografts. METHODS: C3H He/J (C3H;H2k) mice received 2 x 106 freshly isolated, immunobead-purified (>90%) fms-like tyrosine kinase 3 ligand-mobilized C57BL/10 (B10;H2b) CD11c+ DC intravenously (IV), together with 500 microg of anti-CD40L mAb (MR1) intraperitoneally (IP) on days -7, 0, 4, and 10. Controls received either no donor cells, no mAb, or were untreated. B10 aortic grafts were transplanted in the abdominal aorta on day 0. At day 30, antidonor T-cell proliferative and cytotoxic responses and both complement fixing and immunoglobulin (Ig)G alloantibody levels were determined. Grafts were harvested on days 30 and 60 and examined by histology and immunohistochemistry. RESULTS: DC infusion alone enhanced ex vivo antidonor proliferative and cytotoxic T-cell activity. By contrast, complement-fixing alloantibody levels were reduced. Anti-CD40L mAb alone strongly suppressed each of these responses. Graft inflammatory cell infiltration, intimal smooth muscle cell proliferation, fibrosis, and elastic lamina disruption observed in untreated animals were reduced in response to anti-CD40L mAb or donor DC alone. Antidonor immune reactivity, including IgG levels, and intimal proliferation were all markedly suppressed to an overall greater extent in mice given both treatments. CONCLUSION: Whereas blockade of the CD40-CD40L pathway ameliorated transplant vasculopathy, preservation of near-normal vessel architecture was achieved by simultaneous administration of donor DC. This strategy represents a novel application of DC for suppression of chronic rejection.     

Inhibition of chronic vascular rejection by donor-specific blood transfusion is associated with a reduction in transforming growth factor-beta1 expression

BACKGROUND: Concentric intimal thickening and the infiltration of inflammatory cells in cardiac allografts are the pathological hallmark characteristics of chronic vascular rejection (CVR), the leading cause of long-term graft failure. The precise mechanisms involved in the development and pathogenesis of CVR remain elusive. In the PVG-R23 to PVG-RT1u rat model of CVR, prior administration of a donor-specific transfusion (DST) was previously shown to prolong graft survival indefinitely and abolish the vascular lesions associated with CVR. The present study investigates in more depth the underlying mechanisms involved in the subsequent prolongation of allograft survival and inhibition of CVR by DST. METHODS: R23 heart grafts were monitored in nontransfused and transfused RT1u recipients injected 2 weeks before transplantation with 1.5 ml of R23 blood. Severity of arteriosclerosis, transplant infiltrate, transforming growth factor (TGF)-beta1 protein expression within the graft, plasma TGF-beta1 levels, class II MHC expression, tenascin protein expression, and serum alloantibody levels were measured. RESULTS: There was no significant difference in donor MHC class II, myocardial TGF-beta1, or tenascin expression between DST and non-DST-treated recipients. However, DST-pretreated recipients showed greatly reduced histological evidence of CVR and had lower titers of R23-specific IgG subclasses. Furthermore, DST-treated allograft recipients showed significant decreases in circulating TGF-beta1 levels and a reduction in TGF-beta1 and tenascin expression within coronary arteries of the allografts. CONCLUSION: The results suggested that DST inhibited CVR by altering and regulating the expression of TGF-beta1, thereby preventing the fibrogenic effects associated with TGF-beta1.     

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody

BACKGROUND: Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. METHODS: Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. RESULTS: NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. CONCLUSIONS: Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.     

Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms

BACKGROUND: Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4 cells and deletion of alloreactive CD8 cells. The specific roles of CD4 and CD4CD25 cells and the mechanism(s) by which they act are not fully understood. METHODS: We used skin and islet allografts, a CD8 T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4 cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. RESULTS: Deletion of CD4 or CD25 cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4 or CD25 cells had no effect upon survival of healed-in islet allografts, and CD25 cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8 T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8 T cell deletion. CONCLUSIONS: These data document that (1) both CD4CD25 and CD4CD25 cells are required for induction of skin allograft survival, (2) CD4CD25 T cells are not required for alloreactive CD8 T cell deletion, and (3) CD4CD25 regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4 cell subsets and different mechanisms.     

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.