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.     

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.     

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.     

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.     

Viral infection abrogates CD8(+) T-cell deletion induced by costimulation blockade

BACKGROUND: Treatment with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. It is known that prolongation of allograft survival by this method depends in part on deletion of alloreactive CD8(+) T cells at the time of tolerance induction. Recent data suggest that infection with lymphocytic choriomeningitis virus (LCMV) abrogates the ability of this protocol to prolong graft survival. METHODS: To study the mechanism by which viral infection abrogates allograft survival, we determined (1) the fate of tracer populations of alloreactive transgenic CD8(+) T cells and (2) the duration of skin allograft survival following treatment with DST and anti-CD154 mAb in the presence or absence of LCMV infection. RESULTS: We confirmed that treatment of uninfected mice with DST and anti-CD154 mAb leads to the deletion of alloreactive CD8(+) T cells and is associated with prolongation of skin allograft survival. In contrast, treatment with DST and anti-CD154 mAb in the presence of intercurrent LCMV infection was associated with the failure to delete alloreactive CD8(+) T cells and with the rapid rejection of skin allografts. The number of alloreactive CD8(+) cells actually increased significantly, and the cells acquired an activated phenotype. CONCLUSIONS: Interference with the deletion of alloreactive CD8(+) T cells mediated by DST and anti-CD154 mAb may in part be the mechanism by which viral infection abrogates transplantation tolerance induction.     

Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell costimulation

Allorejection and recurrence of autoimmunity are the major barriers to transplantation of islets of Langerhans for the cure of type 1 diabetes in humans. CD40-CD154 (CD40 ligand) interaction blockade by the use of anti-CD154 monoclonal antibody (mAb) has shown efficacy in preventing allorejection in several models of organ and cell transplantation. Here we report the beneficial effect of the chronic administration of a hamster anti-murine CD154 mAb, MR1, in prolonging islet graft survival in NOD mice. We explored the transplantation of C57BL/6 islets into spontaneously diabetic NOD mice, a combination in which both allogeneic and autoimmune components are implicated in graft loss. Recipients were treated either with an irrelevant control antibody or with MR1. MR1 administration was effective in prolonging allograft survival, but did not provide permanent protection from diabetes recurrence. The autoimmune component of graft loss was studied in spontaneously diabetic NOD mice that received syngeneic islets from young male NOD mice. In this combination, a less dramatic yet substantial delay in diabetes recurrence was observed in the MR1-treated recipients when compared with the control group. Finally, the allogeneic component was explored by transplanting C57BL/6 islets into chemically induced diabetic male NOD mice. In this setting, long-term graft survival (>100 days) was achieved in MR1-treated mice, whereas control recipients rejected their grafts within 25 days. In conclusion, chronic blockade of CD154 results in permanent protection from allorejection and significantly delays recurrence of diabetes in NOD mice.     

CD4+ T cells are sufficient to elicit allograft rejection and major histocompatibility complex class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice

BACKGROUND: We characterized the role of T cell subsets and major histocompatibility complex molecules in allograft rejection and recurrence of autoimmune diabetes. METHODS: Adoptive cell transfer and vascularized segmental pancreas transplantation were performed in mice. RESULTS: In an alloimmune response model, transfer of nondiabetic CD4, but not CD8 T cells, elicited pancreas allograft rejection in streptozotocin (STZ)-induced diabetic NOD/scid mice. Pancreas allografts were acutely rejected in STZ-induced diabetic NOD/beta2m mice (confirmed the absence of major histocompatibility complex [MHC] class I and CD8 T cells) and permanently accepted in NOD/CIIT mice (confirmed the absence of MHC class II and CD4 T cells). The results suggest that rejection of pancreas allograft is CD4-dependent and MHC class I-independent. In the autoimmune diabetes model, whole spleen cells obtained from diabetic NOD mice induced autoimmune diabetes in NOD/scid and NOD/CIIT mice, but the onset of diabetes was delayed in NOD/beta2m mice. However, the purified diabetic T cells failed to elicit autoimmune diabetes in NOD/beta2m mice. NOD/scid and NOD/CIIT pancreas grafts were acutely destroyed whereas four of six NOD/beta2m pancreas grafts were permanently accepted in autoimmune diabetic NOD mice. CONCLUSION: CD4 T cells are sufficient for the induction of allograft rejection, and MHC class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.     

Depleting anti-CD4 monoclonal antibody cures new-onset diabetes, prevents recurrent autoimmune diabetes, and delays allograft rejection in nonobese diabetic mice

BACKGROUND: The prevention of recurrent autoimmunity is a prerequisite for successful islet transplantation in patients with type I diabetes. Therapies effective in preserving pancreatic beta-cell mass in patients with newly diagnosed diabetes are good candidates for achieving this goal. Anti-CD3 monoclonal antibody (mAb) and antilymphocyte antisera are the only therapies to date that have cured early diabetic disease in the nonobese diabetic (NOD) mouse. We investigated whether other immunosuppressive therapies, including short-term depleting anti-CD4 mAb or costimulation blockade, would affect the disease progression in recently diabetic NOD mice. We also evaluated the effect of the anti-CD4 mAb on syngeneic and allogeneic graft survival in diabetic NOD recipients. METHODS AND RESULTS: We demonstrate that a short course of anti-CD4 mAb early after hyperglycemia onset cured diabetes. Normal islets and islets with CD4+ and CD8+ T-cell peri-insulitic infiltrate were found in the pancreata of cured NOD mice. A similar regimen prevented the recurrence of autoimmune diabetes in NOD/severe combined immunodeficient disease (SCID) islet isografts and delayed the rejection of allogeneic C57BL/6 islet allografts in diabetic female NOD mice. The co-transfer of diabetogenic splenocytes with splenocytes from anti-CD4 mAb-treated and cured NOD mice into 7-week-old, irradiated, NOD male mice was not able to protect from diabetes occurrence. This indicates that an anti-CD4-mediated cure of diabetes is independent of the induction of immunoregulatory T cells. Anti-CD154 mAb and cytotoxic T-lymphocyte antigen 4 immunoglobulin were ineffective in early-onset diabetes. CONCLUSION: Our results provide the first evidence that newly established autoimmune islet destruction in NOD mice responds to a short course of anti-CD4 mAb. In contrast, costimulation blockade is ineffective in this clinically relevant model.     

Islet allograft survival induced by costimulation blockade in NOD mice is controlled by allelic variants of Idd3

NOD mice develop type 1 autoimmune diabetes and exhibit genetically dominant resistance to transplantation tolerance induction. These two phenotypes are genetically separable. Costimulation blockade fails to prolong skin allograft survival in (NOD x C57BL/6)F1 mice and in NOD-related strains made diabetes-resistant by congenic introduction of protective major histocompatibility complex (MHC) or non-MHC Idd region genes. Here, we tested the hypothesis that the genetic basis for the resistance of NOD mice to skin allograft tolerance also applies to islet allografts. Surprisingly, costimulation blockade induced permanent islet allograft survival in (NOD x C57BL/6)F1 mice but not in NOD mice. After costimulation blockade, islet allograft survival was prolonged in diabetes-resistant NOD.B6 Idd3 mice and shortened in diabetes-free C57BL/6 mice congenic for the NOD Idd3 variant. Islet allograft tolerance could not be induced in diabetes-resistant NOD.B10 Idd5 and NOD.B10 Idd9 mice. The data demonstrate that 1) NOD mice resist islet allograft tolerance induction; 2) unlike skin allografts, resistance to islet allograft tolerance is a genetically recessive trait; 3) an Idd3 region gene(s) is an important determinant of islet allograft tolerance induction; and 4) there may be overlap in the mechanism by which the Idd3 resistance locus improves self-tolerance and the induction of allotolerance.     

The effect of simultaneous CD154 and LFA-1 blockade on the survival of allogeneic islet grafts in nonobese diabetic mice

BACKGROUND: The rate of success in clinical transplantation of islets of Langerhans has dramatically improved with perspectives of wide-scale applicability for patients with type 1 diabetes. One drawback is the need for lifelong immunosuppression, which is associated with significant side effects. Immunomodulatory strategies devoid of side effects and with tolerogenic potential, such as co-stimulatory blockade, would be a great improvement if successful. In this study, the authors have explored the effect of simultaneous blockade of CD40/CD154 and intercellular adhesion molecule (ICAM)/lymphocyte function-associated antigen (LFA)-1 interactions. METHODS: Spontaneously diabetic nonobese diabetic (NOD) mice underwent transplantation with allogeneic (C57BL/6) islets and were treated with anti-CD154 monoclonal antibody (mAb) (500 microg, three doses), anti-LFA-1 mAb (100 microg, three doses), or a combination of both in the early peritransplant period. In another set of experiments, LFA-1 engagement was impaired by transplanting islets isolated from ICAM-1-knockout (KO) mice. RESULTS: Untreated animals rejected their grafts within 10 days. LFA-1 blockade alone did not result in improved islet graft survival, whereas CD154 blockade alone increased graft survival to 18 days. Simultaneous blockade of both pathways led to significantly improved islet graft survival to 30 days (ICAM-1-KO islets plus anti-CD154), 35 days (anti-LFA-1 plus anti-CD154), and 44 days (ICAM-1-KO islets plus anti-LFA-1 plus anti-CD154). CONCLUSIONS: These data suggest that a synergistic effect for prolonged graft survival can be obtained by simultaneously targeting CD154 and LFA-1 in the challenging model of islet allotransplantation in NOD mice. The observation of similar results with anti-LFA-1 mAb and with ICAM-1-KO grafts suggests a key role of direct antigen presentation for the activation of LFA-1-driven signaling.     

NOD congenic mice genetically protected from autoimmune diabetes remain resistant to transplantation tolerance induction

The loss of self-tolerance leading to autoimmune type 1 diabetes in the NOD mouse model involves at least 19 genetic loci. In addition to their genetic defects in self-tolerance, NOD mice resist peripheral transplantation tolerance induced by costimulation blockade using donor-specific transfusion and anti-CD154 antibody. Hypothesizing that these two abnormalities might be related, we investigated whether they could be uncoupled through a genetic approach. Diabetes-resistant NOD and C57BL/6 stocks congenic for various reciprocally introduced Idd loci were assessed for their ability to be tolerized. Surprisingly, in NOD congenic mice that are almost completely protected from diabetes, costimulation blockade failed to prolong skin allograft survival. In reciprocal C57BL/6 congenic mice with NOD-derived Idd loci, skin allograft survival was readily prolonged by costimulation blockade. These data indicate that single or multiple combinations of evaluated Idd loci that dramatically reduce diabetes frequency do not correct resistance to peripheral transplantation tolerance induced by costimulation blockade. We suggest that mechanisms controlling autoimmunity and transplantation tolerance in NOD mice are not completely overlapping and are potentially distinct, or that the genetic threshold for normalizing the transplantation tolerance defect is higher than that for preventing autoimmune diabetes.     

Evaluation of donor-specific transfusion sources: unique failure of bone marrow cells to induce prolonged skin allograft survival with anti-CD154 monoclonal antibody

BACKGROUND: Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8(+) T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. METHODS: Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day -7 plus 4 doses of anti-CD154 mAb on days -7, -4, 0, and +4. Deletion of CD8(+) alloreactive cells was measured using "synchimeric" CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8(+) T cells. RESULTS: Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8(+) T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. CONCLUSIONS: Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8(+) T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.     

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.     

Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection.

BACKGROUND: T-cell activation and the subsequent induction of effector functions require not only the recognition of antigen peptides bound to MHC molecules by T-cell receptor (TCR) for antigen but also a costimulatory signal provided by antigen presenting cells. CD4 T-cell activation and function require the CD4 molecule as a coreceptor of TCR. The CD28/B7 pathway is a major costimulatory signal for T-cell activation and differentiation. METHODS: The effect of targeting CD4 by nondepleting anti-CD4 monoclonal antibodies (mAbs) versus blocking CD28/B7 by CTLA4Ig, anti-CD80 mAbs, and anti-CD86 mAbs on the prevention of recurrence of autoimmune diabetes after MHC-matched nonobese diabetes-resistant (NOR) islet transplantation in nonobese diabetic (NOD) mice were compared. Whether nondepleting anti-CD4 mAbs prolong allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice were studied. Furthermore, the effect of nondepleting anti-CD4 mAbs combined with CTLA4Ig on allogeneic islet graft survival in NOD mice was investigated. RESULTS: Recurrence of autoimmune diabetes can be prevented by nondepleting anti-CD4 mAbs. Blocking the CD28/B7 costimulatory pathway by CTLA4Ig or by anti-CD80 mAbs and anti-CD86 mAbs cannot prevent recurrence of autoimmune diabetes after islet transplantation. Short-term treatment with nondepleting anti-CD4 mAbs significantly prolongs allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice. But nondepleting anti-CD4 mAbs combined with CTLA4Ig decreased allogeneic islet graft survival. CONCLUSIONS: Nondepleting anti-CD4 mAbs but not CD28 antagonists protect islet grafts in diabetic NOD mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. The efficacy of nondepleting anti-CD4 mAbs is compromised when it combines with CTLA4Ig.     

Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into na?ve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.     

Autoimmune diabetes and resistance to xenograft transplantation tolerance in NOD mice

Costimulation blockade induces prolonged rat islet and skin xenograft survival in C57BL/6 mice. Nonobese diabetic (NOD) mice, which are used to model human autoimmune diabetes, are resistant to costimulation blockade-induced allograft tolerance. We tested the hypothesis that NOD mice would also be resistant to costimulation blockade-induced rat xenograft tolerance. We report that rat islet xenograft survival is short in spontaneously diabetic NOD mice treated with a tolerizing regimen of donor-specific transfusion and anti-CD154 antibody. Rat islet xenograft survival is only marginally longer in chemically diabetic NOD mice treated with costimulation blockade but is prolonged further in NOD Idd congenic mice bearing C57-derived chromosome 3 loci. Reciprocally, the presence of NOD-derived chromosome 3 loci shortens islet xenograft survival in tolerized C57BL/6 mice. Islet xenograft survival is longer in tolerized NOD.CD4a(-/-) and (NOD x C57BL/6)F1 mice than in NOD mice but still much shorter than in C57BL/6 mice. Skin xenograft survival in (NOD x C57BL/6)F1 mice treated with costimulation blockade is short, suggesting a strong genetic resistance to skin xenograft tolerance induction. We conclude that the resistance of NOD mice to xenograft tolerance induction involves some mechanisms that also participate in the expression of autoimmunity and other mechanisms that are distinct.     

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.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

The role of autoimmunity in islet allograft destruction: major histocompatibility complex class II matching is necessary for autoimmune destruction of allogeneic islet transplants after T-cell costimulatory blockade

Although it has often been assumed that transplanted allogeneic islets can be destroyed by recurrent autoimmunity in recipients with type 1 diabetes, definitive evidence is lacking and the settings in which this may occur have not been defined. To address these issues, we compared the survival of islet transplants (subject to tissue-specific autoimmunity) with cardiac transplants (not subject to tissue-specific autoimmunity) from various major histocompatibility complex (MHC)-matched and -mismatched donors transplanted into autoimmune NOD recipients. We found that when recipients were treated with combined B7 and CD154 T-cell costimulatory blockade, hearts survived best with better MHC matching, whereas islets survived worst when the donor and recipient shared MHC class II antigens. In the absence of full or MHC class II matching, there was no difference in the survival of islet and cardiac allografts. We also found that the tendency of NOD mice to resist tolerance induction by costimulation blockade is mediated by both CD4+ and CD8+ T-cells, not directly linked to the presence of autoimmunity, and conferred by non-MHC background genes. These findings have clinical importance because they suggest that under some circumstances, avoiding MHC class II sharing may provide better islet allograft survival in recipients with autoimmune diabetes, since mismatched allogeneic islets may be resistant to recurrent autoimmunity. Our results may have implications for the design of future clinical trials in islet transplantation.     

Interferon-gamma is not a universal requirement for islet allograft survival

BACKGROUND: Although many transplantation studies have implicated a graft-destructive role for T helper (Th)1 cytokines and a graft-protective role for Th2 cytokines, more recent studies have challenged this paradigm by showing that long-term allograft survival can actually require the presence of Th1 cytokines, such as interleukin 2 and interferon (IFN)-gamma. The purpose of this study was to examine the requirement for IFN-gamma in the induction of islet allograft acceptance after monoclonal antibody therapy targeting conceptually distinct molecular pathways: the costimulatory molecule CD154, the CD4 coreceptor, or the beta2 integrin lymphocyte function-associated antigen (LFA)-1 (CD11a). METHODS: Diabetic C57Bl/6 (B6; H2b) mice were grafted with fully MHC mismatched BALB/c (H2d) islets, or reciprocally, diabetic BALB/c mice underwent transplantation with B6 islets and were treated with anti-CD154, anti-CD4, or anti-LFA-1. RESULTS: When IFN-gamma gene knockout mice were used as graft recipients, the requirement for IFN-gamma in allograft survival was found to be highly conditional, depending on both the host strain and the induction therapy used. In both strain combinations studied, anti-CD154 was effective in the presence or absence of IFN-gamma, whereas anti-CD4 lost therapeutic potential in the absence of this cytokine. Alternatively, the requirement for IFN-gamma for allograft prolongation by anti-LFA-1 therapy was noted only in B6 transplant recipients. CONCLUSIONS: IFN-gamma is not always requisite in islet allograft survival but rather varies according to the molecular target of induction therapy and the genetic background of the transplant recipient.