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Background: Because CD4CD25Foxp3 regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs., Methods: To identify generation of aTregs, purified graft-reactive CD4CD25 T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4CD25 T cells; half of the recipients undergo tolerance induction treatment., Results: By tracking adoptively transferred cells, we show that purified graft-reactive CD4CD25 T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data-Foxp3 cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance., Conclusions: We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.

Background: Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment of graft-versus-host disease, solid organ graft rejection, and other T-cell-mediated diseases. The mechanisms of action of ECP include lymphocyte apoptosis, cytokine modulation, and the induction of regulatory T cells. It has been suggested that dendritic cells (DCs) are more resistant to ECP-induced apoptosis and might be directly modulated by ECP. We tested this hypothesis using in vitro Psoralen/UVA (PUVA) treatment as an in vitro model of ECP., Methods: Monocyte-derived DCs (mo-DCs) were treated with 8-methoxypsoralen /UVA and analyzed for surface molecule expression, apoptosis markers, endocytosis, and migratory and immunostimulatory capacity. Mo-DC phenotype and cytokine secretion was tested after CD40L stimulation. Naive T cells stimulated with PUVA-treated mo-DCs were tested for Th1/Th2 cytokine secretion and associated chemokine receptor patterns., Results: DCs underwent apoptosis after in vitro PUVA and in vivo ECP. In vitro, the induction of apoptosis was preceded by partial maturation of immature mo-DCs. PUVA-treated immature mo-DCs also exhibited enhanced migratory and immunostimulatory capacity. However, mo-DCs stimulation through CD40 ligation was abrogated and interleukin (IL)-12 secretion was abolished 24 hr after PUVA treatment. PUVA-treated mo-DCs skewed naive T cells toward a Th2 response as defined by increased IL-4, IL-10, and IL-13 and decreased interferon-gamma levels, and the expression of the Th2-associated chemokine receptors CCR4 and CCR10. The observed Th2 shift was partially reversed by exogenous IL-12., Conclusion: These data suggest that direct modulation of DC function as well as apoptosis contribute to the immunoregulatory effects of ECP.

Background: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade., Methods: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules., Results: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production., Conclusions: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.

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.

Background: The administration of donor specific bone marrow (DSBM) to mice conditioned with antilymphocyte serum (ALS) and sirolimus can result in stable multilineage mixed chimerism and long-term graft survival. This study seeks to determine if either the targeted depletion of CD4 and/or CD8 pos T cells or costimulation blockade can substitute for ALS and preserve the efficacy of this regimen., Methods: C57BL/6 recipients of BALB/c skin allografts were treated with DSBM (150 x 10(6) cells), sirolimus (24 mg/kg intraperitonealy), and either ALS or various monoclonal antibodies (alphaCD4, alphaCD8, alphaCD154 alone or in combination). Recipient peripheral blood mononuclear cell (PBMC) depletion, donor chimerism, and deletion of donor reactive T cells were assessed using flow cytometry. The specificity of immunologic nonreactivity and the presence of immunoregulatory activity were assessed through a mixed lymphocyte reaction assay., Results: The administration of ALS, sirolimus, and DSBM resulted in sustained recipient PBMC depletion, transient chimerism, and prolonged graft survival. The substitution of an equivalent degree and duration of targeted depletion of either CD4 or CD8 pos T cells alone for ALS failed to produce chimerism or prolonged graft survival. In contrast, depletion of both CD4 and CD8 pos T cells resulted in durable multilineage chimerism, indefinite allograft acceptance (>350 days), and donor-specific tolerance to secondary skin grafts. Substitution of alphaCD154 monoclonal antibody for ALS also resulted in a state of mixed chimerism and donor specific tolerance. This tolerant state appears to be maintained at least partially through clonal deletion and suppression., Conclusion: Either combined CD4 and CD8 T-cell depletion or alphaCD154 blockade can effectively substitute for ALS in producing chimerism and tolerance in this model.

Background: We previously demonstrated in vitro and in vivo that an IkappaB kinase (IKK) inhibitor blocks cytokine production and suppresses immune responses. These results indicate that a potent IKK inhibitor may have the potential of being a novel therapeutic agent for the prevention of graft rejection., Methods: The IKK inhibitor BMS-345541 was tested in mice for its ability to inhibit anti-CD3-induced interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha production and T-cell proliferation in an in vivo mixed lymphocyte reaction. BMS-345541 was further tested for its ability to suppress graft rejection in a murine nonvascularized heterotopic cardiac allograft model. BMS-345541 was tested as a single agent and in combination with other immunomodulators for inhibition of T-cell proliferation and graft rejection in vivo., Results: BMS-345541 suppressed, in a dose-dependent manner, the production of both IL-2 and TNF-alpha in mice stimulated with an injection of anti-CD3 antibody. Approximately 70% inhibition of both IL-2 and TNF were observed at a dose of 100 mg/kg. When BMS-345541 was administered at 100 mg/kg as a single agent, in vivo T-cell proliferation was not inhibited. However, when combined with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin (200 microg), a synergistic antiproliferative effect was observed, resulting in 77% inhibition of CD4+ T-cell proliferation. In the murine heterotopic heart transplant model, BMS-345541 did not prolong graft survival when administered at 50 mg/kg as a single agent. However, when administered with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin or cyclosporine A (15 mg/kg), graft survival was significantly increased compared with either agent alone., Conclusions: These results indicate that inhibition of IKK may serve as novel adjunctive therapy for the prevention of graft rejection.

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.

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.

Background: Intestinal transplantation has been hampered by high rates of intestinal allograft rejection. One mechanism of altering rejection in other organ transplant models has been blockade of second set T-cell costimulatory signals. AH.F5, a novel hamster anti-rat monoclonal antibody to CD154, blocks CD40-dependent T-cell costimulation. We hypothesized that blockade of this pathway might abrogate rejection in a rodent orthotopic survival model of intestinal transplantation., Methods: Eight groups were studied with different dosing schema, including syngeneic transplants (group 1), untreated allogeneic transplants (group 2), allogeneic transplants plus multiple doses of AH.F5 alone given IV or s.c. (groups 3 and 4), allogeneic transplants plus donor splenocyte preconditioning with and without single dose AH.F5 (groups 5 and 6), and donor splenocyte preconditioning followed by multiple doses of AH.F5 with and without thymectomy (groups 7 and 8)., Results: Control animals all died within 12 days of transplantation, whereas antibody-alone and splenocytes-alone resulted in modest prolongation of survival to 16 days. Only animals treated with splenocytes before transplantation and AH.F5 survived long-term (>60 days, group 8). These animals tolerated donor-specific skin grafts, rejected third-party grafts, and fed normally. However, their weight gain was subnormal and they demonstrated intestinal muscular thickening, which might represent chronic rejection. Thymectomy prevented the induction of tolerance., Conclusions: AH.F5 prevents acute intestinal allograft rejection in combination with donor-specific splenocyte preconditioning. We achieved long-term survival and the animals appeared tolerant. Central conditioning is essential for success with this antibody when used alone. Further studies with different dosing regimens or second agents seem warranted.

Background: CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs)., Methods: Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation., Results: Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity., Conclusions: These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.

Background: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients., Methods: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4., Results: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers., Conclusions: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.

The CD154-CD40 pathway is one of the critical costimulatory pathways that are required for full activation of T cells during alloimmune responses. Blockade of this pathway with anti-CD154 antibodies has been reported to prolong allograft survival in experimental transplantation models and to induce tolerance in some instances. However, anti-CD154 monotherapy cannot induce tolerance in "stringent" models such as skin and islet transplantation and is not sufficient to prevent chronic graft vasculopathy in vascularized organ transplantation. Therefore, combined therapies of anti-CD154 antibodies plus donor-specific transfusion, bone marrow infusion, or B7 blockade by CTLA4-Ig have been tried, and synergistic effects for tolerance induction have been reported. Furthermore, the efficacy of CD154 blockade in primate models has been confirmed for islet and kidney transplantation. The mechanisms of CD154 blockade in vivo include CTLA4-dependent anergy or regulation, T-cell apoptosis, and induction of regulatory cells. Finally, anti-CD154 antibody therapy has been reported to result in unexpected thromboembolic complications in both primates and humans, although the etiology of these conditions remains unclear. In addition, not all antibodies cause this side effect. Clinical trials with humanized anti-CD154 monoclonal antibodies are underway in severe autoimmune diseases, but its development in transplantation is unclear at this time.

Background: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40., Methods: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E., Results: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner., Conclusion: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.

Background: Human lung transplantation carries a poor prognosis because of chronic rejection in the form of obliterative bronchiolitis syndrome (OBS). Using the mouse model of heterotopic tracheal transplantation, we examined the role of costimulation in the allograft rejection that characterizes obstructive airway disease (OAD)., Methods: C57BL/6 or BALB/c tracheae were implanted into wild-type control, CD28-/-, muMT (B-cell deficient), or CD40L-/- recipient mice. Grafts were explanted from 7 to 42 days posttransplantation and evaluated., Results: Thickening of the basement membrane and a decrease in patent luminal area were first noted at 2 weeks in wild-type allogeneic trachea recipients and to a slightly lesser degree in CD28-/- recipients. In contrast, CD40L-/- recipient mice showed no evidence of cellular infiltrates or fibrosis in transplanted tracheae. To determine whether CD40L interacted with host or donor CD40, CD40-deficient tracheae were transplanted into CD40L+/+, CD40+/+ wild-type mice. Wild-type mice rejected CD40-/- tracheae. Tracheae were transplanted into B-cell-deficient mice to determine the role of B-cell CD40 in chronic pulmonary allograft rejection. The OAD reaction was identical in wild-type and B-cell-deficient mice., Conclusions: Development of OAD in the mouse trachea transplant model is primarily dependent on CD40L and is relatively CD28 independent. The ability of mice to reject CD40-/- tracheae demonstrated that host, not donor, CD40 is required for rejection. Furthermore, the ability of B-cell-deficient mice to reject allogeneic tracheae demonstrated that B-cell CD40-mediated responses are not required for the development of OAD.

Curative islet transplantation for type 1 diabetes currently requires lifelong systemic immunosuppression. Induction of islet transplantation tolerance would be far preferable. We have previously demonstrated that blockade of costimulation by the administration of a donor-specific transfusion in combination with anti-CD154 monoclonal antibody leads to permanent islet and prolonged skin allograft survival in mice. The protocol requires the presence of CD4+ T cells, interferon-gamma, and CTLA4, and involves the deletion of CD8+ alloreactive T cells. Translation of this strategy into clinical practice will, however, require attention to at least two issues. First, we have observed that the presence of viral infection during tolerance interferes with tolerance induction. Second, we have observed that our tolerance induction protocol is ineffective in autoimmune nonobese diabetic mice. We hypothesize that resistance to tolerance induction in nonobese diabetic mice is due to the presence of memory autoreactive cells. To overcome the deleterious effects of viral infection and of primed memory responses, it may be necessary to modify current tolerance induction strategies based on costimulatory blockade. These modifications may require patient isolation, the generation of hematopoietic chimerism, or treatments that target the specific T-cell populations, cytokines, and/or costimulatory factors responsible for resistance. Such modifications may make it possible to extend tolerance induction to the "real world" situation of individuals with type 1 diabetes who are likely to harbor both memory allo-and autoreactive immune cells.

Background: Disruption of the CD40/CD154 pathway inhibits rejection in numerous models. The importance of this pathway on intestinal allograft rejection was examined in this study., Methods: Intestinal grafts from B6C3F1 mice transplanted into C57BL/6 recipients were assessed histologically for rejection., Results: The monoclonal antibody to CD154, MR1, failed to inhibit rejection in wild-type mice. Similarly, CD154-/- recipient mice rejected intestinal allografts. MR1 did inhibit early rejection in CD8-/- mice, but had no effect in CD4-/- recipients. All MR1-treated CD8-/- recipients eventually developed rejection. No benefit was observed when blockade of the CD40/CD154 pathway by MR1 was combined with blockade of the CD28/B7 pathway by mCTLA4Ig., Conclusions: These data suggest that CD4+ T cells mediating intestinal allograft rejection may be more dependent upon the CD40/CD154 pathway than CD8+ T cells. This finding highlights the importance of identifying agents that suppress CD8+ T cell-mediated rejection.