Selective interference with the CD45RB isoform by mAb (anti-CD45RB) reliably induces donor-specific tolerance. Although previous studies suggest participation of regulatory T cells, a mechanistic understanding of anti-CD45RB-induced tolerance is lacking. We report herein the unexpected finding that tolerance induced by this agent is not established in B cell-deficient mice but can be recovered by preemptive B lymphocyte transfer to B cell-deficient hosts. Using B cells from genetically modified donors to reconstitute B cell-deficient recipients, we evaluate the role of B lymphocyte-expressed CD45RB, T cell costimulatory molecules, and the production of Abs in this novel tolerance mechanism. Our data document an Ab-induced tolerance regimen that is uniquely B lymphocyte-dependent and suggest mechanistic contributions to tolerance development from the B cell compartment through interactions with T cells.

Involvement of Treg in transplant tolerance has been demonstrated in multiple models. During the active process of graft rejection, these regulatory cells are themselves regulated and inactivated, a process termed counter-regulation. We hypothesize that ligation of the costimulatory molecule glucocorticoid-induced TNF receptor-related protein (GITR) on Treg inhibits their ability to promote graft survival, and by blocking GITR ligation graft survival can be prolonged. To this aim, we have designed a soluble GITR fusion protein (GITR-Fc), which binds GITR ligand and inhibits activation of GITR. Here, we show that GITR-Fc prolonged mouse skin graft survival, and this prolongation is dependent on Treg. In a full MHC-mismatched skin graft setting, GITR-Fc significantly improved graft survival when used in combination with MR1, anti-CD40L, while GITR-Fc alone did not demonstrate graft prolongation. These results demonstrate that disruption of binding of GITR with GITR ligand may be an important strategy in prolonging allograft survival.Key words: GITR . Inflammation . Treg . Tolerance . Transplant IntroductionSub-optimal long-term graft survival rates and the need for chronic immunosuppression to sustain survival in virtually all cases of solid organ allotransplantation continue to undermine success in clinical organ transplantation. To improve graft survival rates and eliminate the need for immunosuppression [1], donor-specific transplant tolerance must be achieved.The application of Treg is an area of intense focus in the field of transplant biology. The naturally occurring population of CD4 1 CD251 Treg maintains both self-tolerance and transplant tolerance by suppressing T-cell responses both in vivo and in vitro [2]. Phenotypically, Treg constitutively express the cell surface molecules CTLA-4, certain toll-like receptors, CD103 (aE integrin), and glucocorticoid-induced TNF receptor-related protein (GITR) at high levels [3][4][5][6][7], and they specifically express the transcription factor Foxp3 [8,9]. GITR is a type I transmembrane protein with high homology to other members of the TNFR family, especially to 4-1BB and CD27 [4,7]. GITR is preferentially expressed by Treg; however, like CD25, GITR is also upregulated in conventional effector T cells (T eff ) upon activation [5]. In a screen to identify proteins specific to CD251 Treg, Sakaguchi's group identified a GITRspecific antibody that could block the ability of Treg to suppress SHORT COMMUNICATIONÃ These authors contributed equally to this work. [4,11]. GITRL expression is upregulated on APC by toll-like receptor (TLR) stimulation but is downregulated within 48 h in vitro [4,11]. We have previously demonstrated that GITRL is upregulated by dendritic cells in the draining lymph node within 1 wk of a skin graft procedure, and we have suggested that this upregulation results in the inactivation of Treg [12].In an in vivo transplantation model, adoptive transfer of antigen-specific Treg can suppress skin graft rejection by T eff when co-transferred in the ab...

Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25− and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.

Background Isolated islet transplantation with infusions from 2-3 donor pancreata and Edmonton immunosuppression consistently achieves insulin independence in patients with type 1 diabetes. The success of this protocol has been attributed to a novel combination of immunosuppressive agents and avoidance of steroids, however, the outcome of islet transplantation may differ in kidney transplant recipients who are already immunosuppressed. Methods We compared the metabolic outcomes and graft survival of islet transplantation in our program where 9 patients underwent islet transplantation alone (ITA) treated with Edmonton immunosuppression and 8 patients received islet after kidney (IAK) transplants under standard kidney transplant immunosuppression often including steroids. Results Transplants in the IAK and ITA setting demonstrated similar islet potency (IEq/unit insulin reduction) and recipients from both groups routinely gained insulin independence, functional islet mass and duration of graft survival, however, seemed superior in the IAK group. Conclusions These results suggest that better islet graft function and survival may be attained using non-Edmonton rather than Edmonton immunosuppression and can include maintenance steroid therapy.