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We previously demonstrated that T-regs inhibit proliferation of graft-reactive T cells in the draining lymph node (DLN), suggesting that this site may be important for regulation. TCR transgenic mice (TS1) specific for viral hemagglutinin (HA) provided antigen-specific T cells for adoptive transfer into syngeneic Balb/c hosts bearing HA+ skin grafts. T-regs were obtained from (TS1xHA28)F1 mice known to have an expanded population of HA-specific T-regs. To determine whether the lymph node is an independent site of suppression, we developed a model in which donor antigen that migrates from the allograft to the DLN drives T-cell activation after graft removal. T-regs that did not encounter the allograft itself remained able to inhibit graft antigen-specific T-cell proliferation in the DLN. Alloantigen-induced regulation can occur in the absence of the graft. This finding identifies the DLN as a potentially critical site of regulation in the early posttransplant period.
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.
T-cell activation is essential for acute allograft rejection. However, the biochemical signaling pathways used by T cells mediating rejection have not been extensively investigated. In vitro, T-cell activation is associated with nuclear translocation of specific transcription factors that regulate expression of genes critical for T-cell function. Given the central role of NF-kappaB in T-cell activation In vitro, we examined its role in the acute rejection of skin and cardiac allografts using mice with defective NF-kappaB translocation in T cells due to the presence of a super repressor IkappaBalpha transgene. T-cell-intrinsic NF-kappaB activation was required for cardiac but not skin allograft rejection, suggesting differential T-cell priming by the two tissues. Strikingly, priming with heart allografts induced complete acceptance of subsequently transplanted donor skin grafts, indicating that impaired NF-kappaB activation in T cells facilitates the induction of donor-specific tolerance to highly immunogenic tissues. These data suggest the biochemical pathways necessary for allograft rejection vary, based on the antigen and the context in which it is presented, and that inhibition of T-cell-intrinsic NF-kappaB activation during allogeneic priming may represent a novel strategy whereby tolerance to transplanted organs can be achieved.
We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.
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.
CD40-CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti-CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft-reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40-CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti-CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL-2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti-CD40L treatment. Interestingly, the spleens of mice bearing long-term cardiac allografts following inductive anti-CD40L treatment retained precursor donor alloantigen-reactive CTL, IL-2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor-strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor-strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long-term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long-term graft acceptance. Collectively, these data demonstrate that inductive CD40-CD40L blockade does not result in the deletion of graft-reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen-reactive cells are subject to this regulation.
Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-alpha1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-gamma and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.