B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) mediate CD40-independent help by memory CD4 T cells.
ABSTRACT: Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients.
Project description:Cognate T-B cell interactions and CD40-CD154 costimulation are essential for productive humoral immunity against T-dependent Ags. We reported that memory CD4 T cells can deliver help to B cells and induce pathogenic IgG alloantibodies in the absence of CD40-CD154 interactions. To determine cytokine requirements for CD40-independent help, we used CD40(-/-) mice containing differentiated subsets of donor-reactive memory Th cells as heart allograft recipients. Th1 and Th17, but not Th2, memory CD4 T cells elicited high titers of anti-donor Ab. Abs induced by Th17 memory CD4 T cells had decreased reactivity against donor MHC class I molecules and inferior ability to cause complement deposition in heart allografts compared with Abs induced by Th1 cells, suggesting a requirement for IFN-? during CD40-independent help. IFN-? neutralization inhibited helper functions of memory CD4 T cells in both CD40(-/-) recipients and wild type recipients treated with anti-CD154 mAb. Our results suggest that IFN-? secreted by pre-existing memory helper cells determines both isotype and specificity of donor-reactive alloantibodies and can thus affect allograft pathology. This information may be valuable for identifying transplant patients at risk for de novo development of pathogenic alloantibodies and for preventing alloantibody production in T cell-sensitized recipients.
Project description:CD40/CD154 interactions are essential for productive antibody responses to T-dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor-reactive memory CD4 T cells can deliver help to CD40-deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40-/- heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40-independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40-independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long-lived plasma cells and memory B cells. In summary, our data suggest that while CD40-independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long-lasting anti-donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor-reactive memory T cells.
Project description:Despite advances in immunosuppression, antibody-mediated rejection is a serious threat to allograft survival. Alloreactive memory helper T cells can induce potent alloantibody responses and often associate with poor graft outcome. Nevertheless, the ability of memory T cells to elicit well characterized manifestations of antibody-mediated rejection has not been tested. We investigated helper functions of memory CD4 T cells in a mouse model of renal transplantation. Whereas the majority of unsensitized C57Bl/6 recipients spontaneously accepted fully MHC-mismatched A/J renal allografts, recipients containing donor-reactive memory CD4 T cells rapidly lost allograft function. Increased serum creatinine levels, high serum titers of donor-specific alloantibody, minimal T cell infiltration, and intense C4d deposition in the grafts of sensitized recipients fulfilled all diagnostic criteria for acute renal antibody-mediated rejection in humans. IFN? neutralization did not prevent the renal allograft rejection induced by memory helper T cells, and CD8 T cell depletion at the time of transplantation or depletion of both CD4 and CD8 T cells also did not prevent the renal allograft rejection induced by memory helper T cells starting at day 4 after transplantation. However, B cell depletion inhibited alloantibody generation and significantly extended allograft survival, indicating that donor-specific alloantibodies (not T cells) were the critical effector mechanism of renal allograft rejection induced by memory CD4 T cells. Our studies provide direct evidence that recipient T cell sensitization may result in antibody-mediated rejection of renal allografts and introduce a physiologically relevant animal model with which to investigate mechanisms of antibody-mediated rejection and novel therapeutic approaches for its prevention and treatment.
Project description:Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-?-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.
Project description:Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC-mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen-4 (VLA-4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti-VLA-4 mAb given to C57BL6 (H-2b ) recipients of AJ (H-2a ) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra-allograft IFN-?-induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA-4Ig treated recipients. Anti-VLA-4 mAb also inhibited priming of donor-specific T cells producing IFN-? until at least day 7 posttransplant. Peritransplant anti-VLA plus anti-CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti-VLA-4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.
Project description:Allosensitization constitutes a major barrier in transplantation. Preexisting donor-reactive memory T and B cells and preformed donor-specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long-term immunosuppression-free allograft survival. We demonstrate that donor-specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti-CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen-presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti-CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long-term islet allograft survival in such recipients.
Project description:Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4(+) and CD8(+) T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4(+) and CD8(+) TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8(+) T-cell expansion, delaying differentiation into IFN-?(+) TNF(+) multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4(+) CD25(hi) Foxp3(+) T-cell population, which did not arise from endogenous natural T(reg) but rather were peripherally generated from naïve Foxp3(-) precursors.
Project description:After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8(+) T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4(+) and CD8(+) donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4(+) T cells to approximately 0.5% and CD8(+) T cells to approximately 5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8(+) T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.
Project description:Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60 days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30 days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1- / - cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.
Project description:Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or <i>Candida albicans</i> Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.