Identification of Novel Mechanisms of Tolerance Resistance Induced By OX40.

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Induction of donor specific tolerance remains an important goal in transplantation and to identify and overcome barriers to tolerance will significantly improve transplant outcomes in the clinic. In the present study, we examined the effector programs that evade tolerance induction by costimulatory blockade in a mouse heart transplant model. Blocking CD40-CD40L costimulation using an anti-CD40L monoclonal antibody (MR1) induced prolonged survival of C57BL/6 heart allografts in BALB/c recipients (MST>60 days). However, we found that MR1 completely failed to prolong survival of heart allografts that express the OX40L transgene (OX40Ltg grafts) (MST=9 days), indicating that OX40 signaling evades tolerance induction by costimulatory blockade. Histologically, rejection of OX40Ltg heart allografts was associated with extensive lymphocyte infiltration, and a significant proportion of graft infiltrating T cells produced IL-9 (40%), while few of them produced IL-4 or IFN-γ, suggesting that OX40 may drive a new mechanism of tolerance resistance. In an in vitro T cell polarization assay, we showed that OX40 engagement inhibited the induction of TH17 cells by TGF-β/IL-6 and that of inducible Treg cells by TGF- β/IL-2. Instead, OX40 diverted CD4+ T cells to IL-9 producing TH9 cells. Mechanistically, OX40 signaling mediated the induction of TH9 cells by triggering noncanonical NF-κB activation in activated T cells. In an in vivo CFSE model in which CD4+ T cells were stimulated by alloantigens, blocking CD40-CD40L costimulation induced TH9 cells only in the presence of OX40 signaling. These data suggest that induction of TH9 cells may constitute a novel mechanism of tolerance resistance. Interestingly, blocking OX40 costimulation induced long-term heart allograft survival (MST>100 days) and markedly reduced transplant vasculopathy (chronic allograft rejection) in CD40L-/- recipient mice. In conclusion, we identified that OX40 dramatically induces the generation of TH9 cells both in vitro and in vivo, and TH9 cells are closely involved in tolerance resistance. These findings may lead to the development of new therapeutics to promote graft tolerance in transplantation.