The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2Xs) sense the ATP, released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2Xs during islet allograft rejection and to establish a novel anti-P2X strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet graft-infiltrating T-cells in a model of allogeneic islet transplantation (BALB/c islets transplanted into hyperglycemic C57BL/6 mice), but only P2X7R is increasingly expressed during alloimmune response, while P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP], 250 Μg day 0 to day 15 i.p.) delayed islet allograft rejection (Mean Survival Time [MST]: oATP treated=22 days vs. Untreated=14 days, n=10; p=0.0001) with indefinite graft survival achieved in 3 out of 10 mice, reduced the frequency of Th1/Th17 cells, and induced hyporesponsiveness toward donor antigens (IFN-Γ-producing cells: oATP-treated day 14=117±9 vs. untreated day 14=418±26, n=5; p<0.0001). oATP-treated mice also displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and Rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. PBMC obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R⁺CD4⁺ T-cells compared to controls and in particular P2X7R expression increased in memory T-cells (CD45RO⁺ T-cells); in vitro, a reduction in the number of IFN-Γ-producing cells in response to allogeneic stimulation was observed with the human specific P2X7R-inhibitor CE-224535 in human PBMC (IFN-Γ-producing cells: CE-224535-treated= 29±1 vs. control= 51±3, n=5; p=0.003) confirming potential for clinical islet transplantation. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function.

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