Despite remarkable improvements in short term transplant survival, the long-term outcome of allografts remains poor in the clinic, and most transplants are eventually lost due to chronic rejection. This is a significant issue in transplant medicine, but the underlying mechanisms remain incompletely defined.

We used a mouse heart transplant model to examine cell types that infiltrate chronically rejected allografts and their roles in chronic allograft loss. We found that treatment of B6 recipients with a single dose of CTLA-4Ig consistently prevented acute rejection of Balb/c heart allografts, but allowed chronic rejection to develop in the grafts. Histologically, the CTLA-4Ig treated grafts showed signs of typical chronic rejection, characterized by prominent neointima formation and perivascular leukocyte infiltration. Immunohistochemistry analysis revealed extensive macrophage infiltrates, and most of the infiltrating macrophages exhibited a M2 phenotype in situ in the chronic rejection lesions, as they showed strong F4/80 and CD206 double staining, while the typical M1 marker iNOS is unremarkable. Screening for M2 surface markers by immunofluorescence and flow cytometry identified preferentially expression of P2X7R by M2 cells. In vitro analysis using bone marrow-derived macrophages where they can be selectively polarized to M1 or M2 cells, we found that P2X7R is also preferentially expressed on polarized M2 cells, and an P2X7R antagonist oxidized ATP (oATP) strongly inhibited the polarization of M2 cells. Interestingly, oATP inhibited the expression of all M2 makers in vitro (Arg-1, Ym1, Fizz) in a dose dependent fashion. Importantly, in the heart transplant model in which the recipient mice were treated with oATP in addition to CTLA-4Ig, all heart allograft survived long-term (MST>90 days), with no signs of neointima formation in the grafts, demonstrating the inhibition of chronic allograft rejection. Histologically, inhibition of chronic rejection was associated with markedly reduced M2 cells in the heart transplants. Thus, our studies identified a previously unknown mechanism of transplant vasculopathy and pinpointed P2X7 as a potential molecular target in the inhibition of M2 cells and chronic rejection.

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