*Purpose: Erythropoietin (EPO) ligation of the EPO receptor (EPO-R) homodimer and EPO-R/CD131 heterodimer has protolerogenic effects on alloreactive T cell responses. While prior work, largely performed in vitro, showed that EPO-R signaling on T cells suppresses IL-2-induced proliferation and EPO-R signaling on monocytes promotes TGF-β secretion that in turn drives regulatory T cell (Treg) generation, whether/how these effects impact allograft survival in vivo have not been addressed.

*Methods: To test this, we generated EPO-R^fl/fl mice and transplanted groups of EPO-R^fl/fl, EPO-R^fl/fl CD4-cre+ (absent EPO-R on T cells), and EPO-R^fl/fl LysM-cre (absent EPO-R on myeloid cells) B6 recipients with BALB/c hearts. Transplant recipients received a single dose of CTLA4-Ig ± recombinant EPO (rEPO) daily x 3 days.

*Results: Untreated animals of all genotypes rejected their allografts within 8 days (p=ns) and CTLA-Ig alone equivalently prolonged survival of all genotypes to a median survival time (MST) of 20 days (p=ns among groups). Addition of rEPO prolonged graft survival in EPO-R^fl/fl recipients to 35 days (p<0.05 vs. CTLA4-Ig alone). rEPO similarly prolonged graft survival in CTLA4-Ig-treated EPO-R^fl/fl CD4-cre+ recipients (MST 32), indicating that EPO-R signaling on T cells is not required for the effect. In contrast, rEPO administration did not prolong BALB/c heart graft survival in CTLA4-Ig-treated EPO-R^fl/fl LysM-cre+ recipients (MST 16 days, p=ns vs. CTLA4-Ig alone), indicating that EPO-R signaling on myeloid cells is required. We next transplanted additional groups of EPO-R^fl/fl and EPO-R^fl/fl LysM-cre B6 mice (all given CTLA4-Ig+rEPO) and sacrificed them on day 14 post-transplant (all grafts beating) to perform ex vivo mechanistic analyses. These studies revealed a 2-fold increase in donor-reactive IFNγ-producing splenic CD8+ T cells and ~3-fold increase in the ratio of IFNγ-producing T effector cells to Tregs in the EPO-R^fl/fl LysM-cre+ recipients vs the EPO-R^fl/fl controls (p<0.001 for each). When we analyzed intragraft cytokine expression, we observed a 2-fold reduction in the ratio of IL-10 to IL-12 in the EPO-R^fl/fl LysM-cre+ mice vs the EPO-R^fl/fl controls (p<0.05).

*Conclusions: Our data show that rEPO interacts with CTLA4-Ig to prolong allograft survival and that the effects are mediated through EPO-induced alterations in myeloid cells, which in turn enhance Treg function. The findings suggest that post-transplant rEPO therapy could be effective in reducing rejection and improving allograft function in humans.

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