Spontaneous allogeneic kidney transplant tolerance occurs in mice, but the underlying mechanisms are unknown. Building upon recent human data showing that EPO, an erythropoietic hormone produced by the adult kidney, has immunosuppressive effects (JASN 2014), we tested the hypothesis that EPO is required for kidney allograft tolerance. Flow cytometric analyses showed EPO receptor expression on murine CD4⁺ and CD8⁺ T cells. Recombinant EPO inhibited T cell proliferation by 85.4±17.9% , promoted Treg induction by 148.8±39.7%, and increased Treg stability by 291.6±78.6% vs. control (p<0.05 for each). When adoptively transferred into B6 rag1^-/- recipients of BALB/c (I-E^d+) kidney allografts, [sim]4% of naïve CD4⁺Foxp3^GFPneg TEa TCR transgenic T cells spontaneously became FoxP3⁺, whereas <0.5% became FoxP3⁺ in recipients of syngeneic B6 kidney transplant controls, or in recipients of BALB/c hearts (p<0.05, n=3-5/group). Administration of recombinant EPO to B6 recipients of BALB/c hearts induced Foxp3 expression (n=3, p<0.05 vs control), treatment of kidney allograft recipients with acriflavine (a HIF-inhibitor that inhibits EPO transcription), or administration of a blocking anti-EPO mAb, prevented conversion of adoptively transferred FoxP3^neg TEa cells into FoxP3⁺ cells (n=3-4, p<0.05 for each). We next transplanted MHC-disparate A/j kidneys or syngeneic B6 kidneys into B6 hosts (removing both recipient native kidneys). While allogeneic kidneys survived for >30 days, treatment with the anti-EPO mAb (but not an isotype control), or with acriflavine precipitated acute allograft rejection (n=5-7/group p<0.05 vs. untreated allograft recipients). Histology showed CD3 cell infiltrates with fewer FoxP3+ cells in the acriflavine or anti-EPO antibody treated A/J kidneys compared to the untreated allograft controls (p<0.05). Acriflavine treatment had no effect on control isograft survival. Together, our findings indicate that kidney allograft-produced EPO is necessary and sufficient for spontaneous allospecific Treg induction, and identify EPO as the crucial kidney-specific inducer of spontaneous kidney allograft tolerance in mice. The results provide the rationale for testing EPO as a Treg-sparing, anti-rejection therapy in humans.

CITATION INFORMATION: Cravedi P, Purroy C, Madsen J, Fairchild R, Tanaka T, Baldwin 3rd W, Alessandrini A, Heeger P. Erythropoietin (EPO) Mediates Spontaneous Kidney Transplant Tolerance in Mice. Am J Transplant. 2016;16 (suppl 3).

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