Linking erythropoietin to regulatory T-cell-dependent allograft survival through myeloid cells

Erythropoietin (EPO) has multiple functions beyond stimulating erythrocytosis, including modulation of T cell immunity. Effects EPO in transplantation and associated mechanisms remain incompletely understood. We analyzed outcomes and performed cellular and molecular mechanistic assessments in murine wild type (WT) and conditional EPO receptor (EPOR) knockout recipients of cardiac allografts. In translational studies, we tested EPO’s effects in a non-human primate system. B6 mice were immunized with 20x106 BALB/c splenocytes. Recipient mice were treated with CTLA4-Ig (150µg i.p. once on day 2) (Abatacept, Bristol-Myers Squibb, New York, NY) ± recombinant EPO (5000 U/kg i.p. daily) (epoetin alfa, Amgen, Thousand Oaks, CA). On day 7, splenic CD11b+ myeloid cells were isolated by positive selection (kit #18970, STEMCELL, Vancouver, BC, Canada). RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA) and cleaned using RNeasy Mini Kit (Qiagen, Hilden, Germany). Gene expression profiling was then performed using the nCounter® Myeloid Innate Immunity Panel (NanoString Technologies, Seattle, WA).