Recent clinical studies showed that complete correction of anemia with erythropoietin (EPO) therapy improves human kidney transplant outcomes, but mechanisms underlying this protective effect are not known. Based on suggestive results from in murine models by several research groups we tested the hypothesis that EPO has immune suppressive effects on human alloreactive Tcells.

Methods: Human CD14+ monocytes and naive CD45RA+CD45RO-CD4+ Tcells were isolated by magnetic beads/sorting. Monocytes were cultured for 5d with IL-4 and GMCSF to induce DCs and then matured with LPS for an additional 48h. Tcell expansion in response to allogeneic DCs or aCD3/CD28-coated beads was measured by CFSE dilution/flow cytometry. Tcells and monocyte-derived DCs (moDCs) were phenotyped by flow analyses. Experiments were done in the presence of EPO alpha or vehicle.

Results: Monocytes, moDCs (both immature and LPS-matured), and CD4+ Tcells expressed EPO receptor (EPOR) on their surfaces. We observed upregulated EPOR expression on the CD4 Tcells upon aCD3/CD28 stimulation (within 1d and lasting up to 5d). When we added EPO to MLRs (naÏve CD4 Tcells with allogeneic mature moDC) we observed a significant, dose dependent decrease in proliferation vs. vehicle (1000 Uds/mL: EPO: -44.6±22.9%; 2000Uds/mL: EPO: 11.1±4%: p<0.05), 4 experiments) without an effect on cell death (<10% in all circumstances). To test whether EPO directly inhibits T cells in the absence of DCs, we stimulated purified CD4+ T cells with aCD3/CD28 +/- EPO and observed a dose-dependent inhibition of proliferation (at doses 1000 to 2000 Μg/ml: -36.5±20.9% to -90±4.37%; p<0.001). To test effects on moDC we added EPO to cultured monocytes during the 7d in vitro differentiation. At the end of the culture period we observed that addition of EPO resulted in significantly lower levels CD40 expression on the moDC (MFI: 931±259 vs. 1395±376; -66,7%, for EPO vs. vehicle, respectively, p<0.05, 3 experiments), without alterations in moDC surface expression of CD80, CD86, or HLA-DR. In MLRs without EPO in the culture, EPO-treated moDCs induced weaker alloreactive Tcell proliferation compared to vehicle-treated-controls (-26.7±18.3%, ns)

Conclusions: Our data demonstrate the unique finding that EPO exhibits in vitro immunosuppressive properties by diminishing human Tcell proliferation and DC maturation. These results provide one potential mechanism underlying the observed protective effects of EPO on kidney transplant outcome in humans.

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