Extending previous work showing that EPO inhibits alloreactive effector T cell proliferation by uncoupling IL2 receptor signaling (JASN 2014), we tested the effects of EPO on human regulatory T cell (Treg) function and stability.

Whereas EPO reduced the expansion of CFSE-labeled naïve human CD4 T cells stimulated with anti-CD3/anti-CD28 and IL-2 by > 85% (p<0.005), EPO had no effect on proliferation of in vitro induced or peripheral human Treg activated with the same stimuli (p=ns), despite similar levels of EPO receptor expression. EPO did not affect the rate of Treg induction when naive CD4 T cells were incubated with anti-CD3/anti-CD28, IL2 and TGF-β (p=ns vs no EPO), nor did EPO affect their suppressive capacity. EPO promoted TGF-β gene upregulation and protein in human monocytes, which, in co-culture experiments, promoted conversion of naïve CD4 T cells into functional Treg (iTreg) despite absence of exogenous TGF-β (CD4⁺CD25⁺FoxP3⁺ cells:21.4% vs 27.3% with vehicle or EPO, respectively; P<0.05). The effect was abolished by adding a specific TGF-β receptor blocking Ab.

To test the effect of EPO on iTreg stability, we transferred HLA-A2^Pos, in vitro induced iTreg plus HLA-A2^Neg PBMC into NOD scid γc-/- mice. At D3 after injection, animals treated with EPO had significantly more HLA-2^PosFoxP3⁺ T cells than the vehicle treated controls (15% vs 3.7%; P<0.005).

Starting from our data that EPO inhibits AKT and ERK phosphorylation in Teff cells activated with anti-CD3/anti-CD28 and IL2, but does not affect STAT5 phosphorylation, we hypothesized that EPO inhibits IL2R signaling through the β (relevant for Teff function), but not the γ (important for Treg function) chain by activating SHP-1 phosphatase. EPO induced SHP-1 activation in human T cells and SHP-1 inhibition (NSC-87877) overcame the suppressive effects of EPO on anti-CD3/anti-CD28 and IL2 induced T cell proliferation (EPO:48.8% vs. EPO+SHP-1 inhibitor:67.2% of vehicle) and IFN-γ production (EPO:51.1% vs EPO+SHP-1 inhibitor:143% of vehicle).

Overall, our data show for the first time that while EPO inhibits alloreactive Teff cells it spares Treg expansion and function by activating the SHP-1 phosphatase. Simultaneously, EPO induces TGF-β production in monocytes, promoting Treg induction. The results may explain why heart and kidney transplant recipients experience fewer acute rejections that heart transplant recipients alone and support further studies to test the efficacy of EPO as immune modulator in the clinic.

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