EPO directly inhibits inhibitory TFH function independent of TFR. (A) B6 mice received allogeneic immunization with BALB/c splenocytes (20 × 106, i.p.), and after 7 days the animals were euthanized. BALB/c enriched splenic B cells and preimmunized B6 enriched splenic CD4+PD1+CXCR5+ TFH–TFR were stimulated with anti-IgM (10 µg/ml) and anti-CD3 (2 µg/ml). Cells were treated with EPO (1000 IU/ml) or vehicle control, incubated for 2 days at 37°C, and collected to measure IgG+ B cells. Representative plots (B) and quantified percentages (C) of B220+ IgM-IgD- IgG+ B cells from BALB/c B cells cultured alone or with TFH and TFR from preimmunized B6, in the presence of EPO or vehicle. (D)–(E) Same experiment represented in (A), done with EPORfl/flCD4-CrePOS and CD19-CreNEG mice, showing (D) representative plots and (E) quantified percentages of B220+ IgM-IgD- IgG+ B cells. (F) B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J mice received allogeneic immunization with BALB/c splenocytes (20 × 106, i.p.); after 7 days the animals were euthanized, and CD4+PD1+CXCR5+Foxp3- TFH cells were flow sorted. BALB/c enriched splenic B cells and preimmunized B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J TFH were stimulated with anti-IgM (10 µg/ml) and anti-CD3 (2 µg/ml). Cells were treated with EPO (1000 IU/ml) or vehicle control, incubated for two days at 37°C, and collected to measure IgG+ B cells. Representative plots (G) and quantified percentages (H) of B220+ IgM-IgD- IgG+ B cells from BALB/c B cells cultured with preimmunized B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J TFH in the presence of EPO or vehicle control. Dashed line represents the percentage value of B220+ IgM-IgD- IgG+ B cells in a culture with BALB/c B cells and B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J TFH–TFR treated with vehicle control as reference value. Each dot represents a different culture well. Experiments were repeated at least three times. Data represent median ± IQR. *P<0.05; ns: not significant between EPO and vehicle. Data were analyzed using an unpaired t test.