Figure 4. CD25⁺ Tfh cells respond to IL-2 and provide B-cell help.

(A) Purified tonsil CD4⁺ T cells were treated with IL-2 (10U/mL) for 5 min and phosphorylated STAT5 levels were detected by flow cytometry. (B–H) Tonsil CD4⁺T cells were purified and treated with or without IL-2 (100U/mL) for 24 h. ICOS (B) andOX40 (C) expression and IL-21 (D) production among the CXCR5⁺PD1⁺cells were examined. For cytokine detection, cells were stimulated with PMA plus Ionomycin for 4 h, then stained with different antibody combinations against CXCR5, PD1 and CD25. (E) Purified tonsil cells were treated with IL-2 treatment for 24h and the percentage of CD25⁺PD1⁺CXCR5⁺ Tfh cells determined by flow cytometry.(F, G) In IL-2 treated tonsil CD4⁺ T cells, the PD1⁺CD25⁺ Tfh cells had higher expression of both ICOS and OX40 compared to the CD25⁻ subset. MFI was calculated as: [MFI (specific mAb) – MFI (isotype control)]/MFI (isotype control). (H) Similarly, IL-2-treated Tfh cells were analyzed for IL-21, IL-17, IL-10, IL-4, IL-2 and IFN-γ production by intracellular staining and gating on PD1⁺CD25⁺ orPD1⁺CD25⁻ cells.(I) B cells cocultured with Tfh cells in the presence or absence of IL-2 (100U/mL) were analyzed for levels of IgG by ELISA. (J) B cells cocultured with CD25⁺or CD25⁻ Tfh cells were analyzed for levels of IgG by ELISA. Flow cytometry plots are representative of 4 experiments. (B–D, F–H) Data are expressed as mean +SEM (n=7 samples). P < 0.05 was considered to be significant; Student’s t test.