Fig. 6.

The deletion of Blimp-1 rescues the perturbed differentiations of T_FH and GC B cells in Srg3^fl/flCD4Cre⁺ mice. (A) CD4⁺ T cells of Srg3^fl/fl CD4Cre⁺ mice were stimulated for 24 h with anti-CD3 and anti-CD28 in the presence of IL-6, anti–IL-4, anti–IFN-γ, and anti–TGF-β, followed by quantitative RT-PCR analysis for the expression of genes involved in differentiation or function in T_FH cells; results are presented relative to those of CD4⁺ T cells of Srg3^fl/fl mice. Data are presented by mean and SEM; *P < 0.05, **P < 0.01, ***P < 0.001. n = 4–8. (B) CD4⁺ T cells of Srg3^fl/fl or Srg3^fl/flCD4Cre⁺ mice were transduced with Empty-RV or Bcl-6–RV after 18-h activation with anti-CD3 and anti-CD28 and further cultured in condition as described in A. After 48 h, GFP⁺ cells were purified by flow cytometry and expression of Blimp-1, CXCR5, and PD-1 was analyzed by quantitative RT-PCR; results in CD4⁺ T cells transduced with Bcl-6–RV are presented as fold induction relative to those in CD4⁺ T cells transduced with Empty-RV. Data are presented as mean and SEM; *P < 0.05. n = 3–4. (C) Flow cytometry of CXCR5⁺PD-1^hi T_FH cells obtained from the spleen of NP-KLH–immunized mice on day 7. The statistical quantitation of frequency in CD4⁺ T cells and cell numbers for CXCR5⁺PD-1^hi T_FH cells was analyzed. Each individual dot represents one mouse and data are presented as mean and SEM. (D) CXCR5 and PD-1 expressions in CD4⁺ cells. Bar graphs show mean fluorescent intensity of CXCR5 (Left) and PD-1 (Right). (E) Flow cytometry of GC B cells obtained from the spleen of NP-KLH–immunized mice on day 7. Cells gated on CD4⁻B220⁺ were analyzed for the expression of Fas and GL-7 (Upper). Numbers adjacent to outlined area indicate the percentage of populations. The statistical quantitation of frequency in CD4⁻B220⁺ B cells and cell numbers for CXCR5⁺PD-1^hi T_FH cells (Lower) was analyzed.