Increased T_FH cells in DCBlimp-1^ko mice in vivo and in vitro. (A) 6–10-wk-old control and DCBlimp-1^ko mice were sacrificed and T_FH (Lin (B220/CD11b/Gr-1)⁻, TCR-β/CD4/CXCR5/PD-1⁺) were analyzed by flow cytometry. Total T_FH cell number was calculated and graphed on the right. Each dot represents an individual mouse and horizontal bars indicate means of three independent experiments. (B) In vitro differentiation of T_FH cells. Naive CD4⁺ T cells and splenic DCs were co-cultured and activated as described in Materials and methods. After 4 d, cells were harvested and the frequency of T_FH cells was analyzed by flow cytometry. The top panel displays representative flow cytometry and the bottom is a summarized table (five independent experiments, n = 9). Total RNA was prepared from purified CD4⁺ T cells from each culture conditions, and the Bcl-6 level was measured by qPCR. The level of Bcl-6 of in vivo T_FH cells (T_FH cells sorted 12 d after NP immunization) was used as a positive control. Mean ± SD of three independent experiments is shown (n = 6).