Figure 2. Generation of T_FH cells is independent of TH1 and TH2 lineages.

(A). CD4+ T cells from CD45.2/OT-II mice were transferred into C57BL/6 (CD45.1) mice which were subsequently divided into 2 groups (3 mice per group). Mice were immunized subcutaneously with Ova protein emulsified in CFA and treated with a 300 μg of control rat Ig or anti-IFNγand anti-IL-4 mAbs. Seven days after the immunization, experimental mice were sacrificed and splenic CD45.1+ and CD45.2+ CD4 cells were stained with biotinylated anti-CXCR5 mAb, followed by APC-labeled streptavidin. Numbers in dot plot quadrants represent the percentages. CD44hiCXCR5⁺ and CD44hiCXCR5^- cells from immunized mice were purified and real-time RT-PCR analysis of T_FH specific genes were performed. (B-C). IL-4 KO, IFNγ KO (B), STAT6 KO (C) and STAT4 KO (D) and their appropriate controls (WT, 3 mice per group) were immunized with KLH emulsified in CFA. Seven days after the immunization, experimental mice were sacrificed and the germinal center B cells were determined by staining with FITC-labeled PNA and PerCP-labeled anti-B220 mAb. The T_FH cells were analyzed by staining with PerCP-labeled anti-CD4 mAb and biotinylated anti-CXCR5 mAb, followed by APC-labeled streptavidin. Numbers in dot plot quadrants represent the percentages. The experiments were repeated three times with consistent results.