C57BL/6 mice were immunized with KLH in CFA. 7 days later CD4⁺CD44^hiCXCR5⁺ BTLA⁺ (T_FH) cells were sorted and restimulated with anti-CD3 for 4 hours for gene profiling analysis (A) and for real-time PCR analysis (B). OT-II T cells differentiated under various conditions were restimulated with anti-CD3 for 4 hours and were analyzed together with T_FH cells. (A). A distinct gene expression profile of T_FH cells from TH1, TH2, and TH17 cells. Hierarchical clustering and PCA were applied to the expression levels of 8350 probe-sets showing differential expressions among the four types of cells (FDR = 0.1, corresponding to an unadjusted p-value = 0.033 from one-way ANOVA). (B). Real-time RT-PCR analysis of TH specific genes. (C-D). CD4⁺CD44^hiCXCR5⁺ BTLA⁺ and CD4⁺CD44^hiCXCR5^- cells were restimulated with anti-CD3/anti-CD28 for 48 hours for cytokine measurement by ELISA (C) and with PMA/Ion for 6 hours for detection of IL-17 and IFNγ expression by intracellular cytokine staining (D). The data represent one of two independent experiments with similar results.

(A) B7h germline knockout mice and their controls (WT, 3 mice per group) were immunized with KLH in CFA. Seven days after the immunization, experimental mice were sacrificed and spleen cells from immunized mice were stimulated in 96-well plates as triplicates with the indicated concentration of KLH. Effector cytokines (IFN-γ and IL-21) were measured after 4 days of treatment. The germinal center B cells and T_FH cells were analyzed. P values were calculated with the t-test by comparing the CXCR5+ cells and B220/PNA+ cells between wild-type and B7h KO mice and are indicated as followed: * P < 0.005; #, P < 0.001. Numbers in dot plot quadrants represent the percentages. (B). Splenic B220+ B cells from B7h germline KO (B7hKO), B7h B-cell conditional knockout (B7h BKO, cre+) and the cre- controls were analyzed for B7h expression. (C-E). Wild-type (WT) and B7h BKO (3 mice per group) were immunized with KLH in CFA. Seven days after the immunization, experimental mice were sacrificed and analyzed as in A. P values were calculated with the t-test by comparing the CXCR5+ cells and B220/PNA+ cells between wild-type and B7h BKO mice and are indicated as followed: ** P < 0.001; ##, P < 0.001. (C). (D). Anti-KLH antibodies (IgM and IgG) were measured in the sera by ELISA. The sera from WT and B7h BKO mice were subject to a 3-fold serial dilution, and the concentrations of KLH-specific IgM and IgG were analyzed by ELISA and averaged for each group. (E). GC in the spleens of KLH-immunized WT and B7h BKO mice were identified by PNA staining (brown). T and B cells were identified by staining with anti-CD4 (red) and anti-B220 (blue) Abs. The data represent at least three independent experiments with consistent results.

IL-6 KO (A) or STAT3 T-cell specific KO mice (C) and their appropriate controls (WT, 3 mice per group) were immunized subcutaneously with KLH emulsified in CFA. Seven days after the immunization, experimental mice were sacrificed and T_FH cells and the germinal center B cells were analyzed. Numbers in dot plot quadrants represent the percentages. (B) C57BL/6 mice were immunized with KLH in CFA. 7 days later, CD4⁺CD44^hiCXCR5⁺ and CD4⁺CD44^hiCXCR5^- cells were sorted and restimulated with anti-CD3 and anti-CD28 with or without IL-6 or IL-21 for 48 hours. Proliferation was assayed by adding [³H]-thymidine to the culture for the last 8 h. P values were calculated with the t-test by comparing the CXCR5+ cells proliferation in the absence and in the presence of IL6 (*p<0.005) or in the absence and in the presence of IL21 (*p<0.005). CD4⁺CD44^hiCXCR5⁺ and CD4⁺CD44^hiCXCR5^- cells were restimulated with anti-CD3 and anti-CD28 for 24 hours and stained with Annexin V-FITC. The experiments were performed two times with consistent results.

A-F, FACS-sorted CD62^hiCD44^loCD25^negCD4⁺ T cells from CD45.1⁺ OT-II mice were cultured with irradiated splenic APC plus OVA_323-339 peptide under TH0, T_FH (IL-21 plus antibodies to IL-4, IFNγ and TGFβ) or TH17 condition for 5 days. After 5 days, CD4 T cells were restimulated with anti-CD3 for 4 hours for real-time PCR analysis (A) or for 24 hours for cytokine measurement by ELISA (B). (C-F). Five days after in vitro differentiation, cells were adoptively transferred into CD45.2⁺ congenic mice (n=3-4) before the recipient mice were subcutaneously immunized with OVA in IFA. A group of mice that did not receive T cells was used as a control (No transfer). Seven days after immunzation, lymphoid cells from the draining lymph nodes of recipient mice were isolated and T_FH cells and germinal center B cells were analyzed (D). Numbers in the boxes represent the percentages. (E). The sera from the recipient mice were subject to a 3-fold serial dilution, and the concentrations of OVA-specific IgM and IgG were analyzed by ELISA and averaged for each group. (F). Germinal center in the spleens of the recipient mice were identified by PNA staining (brown). The results are a representative of multiple mice of two independent experiments with similar results. (G). CXCR5⁺CD44^hi cells were sorted from CD45.1 mice on day 7 after immunization with KLH and transferred to CD45.2 (n=3) recipient mice following immunization with KLH in CFA. A group of mice that did not receive T cells was used as a control (No transfer). Seven days after immunization, germinal center in the spleens of the recipient mice were identified by PNA staining (brown).

(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.

IL-21+/+, IL-21+/- and IL-21-/- mice (3 mice per group) were immunized subcutaneously with KLH emulsified in CFA. Seven days after the immunization, experimental mice were sacrificed and T_FH cells (A) and the germinal center B cells (B) were analyzed. Numbers in dot plot quadrants represent the percentages. Germinal centers were determined by immunohistochemical analysis (C). Spleen cells from immunized mice were stimulated in 96-well plates as triplicates with the indicated concentration of KLH peptide. Proliferation was assayed after 3 days of treatment by adding [³H]thymidine to the culture for the last 8 h. IFN-γ was measured after 4 days of treatment. The experiments were repeated twice with consistent results.

Rag1-/- reconstituted with WT and RORαsg/sg/RORγ KO bone marrow cells (A) or IL-17 KO and IL-17F KO mice and their controls (C) (3 mice per group) were immunized subcutaneously with KLH in CFA. Seven days after the immunization, experimental mice were sacrificed and the T_FH cells were determined. Numbers in dot plot quadrants represent the percentages. (B). C57BL/6 mice were immunized with KLH emulsified in CFA with 100 μg of isotype control antibodies or anti-TGFβ blocking Abs (3 mice per group). 7 days later, experimental mice were sacrificed and splenic T_FH cells and germinal center B cells were analyzed. Splenocytes were restimulated with KLH for overnight and the production of IL-17 and IFNγ was analyzed in CD4⁺ gate by intracellular cytokine staining. The results represent one of three individuals with similar results.