Figure 7. IL-21, in the absence of IL-4, IFNγ and TGF-b signaling, generates T_FH cells.

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).