Fig. 5.

TLR9 signaling in DCs and B cells determines T_FH cell numbers and phenotype and preferentially favors their expansion over T_FR cells. WT mice (^+/+) and mice deleted for Myd88 selectively in DCs or B cells were immunized with non–CpG-NPCGG (empty circles) or CpG-NPCGG (black circles) as in Fig. 1 and analyzed by flow cytometry 14 d later. Numbers of T_FH cells from unimmunized mice are also shown (gray circles). (A) Total numbers of T_FH cells per LN. (B) Pooled data showing frequency of T_FH cells (PD-1⁺CXCR5⁺) from WT, DC^−/−, and B^−/− mice as a percentage of activated CD4⁺ T cells (CD62L^loCD44^hi FoxP3⁻). (C) Cell surface expression of ICOS (MFI) of T_FH from the draining LNs, displayed as the fold increase relative to WT mice immunized with non–CpG-NPCGG. (D) Percentage of PD-1^hi T_FH cells, gated as in Fig. S2, in WT and MyD88 conditional KO mice. (E) Shown are representative flow cytometry plots of CD4⁺CD44⁺CD62L^loPD-1⁺CXCR5⁺ T_FH cells from draining LNs of WT mice 14 d after the indicated immunization, showing FoxP3 and CD25 expression to define T_FR cells. Numbers indicate the percentage of follicular CD4⁺ T cells that are FoxP3⁺ in individual LNs. FoxP3⁺ T_FR cells as a percentage of follicular CD4⁺ T cells at day 5 (F) or day 14 (G) postimmunization of WT littermate control, DC^−/−, and B^−/− mice with non–CpG-NPCGG and CpG-NPCGG, gated as in E. (H) Total number of T_FR cells per draining LN on day 14. (I) FoxP3⁺ regulatory T (T_reg) cells as a percentage of activated extrafollicular T cells (CD4⁺CD62L^loCXCR5⁻CD44^hiCXCR5⁻) on day 14. Symbols represent individual mice. Statistical analysis was performed as in Fig. 3. A, B, and F–I represent pooled data from two of two replicate experiments; in C, WT, DC^−/−, and B^−/− data are from three replicate experiments; and in D, WT, DC^−/−, and B^−/− data are from four replicate experiments.