Fig. 6. IL-10–producing Tfh cells emerge after vaccination, and IL-10, but neither T_regs nor Tfr cells, limits Tfh-dependent vaccine responses in aged mice.

(A) Plots show IA^b-NP tetramer staining in immunized and unimmunized young (3.5 months, n = 5) and aged (18 months, n = 5) IL-10GFP–FOXP3RFP dual-reporter mice. Graphs show the frequency and number of NP-specific FoxP3⁻CD4⁺ T cells that are IL-10⁺ and the number of NP-specific FoxP3⁻IL-10⁺ T cells that are CXCR5⁺PD1⁺ (means ± SEM). (B) Aged (17 months, n ≥ 7) WT C57BL/6 (isotype or anti–IL-10) and (20 months, n = 5) FoxP3⁻DTR [phosphate-buffered saline (PBS)– or DT-treated] mice were immunized with nitrophenol-keyhole limpet hemocyanin (NP-KLH) in alum. Plots display the frequency of germinal center (GC) B cells (NP-specific) gated on Fas⁺GL7⁺. Graphs show the frequency and number of B cells that are IgG1⁺NP⁺ (means ± SEM) and serum levels of immunoglobulin (Ig) specific for NP (IgG1) (means ± SEM). Data are pooled from two independent experiments. (C to E) Tfh10 cells accumulate during aging in humans. Human spleen cells from young (Y) (median, 18.8; range, 18 to 26 years; three males and five females) and old (O) (median, 62; range, 60 to 67 years; four males and four females) individuals were analyzed by flow cytometry. (C) The frequencies of CD3⁺CD4⁺CD45RO⁺FoxP3⁻ that are CXCR5⁺PD1⁺ (means ± SEM). (D and E) CD4⁺ T cells were bead-purified by negative selection; FACS-sorted memory CD4⁺ T cells (CD45RO⁺) into Tfh cells (CD25⁻CD127⁺PD1⁺CXCR5⁺), T_regs (CD25⁺CD127⁻PD1⁻CXCR5⁻), and other memory cells (CD25⁻CD127⁺PD1⁻CXCR5⁻) were either stimulated in vitro with anti-CD3/CD28 beads or unstimulated, and cytokines were analyzed by Luminex (means ± SEM). Each individual is represented by a symbol. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, Student’s t test. BSS, balanced salt solution.