Fig. 5

Accumulation of Th-1 cells in aged mice. Single cell suspensions from either lacrimal gland (LG), and cervical lymph nodes (CLN) were prepared for flow cytometry and stained with CD4 followed by IFN-γ or IL-17A intracellular staining. A separate group of mice was used for conjunctiva (CJ), LG and CLN flow cytometry for evaluation of CXCR3  and IFN-γ and CCR6  and IL-17A frequencies. a Representative dot plots showing frequency of CD4⁺, CD4⁺IFN-γ⁺, and IFN-γ⁺ or CD4⁺IL17A⁺ and IL-17A⁺ cells gated from alive single CD45⁺ cells (n = eight to nine/age). b Accumulative data showing the frequency of CD4⁺IFN-γ⁺ or CD4⁺IL17A⁺ cells gated on live single CD45⁺ cells. Means ± SEM, n = eight to nine/age; biological replicates from four independent experiments were averaged. c Th-1/Th-17 ratio with aging in LG and CLN. n = eight to nine/age; biological replicates from four independent experiments were averaged. d Histograms showing positive CXCR3 and IFN-γ and CCR6 and IL-17 staining in CLN compared to fluorescence minus one (FMO) controls. Representative dot plots of flow cytometry analysis of conjunctiva, CLN and LG from young and aged B6 mice stained with CD4, followed by CXCR3, and IFN-γ or CCR6 and IL-17A staining (n = four). Numbers are percentiles of positive cells gated on CD4⁺T cells. e Accumulative data showing the frequency of CXCR3⁺IFN-γ⁺ and CCR6⁺IL-17⁺ cells gated on live CD45⁺CD4⁺ T cells. Means ± SEM, n = four mice/age; biological replicates from two independent experiments were averaged. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 8 W vs. 24 M age comparison within each cell type. Two-way ANOVA followed by Sidak’s multiple comparison tests