Kinetics of antigen-specific CD4+ T cell expansion after C. muridarum intravaginal (i.vag.) infection.(A) C57BL/6 mice were immunized subcutaneously with 100 µg of C. muridarum peptide (RplF51–59, Aasf24–32 or PmpG-1303–311) in the presence of CFA or infected intravenously with either 1×105C. muridarum or 5×105S. typhimurium. Seven days post immunization/infection, axillary, brachial and inguinal lymph nodes were isolated from peptide-immunized mice, spleen and lymph nodes were harvested from C. muridarum or S. Typhimurium infected mice and endogenous RplF, Aasf or PmpG-1-specific CD4 T cells were detected with RplF51–59:I-Ab, Aasf24–32:I-Ab or PmpG-1303–311:I-Ab tetramers, respectively. FACS plots showing representative results of tetramer staining after fluorescent microbeads enrichment. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells except CD8+ T cells (bottom row), which were gated on CD11b−CD11c−F4/80−B220−CD3+CD8+ cells. (B–D) C57BL/6 mice were infected i.vag. with 1×105C. muridarum. (B) At various time points post infection, bacteria burden at lower genital tract was measured by vaginal swabs. (C) Endogenous PmpG-1-specific CD4 T cells in the spleen, draining lymph nodes and non-draining lymph nodes were detected by PmpG-1303–311:I-Ab tetramers. FACS plots showing representative results of tetramer staining after magnetic enrichment at day 0, 7, 14 and 21 days post infection. All plots were pre-gated on CD11b−CD11c−F4/80−B220−CD3+CD4+ cells. (D) Total cell number of endogenous PmpG-1-specific CD4 T cells calculated based on FACS analysis. Data shown are representative results from two independent experiments with three to five mice per time point. Bar graphs show mean number ± SEM.
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