Fig. 4: CCR2-dependent monocytes are necessary for IFNγ production by CD4+ T cells after Ms immunization.

C57BL/6 (WT) and CCR2-KO mice were immunized i.d. with Ms, Nb, Ca, or PBS as a control. LN cell suspensions were examined by flow cytometry on day 2 (a, b) or 5 (c–k) as indicated. a, b Cellular composition (a) and Ag median fluorescence intensity (MFI) (b) of LN Ag+ populations after immunization with AF488-labeled Ms, Nb or Ca. Monocyte MFI values for CCR2-KO mice are not shown due to low monocyte counts. Graphs show mean ± SEM for groups of n = 8 (4 female + 4 male, Ms WT and Ms KO), 8 male (Nb WT, Ca WT and Ca KO), or 10 male (Nb KO) mice over two independent experiments. Statistical significance was assessed using a two-sided Student’s t test for each cell population. NS: not significant, ***p < 0.001, ****p < 0.0001. Exact values are shown for 0.05 > p > 0.01. c–k CD4+ T cell responses as assessed by flow cytometry. Intracellular cytokine staining was after in vitro restimulation, transcription factor (TF) expression was assessed without in vitro restimulation. c Numbers of CD4+ T cells per LN and (d) frequencies of CD44+ cells in the CD4+ T cell population. Bar graphs show mean values ± SEM, each symbol corresponds to one mouse. For n values please refer to the legends to (f), (i), (k). e Contour plots showing IFNγ staining of CD4+ T cells from Ms-immunized mice. Data are concatenated from 5 mice. f Frequencies of total cytokine+ and Tbet+ CD4+ T cells or (g) IFNγ^hi (red gates in (e)) and Tbet^hi CD4+ T cells in Ms-immunized mice. h, j Contour plots showing IL-4 and IL-17A staining of CD4+ T cells from Nb- or Ca-immunized mice, respectively. Data are concatenated from 4 mice. i, k Frequencies of total cytokine+ and TF+ CD4+ T cells in Nb- or Ca-immunized mice. Cytokine graphs in (f, g) show mean ± SEM for groups of n = 9 (5 males + 4 females, PBS WT), 11 (6 males + 6 females, PBS KO), 10 (5 males + 5 females, Ms WT), and 12 (5 males + 7 females, Ms KO) mice over two independent experiments; the same n values also apply to the Ms bar graphs in (c, d). TF graphs show mean ± SEM for groups of n = 10 (PBS and Ms WT) or 9 (Ms KO) female mice over two independent experiments. Cytokine graphs in (i) show mean ± SEM for groups of n = 10 female mice over two independent experiments. The same n values also apply to the Nb bar graphs in (c, d). The TF graph shows mean ± SEM for n = 10 (PBS and Nb WT) or 8 (Nb KO) female mice examined over two independent experiments. Cytokine graphs in (k) show mean ± SEM for groups of n = 10 (PBS WT), 11 (PBS KO), 9 (Ca WT), or 14 (Ca KO) female mice over two independent experiments. The TF graph shows mean ± SEM for n = 8 (PBS WT and KO), 11 (WT Ca), or 10 (KO Ca) female mice over two independent experiments; the same n values also apply to the Ca bar graphs in (c, d). Statistical significance was assessed using a One-Way ANOVA with Holm-Sidak’s post-test (Ms and Nb TF) or Two-Way ANOVA with Sidak’s post-test (all remaining panels in c–k). Hash symbols refer to comparisons between PBS and immunized mice of the same genotype. Asterisks refer to comparisons between similarly immunized WT or CCR2-KO mice. NS: not significant; ^### p < 0.001; ^####,****p < 0.0001. Exact values are shown for 0.05 > p > 0.01. Source data are provided as a Source Data file.