Figure 2
(a, b) Flow cytometry analysis was conducted to determine the location of CD19+ B cells after s.c./i.n. and s.c./s.c. immunization with ScpA/CAF01 (n=6). Lung cells were examined for labeling with iv injected FITC-conjugated anti-CD45.2 mAb 14 and 42 days post immunization. (a) The CD19+ B-cell staining is indicated. Dot plot shows IgA expression in iv.CD45+ (intravascular) and iv.CD45− (parenchymal) cells. Figure shows concatenated data within groups (‘digital pooling’) from day 14. (b) Boxplot (min/max; median w. s.e.m.) showing number of IgA+CD19+ B cells within the iv.CD45− subset at day 14 after s.c./s.c. or s.c./i.n. immunization. Statistical analysis: one-way ANOVA and Tukey’s post-test *P<0.05. (c–f). IC Flow cytometric analysis of cytokine expression in CD4 T cells after s.c./i.n. and s.c./s.c. immunization with ScpA/CAF01. (c) Gating strategy for quantification and localization of lung-associated CD4 T cells in immunized mice (exemplified by day 14). Data are concatenated within groups. Lung cells were examined for labeling with iv-injected FITC-conjugated anti-CD45.2 mAb 0, 3, 7, and 14 days post immunization by IC Flow cytometry analysis after stimulation with ScpA. Dot plot shows IL-17 or IFN-γ expression in iv.CD45+ (intravascular) and iv.CD45− (parenchymal) cells. (d) Number of CD4+ T cells in s.c./s.c. (•) and s.c./i.n. (○) immunized mice (n=8). Statistical analysis: unpaired T tests at each individual time point *P<0.05, **P<0.01. (e) Number of cyt+CD44+CD4+ T cells (any cytokine) in s.c./s.c. (•) and s.c./i.n. (○) immunized mice (n=8). Statistical analysis: unpaired T tests at each individual time point ***P<0.001. (f) % IL-17- or IFN-γ-expressing cells in the iv.CD45− CD44+CD4+ T cell population, in s.c./i.n. immunized mice. Statistical analysis: unpaired T tests at each individual time point *P<0.05, **P<0.01. (g) Histogram depicting expression of CCR6 on Cyt-iv.CD45-CD44+CD4+ T cells (dashed) IL17+iv.CD45-CD44+CD4+ T cells (transparent) and IFN-γ+iv.CD45-CD44+CD4+ T cells (dark) after s.c./i.n. immunization (exemplified by day 14, data concatenated within groups). (h–j) Number of IL-17+CD44+CD4+ T cells in i.n./i.n., s.c./i.n., and s.c./s.c. immunized mice (n=6) in lung parenchyma (iv.CD45−; h), lung draining tracheobronchial and mediastinal LNs (TLN+MLN; i) and parenteral immunization draining ILN (j) 14 days post booster immunization. Statistical analysis: one-way ANOVA and Tukey’s post-test *P<0.05, **P<0.01. ANOVA, analysis of variance; FITC, fluorescein isothiocyanate; IC, intra cellular; IFN, interferon; IL, interleukin; ILN, inguinal lymph node; i.n., intranasal; iv, intravenously; LN, lymph node; mAb, monoclonal antibody; MLN, mediastinal lymph node; s.c., subcutaneous; ScpA, streptococcal C5a peptidase; TLN, tracheobronchial lymph nodes.
