Figure 5
(a) IC Flow cytometry analysis of parenchymal T cells 42 days after s.c./i.n. immunization with ScpA/CAF01. Mice were intravascular stained by iv injection of FITC-conjugated iv.CD45, 3 min before being killed. CD44+CD4+ T cell populations were subdivided according to their iv.CD45 staining and expression of IL-17. iv.CD45− and iv.CD45+ IL17+CD44+CD4+ T cells were further subdivided according to their CD62L vs. CD69 expression. (b, c) Mice immunized either s.c./s.c. (•), s.c./i.n. (○) or i.n./i.n. (•) received a second boost of ScpA/CAF01 administered i.n. 70 days later and mice were killed before and 7 and 14 days after the second booster immunization. (b) IgA end point titers were determined in supernatants of perfused and homogenized lungs from individual mice. The amount of secreted antibody was analyzed by ELISA. (c) Lung cells from individual mice (n=4) were harvested and stimulated with ScpA for 72 h and the IL-17 levels determined by MSD analysis of culture supernatants. Statistical analysis: one-way ANOVA using Tukey’s post-test. (d, e) In a similar study, half of the mice that received the second boost (n=12) received FTY720 treatment by an oral stomach tube, from 1 day before until termination of the experiment day 7 after a second booster immunization. The mice were intravascular stained by iv injection of FITC-conjugated iv.CD45 and (d) Th17 cells and (e) IgA+ B cells in the parenchyma were analyzed by IC flow cytometry. Statistical analysis: one-way ANOVA using Tukey’s post-test. *P<0.05. ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; IC, intra cellular; IL, interleukin; i.n., intranasal; MSD, Meso Scale Discovery; s.c., subcutaneous; ScpA, streptococcal C5a peptidase.
