A. Mice defective in IFN-γ, IL-4 or IL-17A receptor were immunized as described, then challenged with pneumococcal strain 0603. Mice with IFN-γ or IL-4 deficiency were significantly protected by WCV (P<0.001 vs. respective CT controls) whereas IL-17A receptor deficient mice were not protected (P>0.5 vs. CT). Dashed line represents the lower limit of detection of bacterial colonization. B. Expression of IL-17A from splenocytes of WCV-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone, Concanavalin A (5 µg/ml), WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice, although response to concanavalin A was similar. C. Effect of CD4+ T cell depletion upon IL-17A expression from splenocytes. Splenocytes (without or with CD4+ T cell depletion) from mice immunized with WCV were stimulated for 72 hours with medium alone or WCA after which IL-17A was measured by ELISA. IL-17A expression in splenocytes following WCA stimulation was significantly higher in the presence of CD4+ T cells compared to stimulation with medium alone or when CD4+ T cells were depleted. Repletion of CD4+ T cells restored the response. ** P<0.01 compared to cells stimulated with medium alone. D. IL-17A intracellular staining of splenocytes from WCV immunized mice. Splenocytes from WCV immunized mice were stimulated with WCA, blocked with monensin, harvested and stained for CD4+ and intracellular IL-17A as described. There is a statistically significant increase in CD4+ IL-17A positive cells following stimulation with WCA, which is not observed in the CD4- population. No increase in IL-17A positive cells could be detected in cells from unimmunized mice (data not shown). **P = 0.008 for comparison of frequency of IL-17A-positive cells in absence and presence of WCA stimulation among CD4+ cells. Data shown here are representative of three experiments, including at least 5 mice per experiment. E. Expression of IL-17A from NALT of WCV- vs. CT-immunized mice. Cultured splenocytes from mice immunized with WCV (black columns) or CT alone (white columns) were stimulated for 72 hours with medium alone or with WCA (10 µg dry weight) after which IL-17A production was measured by ELISA. Significantly more IL-17A was expressed following WCA stimulation of WCV-immunized vs. CT-immunized mice. **P<0.01 for comparison of IL-17A in WCV vs. CT-immunized mice following stimulation with WCA.

A. Effect of neutrophil depletion on WCV-induced protection from nasopharyngeal colonization. Each data point represents the density of nasopharyngeal colonization in cfu/nasal wash for each mouse. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line shows the lower limit of detection of bacterial colonization. Mice were immunized with CT or WCV as indicated; just prior to the time of challenge, mice were randomized to receive antineutrophil antibody vs. saline. Proportion of colonized mice and density of colonization was determined 7 days post challenge. WCV-immunized mice that received saline treatment were significantly better protected than WCV-immunized mice that received antineutrophil antibody, with a lower proportion of colonized mice (P = 0.025 by Fisher's Exact) and density of colonization (P = 0.05 by Mann-Whitney U). B. Correlation between neutrophil count and density of pneumococcal colonization. Neutrophil counts following neutrophil depletion were assayed at the time of sacrifice and plotted against density of colonization. There was a strong negative association between neutrophil counts and colonization density (Spearman ρ = −0.75). C. Histopathology of nasopharyngeal tissue following nasopharyngeal challenge of CT- (left panel) and WCV-immunized (right panel) mice. Seven days post pneumococcal challenge, mice were euthanized, heads stored in formalin, and H&E sections of nasopharyngeal tissue prepared and examined under light microscopy at 60× magnification. The presence of a dense neutrophilic infiltrate in the submucosa at the junction of the olfactory and respiratory epithelium was noted in WCV-immunized mice following pneumococcal nasopharyngeal challenge but not in CT-immunized mice. The two slides shown are representative of a total of 15 examined specimens (8 WCV-immunized and 7 CT controls, all at day 7 post pneumococcal challenge). Lesions like those represented here were observed in 6/8 immunized mice and 0/7 controls.

A and B. Effect of human IL-17A on surface phagocytic killing of S. pneumoniae. A. Isolated neutrophils from healthy adult volunteers were incubated with recombinant human IL-17A at the indicated concentrations and evaluated in a surface phagocytic killing assay with pneumococcal strain 0603; colonies were counted after overnight incubation at 37°C with 5% CO₂. IL-17A induces a dose-dependent enhancement of neutrophil killing of S. pneumoniae (P = 0.01 for 1 or 10 µg of IL-17A vs. no added IL-17A). B. Supernatant obtained from neutrophils after incubation with IL-17A did not have any demonstrable antipneumococcal effect, whereas washed neutrophils after incubation with IL-17A demonstrated enhanced killing. C. Effect of human IL-17A on opsonophagocytic killing of S. pneumoniae. Neutrophils purified from the peripheral blood of healthy adult volunteers were incubated with pneumococci anticapsular antibodies, complement, and a range of concentrations of IL-17A as indicated for 90 minutes, following which viable counts were obtained by plating on blood agar plates. Each line represents a different volunteer. IL-17A enhanced killing of pneumococci in a dose-dependent fashion in 6/6 subjects. *P = 0.016 by Wilcoxon matched pairs test.

A. Effect of intranasal immunization with WCV or live exposure upon density of pneumococcal colonization in C57BL/6 mice. Density of colonization in mice immunized with WCV vs. CT alone or repeatedly exposed to live pneumococcal strain 0603 vs. saline alone at various time points (n = 4 per time point) following challenge. By day 4, both the incidence and density of carriage were significantly lower in mice immunized with WCV or exposed to live pneumococcus compared to mice immunized with CT or saline, respectively. * P<0.05. The dashed line represents the lower limit of detection of bacterial colonization. B. Effect of adoptive transfer of CD4+ T cells from WCV-immunized mice on pneumococcal colonization of recipient, unimmunized RAG1^−/− mice. Each data point represents the density of nasopharyngeal colonization in cfu/ nasal wash for each mouse one week post-challenge. The horizontal bar shows the geometric mean cfu/nasal wash for each group and the dashed line represents the lower limit of detection of bacterial colonization. C57BL/6 mice were immunized with WCV or CT as indicated. Four weeks after the last immunization, CD4+ T cells were harvested from splenocytes of mice and infused into naïve, unimmunized RAG1^−/− mice; challenge of these mice and quantification of colonization was then performed. RAG1^−/− mice that received CD4+ T cells from WCV-immunized mice had significantly lower density of colonization than mice that received cells from CT-immunized mice (*** P<0.0001 by Mann-Whitney U).

Three weeks after immunization of mice (n = 90) with CT with doses of WCA ranging from 1 to 100 µg, and one week before pneumococcal challenge, blood samples were obtained and stimulated with WCA (10 µg) for 6 days, after which supernatants were collected and assayed for IL-17A concentration. The correlation between density of colonization (cfu/nasal wash) 7 days after challenge and pre-challenge IL-17A expression was evaluated. IL-17A expression was significantly correlated with density of colonization.

A. Expression of IL-17A from tonsillar mononuclear cells from children. Tonsillar cells (n = 8) were cultured as described and stimulated with WCA or WCA derived from an isogenic, pneumolysin-negative strain (WCAply-). Stimulation with WCA was associated with significantly increased IL-17A expression compared to exposure to medium alone (P = 0.008 by Wilcoxon signed rank test), whereas stimulation with WCAply- did not increase IL-17A production. B. Expression of IL-17A from peripheral blood of adults and umbilical cord blood. Peripheral blood samples from adults (healthy adult volunteers (n = 7), parturient women (n = 11) and umbilical cord blood (n = 11) were stimulated with WCA for 6 days after which IL-17A concentration was assayed by ELISA. IL-17A production was significantly greater in adults than cord blood (P<0.001 by Mann-Whitney U test).