CFU recovered in BAL cell culture supernatant following OPKA.

†<p>Adjusted for clustering within participants.</p

rhIL-17A stimulated alveolar macrophages show increased killing of opsonised pneumococci.

<p>Human alveolar macrophages from non-colonised volunteers were exposed to opsonised D39 pneumococci in the presence of rhIL-17A or a vehicle control (v/v). Percentage changes in CFU following rhIL-17A (<i>y</i>-axis) treatment are shown as increase or decrease relative to vehicle, which was set at 0% for comparison (<i>x</i>-axis). * = <i>p</i><0.05, ** = <i>p</i><0.01 vs 12.5 ng/ml of rhIL-17A.</p

Pneumococcal carriage increases the percentage of pneumococcal-responding IL-17A⁺ CD4⁺ memory T-cells in BAL and blood.

<p><b>A</b> BAL CD4⁺ memory T-cell and <b>B</b> Blood CD4⁺ memory T-cell responses from volunteers without (−, BAL n = 9, blood n = 8) or with (+, BAL n = 11, blood n = 10) experimental pneumococcal carriage (indicated on <i>x</i>-axis). Cells were stimulated <i>ex vivo</i> with or without (not shown) influenza or pneumococcal proteins as indicated. TNF⁺, IL-17A⁺ or IFNγ⁺ responses shown are background subtracted and measured as % of total CD4⁺ memory T-cells. Note the change in scale for the <i>y</i>-axis for each cytokine ns = not significant, * = <i>p</i><0.05; ** = <i>p</i><0.01 vs non-colonised volunteers.</p

BAL cells stimulated with pneumococci <i>ex vivo</i> elicit large quantities of IL-17A.

<p>BAL cells from non-colonised (open bars, n = 6) or colonised (closed bars, n = 7, 1x 23F, 6x6B colonised) volunteers were stimulated with pneumococci (6B) or left untreated (NS) for 20 hours. Following stimulation <b>A</b> IL-17A and <b>B</b> TNF were measured in pg/ml (mean±SD) in cell culture supernatant by ELISA. * = <i>p</i><0.05.</p

Pneumococcal carriage increases the percentage of IL-17A⁺/TNF⁺ CD4⁺ memory T-cells in BAL.

<p>Sub-population analysis of 6B stimulated BAL CD4⁺ memory T-cell responses from <b>A</b> non-colonised (n = 9) and <b>B</b> colonised volunteers (n = 11). The percentage of triple, double or single CD4⁺ memory T-cells producing either TNF, IL-17A and/or IFNγ (<i>x</i>-axis) are shown. Pneumococcal-responding double-producing (IL-17A⁺/TNF⁺) CD4⁺ memory T-cells constitute the dominant phenotype following pneumococcal colonisation. Responses shown are background subtracted. * = <i>p</i><0.05; ** = <i>p</i><0.01 vs non-colonised in <b>A</b>.</p

Study volunteer and colonisation details.

<p> <b>N.D. – Not determined.</b></p><p> <b>N/A – Not applicable.</b></p>†<p> <b>- Values given are background (media control) subtracted from cells stimulated with HK-6B.</b></p

Cellular profile of BAL collected from the pneumococcal non-colonised and colonised groups.

<p>Cellular profile of BAL collected from the pneumococcal non-colonised and colonised groups.</p

Nasal wash IgG response to pneumococcal purified protein antigens.

<p>ELISAs were performed using pneumococcal antigens PspA (A), PspC (B), PdB (C) and PsaA (D) to determine specific IgG in subject's pre and post 23F (<i>n</i> = 7) or 6B (<i>n</i> = 8) pneumococcal challenge (x-axis). Values shown are the mean antibody concentration (of triplicates). Antibodies to PspA and PspC are expressed in µg/ml and antibodies to PdB and PsaA are expressed in arbitrary units/ml (y-axis). *represents statistical significance between pre- and post-challenge antibody levels.</p

Frequency of antigen specific CD4 T cell responses before and after 23F or 6B challenge.

<p>Blood (A and C) and BAL (B and D) responses are shown pre and post 23F (A and B) or 6B (C and D) challenge. The proportion of flu and pneumococcal specific (x-axis) CD4+CD45RO+ T cells producing either TNF ± IFNγ ± IL-17 (or combinations thereof) (y-axis) were analysed by multi-parameter flow cytometry. Shown are mean values ± SD of at least 6 paired samples.</p

Experimental human intra-nasal 23F and 6B pneumococcal challenge study design.

<p>(A) 23F challenge and (B) 6B challenge. NW, blood and BAL were obtained from each volunteer prior to (open symbols) and following (closed symbols) challenge with 23F (Arrows in upper panel <i>n</i> = 8) or 6B (Arrow in lower panel <i>n</i> = 11).</p