Baseline demographic and immune parameters of HIV-infected and uninfected Malawian adult study participants.

<p>Median and interquartile ranges (IQR) for age and immunological data are shown. Statistical significance of differences between the HIV negative group and all HIV-infected adults was tested using Fisher’s exact test (for categorical variables) or Mann Whitney <i>U</i> test (for continuous variables). † Data on <i>S. pneumoniae</i> carriage are missing for some patients for technical reasons.</p

Baseline data for all volunteers in study

As reported in Table

Effect of restoration/addition of Tregs (CD25^hi) cells back into CD25^hi cell depleted MNC samples on proliferative responses by pneumococcal specific CD4⁺ T cells.

<p>Tonsil MNCs were depleted of CD25^hi cells and left or CD25^hi cells added back at the original proportion (∼10%) or at three fold the original proportion (i.e. 30%). Cells were obtained from individuals (<b>a</b>) >16years (<i>n</i> = 5) and stimulated with SPNT, (<b>b</b>) <17 years (<i>n</i> = 3) and stimulated with SPNT or individuals (<b>c</b>) >16 years and stimulated with flu (<i>n</i> = 5). Percentage of proliferating CD4⁺ cells are shown for each individual (open circles) as well as mean (black bars) proliferation in undepleted or CD25^hi depleted or CD25^hi depleted with CD25^hi added back at the original proportion or CD25^hi depleted with CD25^hi added back at three times the original proportion. (*  = <i>p</i> <0.05).</p

Correlation between circulating IgM memory B-cells and CD4⁺ T-cells.

<p>(<b>A</b>) Scatter plots showing absolute IgM memory B-cell numbers & CD4⁺ T-cell counts and (<b>B</b>) % IgM memory B-cells & CD4⁺ T-cells measured using flow cytometry. The association between the two parameters (IgM memory B-cells and CD4 counts) was determined by Spearman’s correlation using Stata (Version 10). Each point represents the result from one subject. r_s is the Spearman rank correlation coefficient.</p

Experiment_Figure_3

As reported in Figure 3 - the effect of pre-existing (in vivo) alveolar carbon exposure with subsequent (in vitro) wood smoke particulate exposur

IgM memory B-cells are depleted while isotype-switched memory B-cells are maintained in HIV-infected Malawian adults.

<p>(A) IgM memory B-cells (CD19⁺ CD27⁺ IgD⁺) absolute numbers, (B) percentages and (C) isotype-switched memory B-cell (CD19⁺ CD27⁺ IgD⁻) numbers, (D) percentages in the circulation of HIV⁻ controls (n = 44), HIV⁺ >350 (n = 16) and HIV⁺ ≤350 (n = 24) adult. Horizontal bars represent medium values. Statistical significance of differences between groups was assessed using the Mann Whitney <i>U</i> test.</p

Anti-pneumococcal CD4 T cells proliferative responses in adult tonsils and blood during <i>in vitro</i> pneumococcal peptide antigen challenge.

<p>(<b>a</b>) A typical FACS plot for CFSE staining within CD4⁺ cells post simulation with flu or SPNT compared to unstimulated (media alone) cells. (<b>b</b>) Purified tonsil MNCs (<i>n</i> = 8) were stimulated over 9 days with flu or recombinant Ply peptides, or D39 bacterial SPNT or media. CD4⁺ cells identified by FACS staining were assessed for their proliferative responses by CFSE staining. Percent of proliferating CD4⁺ cells post flu, Ply or SPNT stimulation were all significantly higher than media control (*  = <i>p</i> <0.05). (<b>c</b>) Greater proliferative responses to pneumococcal peptides by tonsil compared to blood CD4⁺ cells. Tonsil MNCs and PBMCs from the same individuals (<i>n</i> = 5), were purified and stimulated <i>in vitro</i> with flu, Ply or SPNT and CD4⁺ cell proliferation assessed after 9 days. No significant difference was observed between tonsil (open bars) and blood (filled bars) CD4⁺ responses to flu but were significant to SPNT (*  =  <i>p</i> <0.05) and almost significant (#  =  <i>p</i> 0.06) for Ply. Values were calculated with the background (i.e. media alone) proliferation subtracted. Error bars show the SEM.</p

Detection of Ply specific CD127^low/- FoxP3⁺ CD4⁺ Treg cells in CD25 enriched tonsil and blood MNC.

<p>CD25 enriched MNC were stained with anti- CD127 and anti FoxP3 to allow identification of Tregs and with (<b>a</b>) strepavidin-PE, (<b>b</b>) negative control Ply tetramer-PE and (<b>c</b>) Ply tetramer-PE and analysed by FACS. A typical example of a FACS plot is shown. While the CD127^low/- FoxP3⁺ CD4⁺ Treg cells show low level staining at 0.0% with strepavidn-PE (<b>a</b>) and negative control Ply-tetramer at 0.05% (<b>b</b>), a significantly higher percentage of Treg cells are bound by Ply-tetramers at 1.96%(<b>c</b>).</p

Experiment_Figure_4

As reported in Figure 4 - the effect of in vitro exposure of alveolar macrophages to BSO 0.2mM as measured by total and oxidised glutathione concentrations in the cell lysat

Mucosal and peripheral blood CD4 T cell responses to pneumococcal, influenza and MTB-PPD antigens in Malawian children and adults.

<p>Mucosal CD4 T cell proliferation in response to pneumoCCS n = 26 (<b>a</b>) and Ply-CCS n = 24 (<b>b</b>) influenza n = 25 (<b>c</b>) and MTB n = 25 (<b>d</b>) in children and adults. Circulating CD4 T cell proliferation in response to pneumoCCS n = 66 (<b>e</b>), Ply-CCS n = 67 (<b>f</b>), influenza n = 65 (<b>g</b>) and MTB-PPD n = 58 (<b>h</b>) in children and adults. Data was analyzed by nonlinear regression modeling, fitted with second order polynomial.</p