Cytokine and Clinical Data.xls

Plasma Cytokine concentrations during an acute malaria episode and clinical data from a small nested sub-study of children in Nagongera, Uganda. Comparisons across age and by parasite density. <br

Whole-transcriptome analysis of atypical and classical MBCs from parasitemic, but asymptomatic, subjects.

<p>Heat map rows represent individual genes, and columns within each MBC grouping represent distinct individuals. Representative genes are depicted based on gene ontology associations with specific functional categories. Average fold difference in expression between atMBCs and classical MBCs pairs is shown, with values in parentheses representing lower expression in atMBCs and all other values representing higher expression in atMBCs. The red and blue heat map is a graphical depiction of the significant differential regulation of each gene in nonclassical memory B cell subsets in the context of HIV infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref018" target="_blank">18</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref019" target="_blank">19</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref045" target="_blank">45</a>], CVID [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref022" target="_blank">22</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref023" target="_blank">23</a>], SLE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref021" target="_blank">21</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref025" target="_blank">25</a>], HCV infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref026" target="_blank">26</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref046" target="_blank">46</a>], and the tonsil [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref027" target="_blank">27</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref028" target="_blank">28</a>], as well as previously reported expression in atMBCs in the context of malaria [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref011" target="_blank">11</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004894#ppat.1004894.ref031" target="_blank">31</a>]. Direction of expression change was assigned based on previously published transcriptome and protein expression profiles as described in the methods, with red representing higher expression in nonclassical subsets, blue representing lower expression, and white representing the lack of any reported change.</p

Recall antibody secretion by different B cell subsets.

<p>Sorted FCRL5⁺ and FCRL5^-, atypical (CD20⁺CD21^-CD27^-IgG⁺) and classical (CD20⁺CD21⁺CD27⁺IgG⁺) MBCs were stimulated for 4 days with CpG, F(ab’)₂ anti-IgG, and autologous T cells. IgG-secreting cells were detected by IgG ELISpot and are reported as the number of IgG secreting cells per 1000 cells sorted on day 0. ASC, antibody-secreting cells. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05.</p

Higher exposure to <i>P</i>. <i>falciparum</i> is associated with a higher proportion of atMBCs that express FCRL5.

<p>The proportion of FCRL5⁺ atypical MBCs from individuals living in high exposure (n = 16; Nagongera, Uganda; annual entomologic inoculation rate = 310) vs. moderate exposure (n = 9; Walukuba, Uganda; annual entomologic inoculation rate = 2.8) is shown, p = 0.004. Statistical significance was determined using the Wilcoxon rank-sum test. Multivariate linear regression, including age of subject, yielded similar results.</p

Spontaneous IgG secretion by different B cell subsets.

<p>(<b>A</b>) Sorted transitional cells (CD19⁺CD10⁺), CD20⁺ atMBCs (IgG⁺CD21^-CD27^-CD19⁺), classical MBCs (IgG⁺CD21⁺CD27⁺CD19⁺), and CD27^- plasmablasts (CD20^-IgG⁺CD21^-CD27^-CD19⁺) were cultured on anti-IgG ELISpot plates for 18 h without additional stimulation. (<b>B</b>) Gating strategy and frequencies of CD38^hi cells in the above plasmablast gating strategy.</p

Phenotypic characterization of surface proteins on IgG⁺ atypical MBCs.

<p>(<b>A</b>) Surface expression, expressed as median fluorescence intensity (MFI), of CD85d, CD120b, CD360, CD11c, and IgG (BCR) on IgG⁺ atMBCs and IgG⁺ classical MBCs. Lines between symbols denote MBC subsets from the same subject. Wedges represent means. (<b>B</b>) Labeling of SVT2 mouse fibroblast cell lines that express full-length human <i>FCRL4</i> or <i>FCRL5</i> protein by monoclonal antibodies 2A6, 1A3, and 7D11. (<b>C</b>) Labeling of human atMBCs with monoclonal antibodies 2A6, 1A3, and 7D11. (<b>D</b>) Isotype-subtracted MFI of FCRL family member expression (“Net MFI”) on atypical and classical MBCs from highly <i>P</i>. <i>falciparum</i>-exposed individuals. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01</p

MOESM2 of Reductions in malaria in pregnancy and adverse birth outcomes following indoor residual spraying of insecticide in Uganda

Additional file 2. Sensitivity analysis: estimation of effect of any IRS vs no IRS protection on outcomes measured at birth

Magnitude of malaria-specific CD4⁺ T cell responses and protection from symptomatic malaria.

<p>Note: HR: Hazard Ratio; IRR: incidence rate ratio; PRR: prevalence rate ratio. Numbered rows refer to cell populations described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003864#ppat-1003864-g002" target="_blank">Figure 2</a>. Associations in row 3 are not applicable because these responses were undetectable.</p><p>Multivariate models controlled for duration since last malaria infection. Similar results were obtained when controlling for cumulative episodes over prior 3 years and for the presence or absence of parasitemia.</p

T cell responses to malaria-infected red blood cells using multiparameter flow cytometry.

<p>A. Gating strategy to identify live CD3⁺ γδ⁻ T cells. B. Intracellular cytokine assay demonstrating the T cell response of one representative malaria-exposed child to Pf-infected RBC (iRBC; bottom row), with negative controls (uRBC and media) and positive control (PMA/Io) shown in rows above. Shown are CD8 (first column) and CD4 (right 3 columns) production of IFNγ (y-axis, columns 1–3), TNFα (x-axis, columns 1–2; y-axis, column 4), and IL-10 (x-axis, column 3–4). C. The overall malaria-specific CD4⁺ T cell response (left column) is followed by the overall frequency of CD4⁺ T cells producing IFNγ, IL-10, and TNFα in all participants (n = 78, horizontal black lines indicate the median response for each group, *** <i>P</i><0.001, Wilcoxon Rank-Sum).</p

Prior malaria incidence influences function of malaria-specific CD4⁺ T cell response.

<p>A. The overall malaria-specific CD4⁺ T cell response (left column) is followed by the overall frequency of CD4⁺ T cells producing IFNγ, IL-10, and TNFα stratified by prior malaria incidence. Blue dots represent responses from children with lower prior malaria incidence (<2 episodes ppy, n = 10) and red dots represent responses from children with higher prior malaria incidence (≥2 episodes ppy, n = 68,* <i>P</i><0.05, *** <i>P</i><0.001, Wilcoxon Rank-Sum. Horizontal black lines indicate the median response for each group). Median frequencies of cytokine producing cells were similar in children with ≥2–5 and >5 episodes ppy (data not shown). B–C. Boolean gating of malaria-specific CD4⁺ T cells reveals 7 distinct cytokine-producing populations. Shown are the absolute frequency (B) and the relative proportion (C) of each individual combination of IFNγ, IL-10, or TNF-producing cells. Blue dots again represent responses from children with <2 prior episodes ppy, and red dots represent responses from children with ≥2 episodes ppy (* <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, Wilcoxon Rank-Sum. Horizontal black lines indicate the median response for each group). For pie charts, blue arcs represent total proportion of CD4⁺ T cells producing TNFα; red arcs represent total proportion of CD4⁺ T cells producing IL-10; and green arcs represent total proportion of CD4⁺ T cells producing IFNγ. The proportion of IFNγ⁻/IL-10⁺/TNFα⁻ (population 3) producing cells is <0.01% of the total malaria-specific response, and thus does not have a visible corresponding pie slice.</p