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

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

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

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

CD4⁺ T cell proliferation impaired in setting of heavy prior exposure.

<p>A. The proliferation fold change (fraction of CFSE-lo cells following PfSE stimulation vs uRBC stimulation) is significantly reduced in children with higher prior malaria exposure (≥2 episodes ppy, n = 33) vs children with low malaria exposure (<2 episodes ppy, n = 9, <i>P</i> = 0.007, Wilcoxon Rank Sum. Horizontal lines show medians for each group with 95% CI). B. Impact of IL-10 blockade on CD4⁺ T cell proliferation following PfSE stimulation in one representative subject. The left panel shows CFSE dilution following PfSE stimulation with addition of isotype control, and the right panel shows CFSE dilution following PfSE stimulation with addition of anti IL-10 receptor α blocking antibody. C. Change in the percent of CD4⁺ T cells divided following isotype control vs anti IL-10 receptor α blocking antibody in a subset of 9 children from whom additional cells were available (fold change 1.7, <i>P</i> = 0.01).</p

CD4⁺ T cell functions and relationship with recent and cumulative malaria infection.

<p>The frequencies of CD4⁺ T cells producing any IL-10 (A) and any IFNγ (B) are inversely associated with days since last malaria episode (Spearman's <i>Rho</i> = −0.39, <i>P</i><0.001; <i>Rho</i> = −0.23, <i>P</i> = 0.046, respectively). Frequencies of CD4⁺ T cells producing any TNFα (C) are positively correlated with days since last malaria episode infection (Spearman's <i>Rho</i> = 0.23, <i>P</i> = 0.041). Frequencies of IFNγ⁺/IL-10⁺/TNFα⁻ CD4⁺ T cells are inversely associated with days since last malaria episode (D, Spearman's <i>Rho</i> = −0.39, <i>P</i><0.001) and positively associated with the cumulative number of episodes in the prior 3 years (E, Spearman's <i>Rho</i> = 0.26, <i>P</i> = 0.023).</p