Figure 5. Subsets of classical and atypical class-switched MBCs associate with reduced P. vivax parasite burden.

PBMCs from P. vivax symptomatic (n = 11) and asymptomatic (n = 19) infected individuals as well as healthy immune controls (n = 24) were stained with a panel of metal-labeled antibodies and analyzed by CyTOF. Percentages of classical MBC (A), activated MBC (B), and atypical MBC (C) subpopulations identified by CyTOF after FlowSOM analysis. Boxes represent the 25th to 75th percentile, whiskers show the range (minimum to maximum), and lines represent the median. The relationship between MBC subpopulations and clinical parameters was determined by Spearman’s rank correlation (D). P. vivax symptomatic (n = 6) and asymptomatic (n = 8) infected individuals as well as healthy immune controls (n = 6) were stained with fluorescent antibodies and analyzed by flow cytometry to assess IgG and IgA expression among IgM^–IgD^–CXCR5^loCCR6^lo classical MBCs (E and F), IgM^–IgD^– activated MBCs (G and H) and IgM^–IgD^–T-bet⁺ atypical MBCs (I and J) as well as FcRL5 expression among IgM^–IgD^–CCR6⁺T-bet⁺ atypical MBCs (K). Representative histograms and contour plots are shown. Stacked bars represent the mean ± SEM. Boxes represent the 25th to 75th percentile, whiskers show the range (minimum to maximum), and lines represent the median. CXCR5 and CCR6 expression was also assessed among gated IgM^–IgD^– classical MBCs (L), and a logistic regression model was used to determine the association between CXCR5 and CCR6 expression levels and the risk of asymptomatic or symptomatic P. vivax infection (M). Symbols represent the odds ratio and vertical lines depict the 95% confidence interval; *P < 0.05, **P < 0.01, ***P < 0.001. Significance was determined by 1-way ANOVA with Holm-Sidak post test or Kruskal-Wallis with Dunn’s multiple-comparison post test (A–C, F, and H), Spearman’s rank correlation (D), or unpaired, 2-tailed t test or Mann-Whitney U test (K). HC, healthy control; AS, asymptomatic; SY, symptomatic.