Descriptive statistics of study cohort.

<p>Note: IQR, interquartile range.</p><p>Asymptomatic parasitemia defined as positive routine blood smear in the absence of fever that was not followed by the diagnosis of malaria in the subsequent seven days. Period prevalence calculated as the number of episodes/total months observed.</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

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

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

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

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

<div><p>FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by <i>P</i>. <i>falciparum</i> both <i>in vivo</i> and <i>in vitro</i>; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced <i>in vitro</i> induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with <i>P</i>. <i>falciparum</i> among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.</p></div

Regulatory T cells decline with increasing prior malaria incidence and mosquito exposure among children in a high transmission setting.

<p>Regulatory T cell frequencies were analyzed as the percentage of FoxP3+CD25+ of CD4+ T cells from <b>(A)</b> fresh whole blood in 2-year old (PROMOTE-cohort, no chemoprevention control arm) and <b>(B)</b> and frozen PBMCs from 4-year olds (TCC) and the association with prior malaria incidence analyzed. In both 2 and 4 year olds, T_reg frequencies declined with increasing prior malaria incidence. <b>(C/D)</b> Regulatory T cell frequencies, analyzed as the percentage of FoxP3+CD25+CD127^dim of CD4+ T cells from 1 to 11 year old children (PRISM cohort, high transmission Nagongera, Tororo District), declined with increasing mean daily household mosquito exposure (from monthly CDC light traps) <b>(C)</b> and age <b>(D)</b>. <b>(E)</b> The relationship between T_reg frequencies and age was analyzed in children from the low transmission Jinja District; there was no decline in T_reg frequencies with age in children from the low malaria transmission settings. For all analyses, Spearman’s rho and p are indicated.</p

TNFR2 expression on FoxP3+ regulatory T cells declines with increasing prior malaria incidence.

<p><b>A.</b> Frequencies of TNFR2 expressing T_regs (FoxP3+CD25+CD127^dim) were compared between children in the high malaria transmission area (Tororo District) and in the low transmission area (Jinja district). TNFR2 expression on T_regs was higher in low compared to high transmission settings. Wilcoxon signed rank test indicated. <b>B.</b> The association between frequencies of TNFR2 expressing T_regs and number of recent malaria episodes was analyzed among children from high malaria exposure area (Tororo District). TNFR2 expression declined with increasing number of malaria episodes in last 90 days. Regression coefficient and p value are indicated.</p

Relationship between T_reg frequencies and prospective risk of malaria.

<p>¹ –Multivariate is adjusted for prior malaria incidence</p><p>Relationship between T_reg frequencies and prospective risk of malaria.</p