The magnitude of CD4 T cell cytokine response is higher in the rural population.

<p>CD4 T cell cytokine responses from adults living in rural Kenya (RA, n = 25) were analysed and compared to those from urban populations of African (UA, n = 8) and European (UE, n = 8) donors. The functional signatures of CD4 T cells were determined after non-specific <i>in vitro</i> stimulation with PdBU and ionomycin via the analysis of an array of functions including IFNγ, IL-2, IL-10, IL-17, TNFα, IL-21, IL-22, IL-4 and IL-9 secretion (panels 1-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055195#pone.0055195.s006" target="_blank">Table S1</a>). (A) The total frequency of CD69 positive CD4 T cells following stimulation was expressed as a percentage of total CD4 T cells. (B) The total frequency of CD4 T cells expressing at least one cytokine was expressed as a percentage of CD4 T cells (%). Horizontal bars indicate the mean (95% confidence intervals are represented) for each group. Nonparametric Mann-Whitney U test was used to analyse differences in the T cell responses between groups. Statistically significant <i>P</i>-values (< 0.05) are indicated by an asterisk (P<0.05 *; P≤0.01 **; P≤0.001 ***).</p

CD4 T cell differentiation and activation profile significantly differs between African and European donors but is similar between African individuals from either rural or urban Kenya.

<p>The differential expression of CCR7, CD27, CD28 and CD45RA by CD4 T cells was analysed by Boolean gating. Each phenotype (defined by a specific combination of markers) is shown under each subset. Each dot denotes the expression of each marker indicated on the bottom left. The differentiation stage is specified when a specific phenotype has been ascribed to a particular stage 4,13,19]. T_CM and T_EM refer to central memory and effector memory cells, respectively. CD4 T cell subsets were then ordered according to the number of markers they express specified by the horizontal bars of different colours showing these combinations of 4, 3, 2, 1 or 0 marker. The frequency of each subset is represented as a percentage of the total CD4 T cells (medians and 95% confidence intervals are shown for each group) and was compared between the three groups (rural African (RA, n = 25), urban African (UA, n = 8), and urban European donors (UE, n = 8)). Differences in CD4 T cell phenotypic profile among groups were tested using Kruskal-Wallis test and where significance was obtained, nonparametric Mann-Whitney U test was used for pair-wise analysis of the differences between groups. Statistically significant <i>P</i>-values (<0.05) are indicated by an asterisk (in red for Kruskal-Wallis or black for Mann-Whitney tests, respectively). *indicates P<0.05, **P≤0.01 and ***P≤0.001.</p

The functionality of CD4 T cell pool differs between rural and urban populations.

<p>CD4 T cell cytokine responses from rural African donors (RA, n = 25) were analysed by assessing simultaneously IFNγ, IL-2, IL-10, IL-17 and TNFα expression using panel 1 (A) or the expression of IFNγ, IL-2, IL-17, IL-21 and IL-22 using panel 2 (B) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055195#pone.0055195.s006" target="_blank">Table S1</a>). These responses were then compared to those from urban African (UA, n = 8) and European donors (UE, n = 8). The frequency of each CD4 T cell subset expressing the particular combination of cytokines, shown in the panel below each plot, is represented as a percentage of total CD4 T cells (mean (%) and 95% confidence intervals are indicated) for each group of donors. Each dot designates positivity for the cytokine indicated on the bottom left. Amongst the array of theoretically possible cytokine combinations, those observed are represented with black dots while those designated with grey dots were not observed. Orange bars represent the subpopulations that were observed among the CD4 T cells of the rural African donors but were completely absent in the CD4 T cells of both urban groups. Horizontal bars of different colors show these combinations of 5, 4, 3, 2 or 1 cytokine.</p

Total frequency of IFNγ and/or IL-10 expressing CD4 T cells correlates with malaria exposure.

<p>The percentage of total CD4 T cells positive for either IFNγ or IL-10 is plotted against either antimalarial Ab response, expressed by the number of Ag for which the Ab reactivity was defined as “high” levels (score between 0 and 4) (A), or malaria exposure indexes (B). Spearman’s correlation coefficients r and <i>P</i>-values are indicated for each plot.</p

Functional analysis of CD4 T cell cytokine response in the three groups of donors.

<p>(A) The frequency of CD4 T cells positive for each cytokine (IFNγ, IL-2, IL-10, IL-17, TNFα, IL-21, IL-22, IL-4 and IL-9, respectively) is represented as a percentage of CD4 T cells (mean (%) and 95% confidence intervals are indicated) and compared between the 3 groups of donors (rural African donors, RA, n = 25; urban African donors, UA, n = 8; urban European donors, UE, n = 8). Nonparametric Mann-Whitney U test was used to analyse differences in the T cell responses between groups. An Asterisk indicates statistically significant <i>P</i>-values (< 0.05) (*P<0.05; **P≤0.01; ***P≤0.001). The pie charts in B show the relative frequency of the different cytokine responses within the overall cytokine producing CD4 T cell population. The mean frequency of CD4 T cells positive for each cytokine is represented as a fraction of the total cytokine response by CD4 T cells (%) and compared between the 3 groups of donors (RA, n = 25; UA, n = 8; UE, n = 8).</p

Areas under the ROC curves for the Multivariable weighted local prevalence based models for the three cohorts.

<p>Areas under the ROC curves for the Multivariable weighted local prevalence based models for the three cohorts.</p

Demographic and parasitological characteristics of the cohorts used in the analysis.

<p>Demographic and parasitological characteristics of the cohorts used in the analysis.</p

Merozoite antibody versus weighted local prevalence based models in predicting malaria infection in a Junju sub-cohort.

<p>*AUC: Area under the curve.</p

Effect of weighted local prevalence of malaria infection from four annuli around each individual on risk of malaria infection.

*<p>AUC: Area under the curve.</p

Demographic and clinical characteristics of participants enrolled in the RTS,S trial.

Demographic and clinical characteristics of participants enrolled in the RTS,S trial.</p