Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion

Abstract

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the "atypical" memory B cells (MBCs) and "exhausted" CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a "natural" experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1-expressing and PD-1/LAG-3-coexpressing CD4 T cells was largely confined to CD45RA(+) CD4 T cells. The percentage of CD45RA(+)CD27(+) CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

FIGURE 1.

Gating strategy for flow cytometric phenotyping of B cells. Total B cells were identified by CD19 expression and then subsets were identified by the expression of CD10, CD20, CD21, and CD27. Shown are plots representative of the malaria-naive controls (top panels) and the exposed groups (bottom panels). All numbers represent the percentage of the parent gate.

FIGURE 2.

Atypical MBCs are significantly expanded in the presence of persistent P. falciparum transmission. (A–F) Comparison of the proportions (of total CD19⁺ B cells) of different B cell subsets as defined in Fig. 1 between the different study groups. Each dot is an individual child, and the solid horizontal lines indicate the median values for the respective groups. Statistical significance between various paired groups was determined with the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001. (G) The relative percentages of the various B cell subsets out of the total CD19⁺ B cells for each of the study groups.

FIGURE 3.

Gating strategy for flow cytometric phenotyping of CD4 T cells. CD4 T cells were identified and selected from the rest of PBMCs, as shown for representatives of the malaria-naive (top panel) and exposed (bottom panel) groups. CD4 T cells were then phenotyped into four different subsets based on CD45RA and CD27 expression. Subsequently, the percentages of total CD4 T cells and the associated CD4 T cell subsets expressing PD-1 and LAG-3 were determined. A similar layout for the phenotyping of CD8 T cells and the determination of frequencies of CD8 T cells expressing inhibitory markers is shown in Supplemental Fig. 4.

FIGURE 4.

CD4 T cells expressing the inhibitory PD-1 and LAG-3 molecules are significantly increased in children exposed to persistent P. falciparum infections. Comparisons of the frequencies of CD4 T cells and CD4 T cell subsets expressing PD-1 (A–C), LAG-3 (D–F), and both PD-1 and LAG-3 (G–I) are shown. Each dot is an individual child, and the solid horizontal lines indicate the median values for the respective groups. Dot plots compare the expression of the inhibitory receptors across the different groups of children for total CD4 T cells (left column), CD45RA⁺CD27⁺ (naive/T_CM late) (middle column), and CD45RA⁺CD27⁻ (T_EM+EFF) CD4 T cells (right column). Statistical significance between pairs of groups was determined with the Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

CD8 T cells expressing the inhibitory PD-1 and LAG-3 molecules are significantly increased in children exposed to persistent P. falciparum infections. Comparison of the frequencies of CD8 T cells and CD8 T cell subsets expressing PD-1 (A–C), LAG-3 (D–F), and both PD-1 and LAG-3 (G–I) are shown. Each dot is an individual child, and the solid horizontal lines indicate the median values for the respective groups. Dot plots compare the expression of the inhibitory receptors across the different groups of children for total CD8 T cells (left column), CD45RA⁻CD27⁻ (T_EM) (middle column), and CD45RA⁺CD27⁻ (T_EM+EFF) CD8 T cells (right column). Statistical significance between pairs of groups was determined with the Wilcoxon rank-sum test. *p < 0.05, ** p < 0.01, ***p < 0.001.

FIGURE 6.

Relationships between the expansion of PD-1 and LAG-3 double-positive CD45RA⁺CD27⁺ CD4 T cells and frequencies of different B cell subsets. Left column, Pooled data points from the Junju exposed-parasitemic group (blue) and the exposed-nonparasitemic group (red). Right column, Malaria-naive children (Ngerenya-naive). The correlation coefficients and associated p values were determined by Spearman rank correlation analysis.