Loss and dysfunction of Vδ2⁺ γδ T cells are associated with clinical tolerance to malaria

Abstract

Although clinical immunity to malaria eventually develops among children living in endemic settings, the underlying immunologic mechanisms are not known. The Vδ2(+) subset of γδ T cells have intrinsic reactivity to malaria antigens, can mediate killing of Plasmodium falciparum merozoites, and expand markedly in vivo after malaria infection in previously naïve hosts, but their role in mediating immunity in children repeatedly exposed to malaria is unclear. We evaluated γδ T cell responses to malaria among 4-year-old children enrolled in a longitudinal study in Uganda. We found that repeated malaria was associated with reduced percentages of Vδ2(+) γδ T cells in peripheral blood, decreased proliferation and cytokine production in response to malaria antigens, and increased expression of immunoregulatory genes. Further, loss and dysfunction of proinflammatory Vδ2(+) γδ T cells were associated with a reduced likelihood of symptoms upon subsequent P. falciparum infection. Together, these results suggest that repeated malaria infection during childhood results in progressive loss and dysfunction of Vδ2(+) γδ T cells that may facilitate immunological tolerance of the parasite.

Fig. 1

Incidence of malaria (left y-axis, red line) and the probability that parasitemia is asymptomatic (right y-axis, blue line) from age one to five years (n=78), visualized using a generalized additive model. Standard errors are indicated by the dotted lines. Blood samples for this study were obtained at 48 months of age (hashed vertical line).

Fig. 2

Loss of Vδ2⁺ γδ^low cells in setting of heavy malaria exposure. (A) Flow cytometric analysis of live CD3⁺ γδ⁺ T cells labeled with a pan-antibody revealed two distinct populations of γδ⁺ T cells, with distinct populations of γδ^high and γδ^low cells. Through co-staining (A) and parallel testing (B-C) of the pan-γδ antibody and the predominant Vδ-chains in humans in a subset of patients (n=55), γδ^low cells were predominantly Vδ2⁺ and γδ^high cells were predominantly Vδ1⁺. (D) Percentages of Vδ2⁺ T cells are inversely associated with prior malaria incidence. (E) No significant association between percentages of Vδ1⁺ T cells and prior malaria incidence. r_s: Spearman correlation coefficient. ppy=per person year. Solid lines in panels B-E represent best fit regression lines. (F) Fold change of Vδ2⁺ T cells between 4-5 years of age in children with either 0-1 episodes or ≥8 episodes of malaria ppy between 4-5 years of age (n=6 per group, Wilcoxon rank-sum. See fig. S2 for individual data).

Fig. 3

Dysfunction of Vδ2⁺ γδ^low cells in setting of heavy prior malaria. Shown are the absolute frequency (A) and the relative proportion (B) of each individual combination of iRBC-stimulated IFNγ, TNFα, or IL-2-producing γδ^low T cells. (C) Percentages of IFNγ⁺/TNFα⁺-producing γδ^low cells are inversely associated with prior malaria incidence (n=78). (D) Heavy prior malaria associated with proliferative defect of Vδ2⁺ T cells. Shown is CFSE dilution following 7 days of P. falciparum stimulation in one representative sample from a child with low prior incidence (left) and one representative sample from a child with high prior incidence (right). (E) Vδ2⁺ proliferation was assessed in a subset of children (n=42) and found to be inversely associated with the prior incidence of malaria. r_s: Spearman correlation coefficient. ppy=per person year. Solid lines in panels C and E represent best fit regression lines.

Fig.4

Differential gene expression in unstimulated Vδ2⁺ T cells from children with low and high prior malaria incidence. (A) Whole transcriptome analysis of sort-purified unstimulated Vδ2⁺ γδT cells from children with <2 episodes ppy vs ≥8 episodes ppy (n=4 per group). Differentially expressed genes (determined using a 5% false discovery rate (FDR), fold change ≥ 2 with two-class unpaired comparisons) are depicted as a heat map of relative expression intensities, log₂ normalized to the median expression across all samples for each gene. Yellow represents higher and blue represents lower expression relative to the median; gene names depicted in red represent genes with previously reported roles in tolerance or immunoregulation, with common protein names provided in parentheses following the official gene symbol. (B) Flow cytometric analyses confirming increased expression of immunoregulatory proteins in children with high prior malaria incidence. (n=4-5 per group, Wilcoxon rank-sum. See table S1 for individual data).

Fig 5

Diminished gene induction in Vδ2⁺ T cells following P. falciparum stimulation in children with high prior malaria incidence. (A) Shown are relative gene expression of significantly induced genes before and after stimulation with iRBCs in children with low prior incidence (n=4, left set of columns, determined using 5% FDR, fold change ≥ 2, two-class paired comparisons). Relative Vδ2⁺ gene expression before/after iRBC stimulation in children with high prior incidence shown for comparison (n=3, right set of columns; one paired sample not analyzed due to amplification failure in stimulated sample). (B) Venn diagram showing number of significantly induced genes following iRBC stimulation in low and high prior incidence children. The full list of significantly induced genes is available in table S2. (C) Significantly induced genes from (A) plotted as fold change gene induction (stimulated/unstimulated) in high prior incidence versus low prior incidence. The identity line (m = 1) is shown. The seven genes with the largest residuals from the identity line are labeled. (D) Fold change gene induction (stimulated/unstimulated) of Vδ2⁺ T cells of the six most significantly induced cytokines in children with low (left, n=4) and high (right, n=3) prior incidence (t test of log₂-transformed fold changes.)

Fig. 6

Lower percentages of P. falciparum-responsive γδ^low T cells in children who later develop asymptomatic infection. Shown are γδ^low T cells producing IFNγ⁺/TNFα⁺ in response to malaria antigen stimulation, as a proportion of the total CD3⁺ T cell population. Subjects are stratified by the absence (n=24) or presence (n=47) of any asymptomatic infection during one year of follow-up, conditional on the presence of any parasitemia during this period (Wilcoxon rank-sum.)

Fig. 7

The probability of parasitemia (A), probability of developing symptoms if parasitemic (B), and incidence of malaria (C) by age in children with the highest and lowest tertile percentages of malaria-responsive IFNγ⁺/TNFα⁺-producing γδ^low T cells. Vertical dashed lines represent time assay performed. Children in the lowest tertile of IFNγ⁺/TNFα⁺-producing γδ^low T cells had a consistently higher monthly probability of parasitemia (A), but lower probability of symptoms if parasitemic (B). Although children in the lowest tertile had a higher incidence of symptomatic malaria early in the study (C), by 48-60 months of age the incidence of symptomatic malaria in the two groups was similar. (D) Associations between the lowest vs. highest (reference group) of γδ-T cell responses and the monthly risk of parasitemia, probability of developing symptoms if parasitemic, and incidence of malaria, stratified by year of age using generalized estimating equations with robust standard errors. OR: Odds ratio.