Cytotoxic T Cell-Derived Granzyme B Is Increased in Severe Plasmodium Falciparum Malaria

Abstract

In Plasmodium falciparum malaria, CD8⁺ T cells play a double-edged role. Liver-stage specific CD8⁺ T cells can confer protection, as has been shown in several vaccine studies. Blood-stage specific CD8⁺ T cells, on the other hand, contribute to the development of cerebral malaria in murine models of malaria. The role of CD8⁺ T cells in humans during the blood-stage of P. falciparum remains unclear. As part of a cross-sectional malaria study in Ghana, granzyme B levels and CD8⁺ T cells phenotypes were compared in the peripheral blood of children with complicated malaria, uncomplicated malaria, afebrile but asymptomatically infected children and non-infected children. Granzyme B levels in the plasma were significantly higher in children with febrile malaria than in afebrile children. CD8⁺ T cells were the main T cell subset expressing granzyme B. The proportion of granzyme B⁺ CD8⁺ T cells was significantly higher in children with complicated malaria than in uncomplicated malaria, whereas the activation marker CD38 on CD8⁺ T cells showed similar expression levels. This suggests a pathogenic role of cytotoxic CD8⁺ T cells in the development of malaria complications in humans.

Keywords: CD8+ T cells; Plasmodium falciparum; granzyme B; malaria; severe malaria.

Figure 1

Granzyme B level in the plasma of P. falciparum infected children is associated with disease severity. Blood samples were taken from healthy, non-infected children (healthy donors, HD; n = 41) and asymptomatically infected children (AS, asymptomatically infected; n = 41) as well as from children with uncomplicated malaria (outpatients, OP; n = 35) and severe malaria (inpatients, IP; n = 32). Granzyme B levels in the plasma were measured by ELISA. Statistical significance was determined using Kruskal–Wallis Test with post-hoc Dunn's test: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 2

CD3⁺CD8⁺ T cells express high amounts of GrzB in P. falciparum infected children. CD3⁺ lymphocytes of all study participants were concatenated group wise (HD n = 41; AS n = 41; OP n = 36; IP n = 32). The cell number of all concatenated groups were downsampled to 10⁶ cells/group. Hierarchical stochastic neighbor embedding (HSNE) analysis of all groups based on the expression of CD3, CD8, CD4, Granzyme B, CD38, and PD-1 were computed. Landmark development and clustering were complete data-driven. Landmark-based clusters were used for heat map generation. Color code represents mean fluorescence intensity (red = high; blue = low). Blue boxes indicate GrzB-producing CD4⁺ T cell subsets, red boxes indicate GrzB-producing CD8⁺ T cells. Orange boxes highlight CD3⁺CD4⁻CD8⁻ T cell subsets, producing GrzB.

Figure 3

Granzyme B expression in CD8⁺ T cells correlates with disease progression. Blood samples from children infected with P. falciparum but lacking symptoms (AS, n = 41), children treated as outpatients for uncomplicated malaria (OP, n = 35), children treated as inpatients for severe malaria (IP, n = 32), as well healthy, non-infected children (HD, n = 41) were stained with fluorescent labeled αCD3, αCD8, αCD4, αGranzyme B, αCD38, and αPD-1 antibodies for flow cytometric analyses. (A) Frequencies of CD8⁺ T cells among CD3⁺ cells were assessed in all 4 groups. (B) The CD8⁺CD3⁺ T cells were further analyzed for the expression of the effector molecule granzyme B, (D) CD38 and (E) PD-1 in all four groups. Statistical significance was determined using Kruskal–Wallis Test with post-hoc Dunn's test: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. (C) The correlation between the frequency of GrzB expression by CD3⁺CD8⁺ T cells and parasitemia was analyzed in the blood of all infected children (n = 89), using Spearman rank correlation.