Fig 1.
CD16+ Vδ2 T cells are more likely to express cytotoxic markers and KIRs.
(A) The percentage of CD16+ and CD16-Vδ2 T cells expressing relevant cytotoxic markers are shown (n = 65; scatter plot with median and IQR). (B) Vδ2 T cells positive for 4 or any combination of 3, 2 or 1 of the cytotoxicity markers listed in A are grouped along the x-axis and their relative percentage of CD16 is shown (n = 65; scatter plot with median and IQR). (C) The percentage of CD16+ and CD16-Vδ2s expressing the listed combinations of the transcription factors Tbet and Eomes are shown. (n = 65). (D) The percentage of CD16+ and CD16-Vδ2 T cells expressing the listed KIR or KIR combinations is shown. Individuals who were genotype negative for the listed KIRs were omitted from this analysis (n = 22 for KIR3DL1 and KIR2DS3/L1/S1/S5; n = 20 for KIR2DL3). (E) The percentage of CD16+ and CD16-Vδ2 T cells expressing IL18Rα and the MFI of IL18Rα on CD16+ and CD16-Vδ2 T cells is shown (n = 10). All Vδ2+ T cells shown were gated on singlets and CD3+ events to exclude NK cells. P values for A, B, D and E determined by Wilcoxon matched pairs signed rank test.
Fig 2.
CD16+ Vδ2 T cells from malaria-exposed individuals downregulate TCR and can be independently stimulated through CD16.
(A)The MFI of the Vδ2 and Vγ9 chains is shown for CD16+ and CD16- Vδ2+Vγ9+ T cells. Data points and accompanying p-value are paired. (n = 39; scatter plots with median and IQR). (B) The percentage of Vδ2 T cells producing IFNγ in response to anti-CD16 crosslinking antibody or isotype is compared (n = 35; box plots with median and Tukey whiskers). (C) The percentage of Vδ2 T cells producting IFNγ in response to plate-bound anti-CD16 crosslinking antibody is graphed against the percentage of Vδ2 T cells expressing CD16 prior to stimulation (n = 35). (D) The percentage of Vδ2 T cells positive for IFNγ or CD107a after stimulation with anti-CD16 crosslinking antibody or iRBC is compared (n = 10) (E) Vδ2 T cells positive for IFNγ or CD107a after iRBC stimulation from the experiment in E are grouped by CD16 expression. All Vδ2+ T cells shown were gated on singlets and CD3+ events to exclude NK cells. P values determined by Wilcoxon matched pairs signed rank test in A, B, D and D, and with Spearman correlation in C.
Fig 3.
Stimulation through CD16 augments Vδ2 cell activation by P.falciparum.
(A) Schematic detailing stimulation conditions for data presented in panel B. (B) Percent of Vδ2 T cells producing IFNγ or positive for CD107a after stimulation of whole PBMC with uRBC, iRBC or IPP, with or without plate bound anti-CD16 crosslinking antibody (n = 9). (C). Percent of Vδ2 T cells producing IFNγ or positive for CD107a after stimulation of negatively selected γδ T cells with uRBC or iRBC, with or without plate bound anti-CD16 crosslinking antibody (n = 4). (D) Live cells positive for γδ TCR after negative selection. All Vδ2+ T cells shown in B-C were gated on singlets and CD3+ events to exclude NK cells. Samples for these experiments were selected from individuals with greater than 30% of their Vδ2 T cells expressing CD16. P values were determined by Wilcoxon matched pairs signed rank test.
Fig 4.
Stimulation by opsonized antigen augments Vδ2 cell activation by P.falciparum.
(A) Schematic detailing the stimulation conditions for data presented in panel B. (B) Percent of Vδ2 T cells positive for IFNγ and CD107a after stimulation of whole PBMC with uRBC, iRBC, iRBC + naïve IgG, or iRBC + hyper-immune IgG. (n = 6). (C) Percent of Vδ2 T cells positive for IFNγ and CD107a after stimulation of negatively selected γδ T cells with uRBC, iRBC, iRBC + naïve IgG, or iRBC + hyper-immune IgG. (n = 9). All Vδ2+ T cells shown were gated on singlets and CD3+ events to exclude NK cells. Samples for this experiment were selected from individuals with greater than 30% of their Vδ2 T cells expressing CD16. P values were determined by Wilcoxon matched pairs signed rank test.
Fig 5.
Blocking BTN3a1 does not inhibit stimulation of Vδ2 T cells by opsonized antigen.
Percent of Vδ2 T cells positive for IFNγ and CD107a after stimulation with iRBC + naïve IgG, or with iRBC + hyper-immune IgG, are compared in the presence or absence of a Butyrophilin 3a1 blocking antibody. (n = 10; p values determined by Wilcoxon matched pairs signed rank test). All Vδ2+ T cells shown were gated on singlets and CD3+ events to exclude NK cells. Samples for this experiment were selected from adults with greater than 30% of their Vδ2s expressing CD16.
Fig 6.
Model of Vδ2 T cells over the course of repeated Pf infection.
As an individual experiences more episodes of Pf, the frequency of Vδ2 T cells in their peripheral blood declines. Remaining Vδ2 T cells become less responsive through their TCRs while the frequency of CD16+ expression increases. These changes occur while an individual’s anti-Pf antibody response is simultaneously growing and diversifying, presumably providing more opsonized parasite targets for CD16+ Vδ2 T cells. TCR-responsive Vδ2s are activated by engagement with BTN3A1 on another cell, probably a lymphocyte, when BTN3A1’s conformation is altered by the intracellular binding of parasite-derived phosphoantigens. CD16-responsive Vδ2 T cells are activated by opsonized parasite antigen and may have their activation threshold modulated by KIR expression and engagement. Created with Biorender.