Granulysin and Vδ2⁺ T-cell phenotype in primary-infected patients. (A) PBMCs from P falciparum primary-infected patients and healthy donors were stained ex vivo for intracellular granulysin and effector/memory surface markers. (i) Representative flow cytometry dot-plot outlining the gating strategy for Vδ2⁺ T cells (CD3⁺Vδ2⁺ lymphocytes). Flow cytometry histograms showing granulysin expression in Vδ2⁺ gated T cells (black line) compared with isotype control antibody (dotted line) in a representative patient and healthy donor (control). The percentage of positive cells for granulysin intracellular staining is indicated in the top right corner of each panel. (ii) Percentages of CD3⁺Vδ2⁺ cells positive for granulysin in malaria patients and healthy donors (controls). The box represents the 75th and 25th percentiles, and the bar represents the median. P value was determined using Mann-Whitney rank sum test to compare patients (n = 7) with controls (n = 10). Data were obtained by collecting 300 000 total events. (Bi) Representative example of flow cytometry data showing the gating strategy for Vδ2⁺ T cells (CD3⁺Vδ2⁺ lymphocytes). Expression of the CD27 and CD45RA cell surface markers in the gated Vδ2⁺ T cells define distinct effector/memory subpopulations (T_NAIVE, T_CM, T_EM, and T_EMRA). (ii) Data represent the mean ± SD of the percentage of Vδ2⁺ T cells of each effector/memory subset from patients or controls (*P ≤ .05 by Mann-Whitney rank sum test comparing patients [n = 6] with controls [n = 10]). Data were obtained by collecting 300 000 total events. (Ci) Plasma granulysin levels in patients and controls (i) were detected by ELISA. Box represents the 75th and 25th percentiles, and the bar represents the median. The P value was determined using Mann-Whitney rank sum test to compare patients (n = 12) with controls (n = 13). (ii) Log-transformed levels of plasma granulysin plotted against the percentage of CD3⁺ T cells expressing Vδ2 in patients. Statistical analysis was performed using Spearman rank correlation test, and the ρ and the P value are indicated (n = 7). (D) Fresh PBMCs from 2 patients (P1 and P2) were cocultured with purified trophozoites (iRBCs) or uiRBCs at a 1/5 E/T ratio. CD107a surface expression detected after 6 hours of coculture was measured. The percentage of CD3⁺Vδ2⁺ T cells or CD3⁺Vδ2⁻ T cells expressing CD107a is shown. Data were obtained by collecting 300 000 total events.