Vδ2 pe

Repeated assessments of T cell absolute counts were performed at the time of symptomatic malaria and 3,6, and 9 weeks following the malaria episode, in a subset of children in the Nagongera cohort (n = 43). Children were enrolled in this “symptomatic malaria cohort” if they met the following criteria: 1) duration of fever <48hrs; 2) P. falciparum infection with a parasite density >5,000/uL; 3) absence of complicated malaria; 4) no clinical suspicion of concurrent non-malarial illness; 5) no malaria in the prior month. Convenience sampling was used to select study participants to ensure that similar numbers of children in each of 3 age groups (0.5 - < 4 yrs, 4 - < 7 yrs, and 7- < 11 yrs) were included. At the time of each blood draw, 3 to 5 mls of blood were obtained in acid citrate dextrose tubes. 25 μl of fresh whole blood was stained with CD3 PerCP, CD4 FITC, Vδ2 PE, and Vγ9 APC (all Biolegend) for 20 minutes in a Trucount Tubes (Becton Dickinson) and then lysed and permeabilized with BD FACS Lyse buffer prior to acquisition on an Accuri C6 Cytometer (BD Biosciences).

All flow cytometry data were acquired on BD LSRII flow cytometer, and results were analyzed using FlowJo software (v.X.0.7, Tree Star, Ashland, OR). Compensation was carried out using single-color controls of either cells or beads (OneComp eBeads, eBioscience). Appropriate isotype controls, fluorescence minus one (FMO), or NTD cells were used to validate gating. All samples were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) or Fixable Viability Dye eFluor 780 (eBioscience) before antibody staining. The following fluorochrome-conjugated mouse anti-human antibodies were used: CD3 PerCP-Cy5.5 (BioLegend, clone UCHT1), Vδ2 PE (BioLegend, clone B6), Vδ2 fluorescein isothiocyanate (FITC) (Miltenyi, clone 123R3), Vδ1 APC-Vio770 (Miltenyi, clone REA173), Vδ1 PE (Miltenyi, clone REA173), anti-TCR γδ PE-Vio770 (Miltenyi, clone 11F2), anti-TCR αβ Brilliant Violet (BV) 421 or PE-Cy7 (both BioLegend, clone IP26), QBend10 APC (R&D Systems, clone 4H11), CD27 BV711 (BioLegend, clone O323), CD45RA PE-Cy7 (BioLegend, clone H100), PD1 FITC (BioLegend, clone EH12.2H7), TIM3 BV605 (BioLegend, clone F38-2E2), and TCR Vβ12 FITC (Abcam, clone S511).