PBMCs from PROMOTE subjects (28 months of age; no chemoprevention control arm) were thawed and rested overnight either in standard media (untreated), 5uM camptothecin (Sigma) or P. falciparum schizont extract (PfSE) or protein extract from uninfected RBCs (uE) at an effector:target ratio of 3:1. To test for induction of apoptosis, stains for AnnexinV (Biolegend) or YoPro (Invitrogen), or activated Caspase 3 FITC (BD) were used according to the manufacturer’s instructions in combination with the following antibodies: AnnexinV and YoPro staining—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) APC-Cy7, CD127 (A019D5) APC, CD25 (BC96) PE-Cy7 from Biolegend; for Caspase3—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) PerCP, CD127 (A019D5) Pacific Blue, CD25 (BC96) PE-Cy7. Tregs were gated as CD3+CD4+CD25+CD127dim. Sensitivity to apoptosis was measured ex vivo (untreated control), after induction with camptothecin (fold change compared to untreated), and after stimulation with P. falciparum schizont extract (fold change comparing PfSE to uE).
Boyle M.J., Jagannathan P., Farrington L.A., Eccles-James I., Wamala S., McIntyre T.I., Vance H.M., Bowen K., Nankya F., Auma A., Nalubega M., Sikyomu E., Naluwu K., Rek J., Katureebe A., Bigira V., Kapisi J., Tappero J., Muhindo M.K., Greenhouse B., Arinaitwe E., Dorsey G., Kamya M.R, & Feeney M.E. (2015). Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria. PLoS Pathogens, 11(7), e1005041.
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