Isolation and Cultivation of Primary CD4+ T Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from anonymous, healthy donors (provided by UK National Health Service Blood and Transplant Service) using Ficoll Paque Plus (Sigma) according to manufacturer’s instructions. CD4+ T cells were isolated by negative selection using MojoSort Human CD4+ T Cell Isolation kit (Biolegend). Primary CD4+ T cells were maintained in RPMI medium 1640 (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Labtech), 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific), and 10 IU/mL IL-2 (Centre for AIDS Reagents [CFAR], National Institute of Biological Standards and Controls [NIBSC]). HEK293T and HeLa TZM-bl cell lines were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% FCS and 100 U/mL penicillin–streptomycin. Proviral constructs carrying functional nef alleles and nef-defective virus were previously described (16 (link)). Viral stocks were generated by transfecting HEK293T cells using Fugene 6 (Promega). After 48 h, virus was harvested, purified by sucrose gradient ultracentrifugation, and titrated by infectivity assay on HeLa TZM-bl reporter cells using Bright-Glo (Promega).