Isolation and Infection of CD4+ T Cells

Human tonsil or splenic tissues were obtained from the National Disease Research Interchange and the Cooperative Human Tissue Network and processed as previously described^{11 (link)}. Dead cells within the complete HLACs were first removed by Ficoll-Hypaque gradient centrifugation. CD4 T cells (CD3+) were isolated from HLACs by positive selection using CD4 microbeads (Miltenyi) as described^{11 (link)}. CCR5-expressing CD4 T cells were positively separated from CD4 T-cell cultures (PlusCellect R&D Systems Cat # PLS180), negatively isolated from complete HLACs (STEMCELL Technologies, EasySep™ Human CD4+ T-cell Enrichment Kit). In shRNA experiments, infections with R5-tropic HIV-1, and when splenic cells that are extremely refractory to HIV-1 infection were used, we modified the infection system by overlaying HLAC cells on a monolayer of 293T cells that had been transfected with HIV-1 proviral clones, as previously described^{11 (link)}. Flow cytometry data were collected on a FACS Calibur (BD Biosciences) and analyzed with FlowJo software (Treestar). HIV-1 viruses were generated by transfection of proviral DNA into 293T cells using calcium phosphate.