High levels of integrated HIV-1 in resting CD4⁺ T cells after incubation with CCL19 and CCL21. (A) Method used to purify resting CD4⁺ T cells. Resting CD4⁺ T cells were purified from peripheral blood mononuclear cells (PBMCs) by negative selection using mouse antibodies to human CD8 (American Type Culture Collection [ATCC], Manassas, VA), CD14 (ATCC), CD16 (ATCC), CD19 (Hedi Zola, Flinders Medical Center, Adelaide, Australia), HLA-DR (Tony D'Apice, St Vincents Hospital, Melbourne, Australia), CD69 (BD Biosciences), and CD11b (ATCC); magnetic beads conjugated with antibodies to mouse immunoglobulin G (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic-activated cell sorting. The mean purity was 97% (range, 95%-98%). In some experiments, resting CD4⁺ T cells were further purified into naive (CD45RO⁻) and memory (CD45RO⁺) CD4⁺ T cells. (B) Method used to infect resting CD4⁺ T cells. Purified resting CD4⁺ T cells were activated for 3 days with CCL19/CCL21 or IL2/PHA or were left unactivated before infection with HIV-1. The cells were then maintained in IL-2 and supernatant and cells were collected after 4 and 7 days. (C) Low-level productive infection after infection of resting CD4⁺ T cells incubated with CCL19. Resting CD4⁺ T cells were cultured for 3 days with CCL19 (○) or IL-2/PHA (□) or were unactivated (◇) and infected with pNL4.3 HIV-1 or mock (△) as described previously in this paragraph. RT activity in the culture supernatants was determined at the indicated time points. Mean plus SD (error bar) for 6 separate experiments is shown. (D) Experiments similar to those in panel C showing productive infection in CD4⁺ T cells infected with AD8. (E) Quantification of integrated HIV-1 DNA. Integrated HIV-1 DNA was quantified by Alu-LTR real-time PCR. Resting CD4⁺ T cells were activated with CCL19 (■), IL-2/PHA (▒), or unactivated (○) and were infected with HIV-1 pNL4.3 (n = 5; median + IQR [error bar]). Infection of CCL19-treated resting CD4⁺ T cells with HIV-1 pNL4.3 D116N (integrase-; □) and HIV-1 pNL4.3 after incubation with CCR7 antibody 3D12 (10 μg/mL; ▩) did not show evidence of integrated HIV-1 DNA. The detection limit of the assay was 330 copies/10⁶ cells and is shown by ----. (F) Phenotype of purified CD4⁺ resting T cells after activation with different stimuli. CCL19 or CCL21 (10 nM), IL-2/PHA, or unactivated cells were labeled after 3, 24, 48, and 72 hours for CD25, CD69, HLA-DR, and CCR7 and examined by flow cytometry (FCM). The percentage of CD4⁺ T cells that express the particular protein after 72 hours in culture is shown for all markers.