Generation of latently HIV-1–infected primary CD4⁺ T cells ex vivo. Cells were primed in NP, Th1-, or Th2-polarizing conditions and 7 days after activation cells were infected with DHIV. (A) At 3 and 5 days after infection, cells were assessed for intracellular p24 gag expression by flow cytometry. The percentage of p24-postive cells is indicated in each panel. The experiment shown is representative of 4 different experiments with 4 different donors. (B) At 5 days after infection, cells were assessed for annexin-PE and DiOC₆(3) by flow cytometry. For each panel, the percentage of apoptotic cells (annexin-PE positive and DiOC₆(3) low) is indicated. The experiment shown is representative of 3 different experiments with 3 different donors. (C) At 7 days after infection, cells were cultured without stimulation (untreated) or costimulated with antibodies to CD3 and CD28 for 3 days (CD3/CD28) and assessed for intracellular p24 gag expression by flow cytometry. The percentage of p24-postive cells is indicated in each panel for this representative experiment. Values corresponding to 7 different donors are shown in panel D, where each symbol represents a different donor and horizontal lines indicate media values. Significance by 2-tailed paired-samples t test analysis (P values provided). (E) Viral integration was analyzed by Alu-PCR 3 days after infection in donors 1 and 2. Horizontal lines indicate media values.