Fig. 4

Replication competent HIV-1 detected by QVOA from AM, MC and T cells of HIV patients under suppressive ART. A Monocytes and CD4⁺ T cells from blood and AMs from BAL fluid were collected from five HIV-infected patients and purified by T cell pan isolation kit. T cells were first cultured for three days using stimulating conditions and then plated in serial dilutions into three to five wells. Monocytes purified by the pan monocyte isolation kit were checked for T-cell contamination by RT-qPCR using TCR-β primer pairs (98.38 ± 0.43% purity). MDM were generated by culturing MC under adherent conditions for seven days in the presence of M-CSF, and the cells were serially diluted into three to five wells. AM were plated directly in 3–5 serial dilution wells. Cells were cultured in the presence of the HIV fusion/entry inhibitor T20 (Enfuvirtide) as indicated. Nonadherent cells and T20 were removed prior to activation with LPS and coculturing with MOLT4/CCR5 cells. B TCR β RNA levels detected in QVOA lysates of T, MC and AM cells were measured by RT-qPCR using a seven-point standard curve established by serial dilution. The relative expression percent was normalized to CD4⁺ T cells. The mean is the average of three donors. Errors are means with standard deviations, N = 3 for AM, T and MC, where N is number of donors. (C) The infectious units per million cells (IUPM) of AM, MC and CD4⁺ T cells were determined using RT-qPCR (N = 3, the error bars are smaller than the data points). The IUPM calculations used the online Infection Frequency Calculator which utilizes limiting dilution Poisson statistics and the number of positive wells and the input number of cells as the input parameters (https://silicianolab.johnshopkins.edu)