Figure 5. HIV was outgrown from brain-derived MG.

(A) Cell-free HIV RNA (gag) in the culture supernatants was measured in the SAHA- and CM272-treated MG cultures (10⁵ cells/well) isolated from the parietal cortex of donor 2 (n = 3). (B) Outgrowth HIV was tracked over time by measuring viral RNA release in the culture supernatant of SAHA- and CM272-treated MG wells (from the indicated brain region of donor 2) after addition of CD8-depleted PHA PBMC blasts (n = 3). (C) MG QVOA and de novo infection by human brain MG–derived HIV. After MG were isolated from the brain of PWH, cells were plated in the 24-well plates with limited dilutions and cultured for 14 days in the presence of ART, allowing the cells to settle down and attach to the surface. The latent HIV was activated with SAHA and CM272 for another 7 days, and then the LRAs were washed out. For the MG QVOA, LRA-treated MG were cultured with CD8-depleted, HIV^– PBMC PHA blasts or MOLT-4/CCR5 cells. Viral outgrowth was measured on day 21 and was further confirmed on day 28. De novo HIV infection was used to assess MG-derived, replication-competent HIV via inoculation of virus from LRA-stimulated MG culture into MG or PBMC blasts isolated from HIV^– donors. HIV replication was assayed by HIV RNA and p24 released into culture supernatants. ART consisted of raltegravir plus darunavir plus nevirapine. (D and E) The IUPM of MG and CNS T cells was calculated by standard viral outgrowth assay (measuring HIV p24 antigen release in the wells) (D) and by RT-ddPCR to measure viral RNA⁺ wells (E) (n = 4).