Fig 8. Inhibition of PI3K and mTORC kinases partially suppresses TCR-induced P-TEFb expression and modestly inhibits TCR-induced proviral reactivation in primary CD4⁺ T cells.

(A) Proposed role of the PI3K/AKT/mTOR signaling pathway in mediating the biogenesis of P-TEFb. Inhibitors that were tested in the current study are shown in black italics. (B) PI3K and mTORC kinase inhibitors partially repress TCR-induced expression of active P-TEFb and albeit with significant variability. Memory CD4⁺ T cells (three experiments) and resting primary Th17 cells (four experiments) were treated or not for 30 min with inhibitors targeting PI3K (LY294002), mTORC1 (Rapamycin) or both mTORC1 and mTORC2 (Torin) at the concentrations shown prior to TCR co-stimulation for 24 h. Thereafter, cells were examined for active P-TEFb expression by flow cytometry following dual immunofluorescence staining for CycT1 and pSer175 CDK9. (C) PI3K and mTORC kinase inhibitors modestly repress proviral reactivation that is in response to TCR co-stimulation. Latently infected Th17 cells prepared using naïve CD4⁺ T cells isolated from three healthy donors were treated or not for 30 min with inhibitors to PI3K (LY294002), mTORC1 (Rapamycin) or both mTORC1 and mTORC2 (Torin) prior to stimulating the cells through the TCR for 24 h. Cells were thereafter analyzed by flow cytometry following immunostaining using a fluorophore-conjugated antibody towards HIV Nef. Statistical significance (p values) in B and C was determined using a two-tailed Student’s t test.