Effect of SAHA on the frequency of latently infected cells. PBMCs from patients on HAART were isolated and cultured in basal medium with or without 500 nM SAHA for six days. Efavirenz, raltegravir and enfuvirtide were added in the culture to prevent further rounds of viral replication. Resting CD4⁺ T cells were then isolated from the cultures. The frequency of cells harboring replication-competent HIV-1 was then measured using a previously described co-culture assay (Siliciano and Siliciano. 2005).

(A) 500 nM SAHA reactivates latent viruses. The effect of SAHA was normalized to effect of co-stimulation with anti-CD3 and anti-CD28 antibodies at day 2. (B) GFP expression is correlated with Gag protein expression. (C) Calculation of residual CD4⁺ GFP⁺ cells. In vitro latently infected CD4⁺ T cells generated from Elite controller 32 were treated with SAHA and cocultured with freshly isolated autologous CD8⁺ T cells at a 1:1 ratio. Numbers in the quadrant indicate the percentage of cells. (D) Effect of CD8⁺ T cells from different healthy donors, ECs, and patients on HAART on the survival of autologous CD4⁺ T cells in which latent HIV-1 infection has been reversed. E:T ratio is 1:1. Data represent the mean ± SEM, N=3. (E) Killing of latently infected cells is observed with higher E:T ratio. Freshly isolated autologous CD8⁺ T cells were co-cultured with SAHA-treated CD4⁺ T cells at different E:T ratio.

(A and B) Proliferative responses. PBMCs from patients on HAART were stained with CFSE and cultured with indicated treatments for 6 days. CD8⁺ T cells were then isolated and analyzed with FACS. Numbers in the quadrant indicate the percentage of cells. (C) Production of granzyme B, interferon-γ, perforin, CD107a and IL-2. PBMCs from two patients were stained with CFSE and cultured with indicated treatments for 6 days. CD8⁺ T cells were then isolated from PBMCs and used for intracellular staining for the indicated proteins.

(A and B) PBMCs from patients on HAART were isolated and cultured in basal medium for 5-6 days to allow the decay of remaining intracellular triphosphorylated nucleoside/nucleotide reverse transcriptase inhibitors, and then stimulated with phytohemagglutinin. CD4⁺ T cells were isolated 2 days after stimulation and infected with autologous viruses. Pre-stimulated autologous CD8⁺ T cells were added into the culture 4 hours after infection at the effector-to-target ratio 1:1. Raltegravir and enfuvirtide were added in the culture to prevent further rounds of viral replication. The fraction of residual Gag⁺ CD4⁺ T cells was measured after 3 days of co-culture. Numbers in the quadrant indicate the percentage of cells. Data represent the mean ± SEM, N=3. (C) Patient autologous viruses or HIV-1 BaL were used to infected CD4⁺ T cells. Infected cells were then cocultured with CD8⁺ T cells as described above. The fraction of residual Gag⁺ CD4⁺ T cells was measured after 3 days of co-culture. Data represent the mean ± SEM, N=3.

(A-C) Resting CD4⁺ T cells are not killed by viral CPE after acute infection. Primary resting CD4⁺ T cells were isolated and treated with 500 nM SAHA or anti-CD3 and anti-CD28 antibodies for one day and then infected with NL4-3-Δnef-EGFP Infected cells were either activated with anti-CD3 plus anti-CD28 antibodies, or left in a resting state. Efavirenz and enfuvirtide were added to block additional rounds of viral replication at day 3. GFP expression and annexin-V staining were analyzed by FACS. (D-F) Resting CD4⁺ T cells latently infected with HIV-1 are not killed by viral CPE after virus reactivation. Bcl-2 transduced CD4⁺ T cells were infected with NL4-3-Δnef-Δpol-EGFP to generate latent infection in vitro. To reactivate latent HIV-1, cells were treated with 500 nM SAHA or anti-CD3 and anti-CD28 antibodies. In D and E, The fraction of remaining GFP⁺ cells and the fraction of annexin-V-positive cells were monitored by FACS analysis. In F, GFP⁺ cells were purified by sorting and cultured for additional 7 days. Cell viability was monitored using forward and side scatter. Numbers in the quadrant indicate the percentage of cells.

Before coculture, CD8⁺ T cells from ECs or patients on HAART were stimulated with B57-restricted Gag peptides KF11 and TW10 (A), or mixture of 129 Gag peptides (B), respectively. Solid lines: unstimulated CD8⁺ T cells; Dashed lines: pre-stimulated CD8⁺ T cells. Data represent the mean ± SEM, N=3.

(A) Reduction of GFP⁺ cells is cell contact-dependent and MHC class-I restricted. Pre-stimulated autologous CD8⁺ T cells from patients on HAART were co-cultured with SAHA-treated CD4⁺ T cells at a 4:1 ratio with 500 nM SAHA and the indicated treatment for 4 days. (B and C) Elimination of latently-infected cells requires viral gene expression. Latently infected cells were generated using indicated viruses. Unstimulated autologous CD8⁺ T cells were co-cultured with SAHA-treated latently infected CD4⁺ T cells at a 4:1 ratio with 500 nM SAHA for 4 days. (D) GFP⁺ cells cannot be recovered after co-culture. CD4⁺ T cells were isolated after 8 days of co-culture with pre-stimulated autologous CD8⁺ T cells at a 4:1 ratio in the presence of 500 nM SAHA, and then co-stimulated with anti-CD3 and anti-CD28 antibodies for 2 days. GFP⁺ cells were analyzed with FACS and normalized to the control culture set up without CD8⁺ T cells. Data represent the mean ± SEM, N=3.