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Steel et al., 2006;

Siliciano et al., 2007;

Archin et al., 2008, 2010;

Sagot-Lerolle et al., 2008). These studies raise a critical issue: the fate of this reservoir after virus reactivation in resting CD4+ T cells. It is generally presumed that infected cells will die after reactivation of virus gene expression either as a result of viral cytopathic effects (CPEs) or host immune responses or both. Because newer approaches for reactivating latent HIV-1 utilize agents that do not induce global T cell activation, it is important to determine whether viral CPE or host responses can eliminate latently infected resting CD4+ T cells after virus reactivation. Direct killing of infected cells by HIV-1 through caspase- dependent or -independent mechanisms has been observed in activated CD4+ T cells (Roshal et al., 2001;

Bolton et al., 2002;

Sakai et al., 2006;

Shedlock et al., 2008). Other studies showed that early events in abortive HIV-1 infection induced cell death in resting CD4+ T cells (Zhou et al., 2008;

Doitsh et al., 2010). However, whether the reversal of viral latency causes cell death in resting CD4+ T cells or not has not been assessed. Besides viral CPE, host immunity is also presumed to eliminate the latently infected CD4+ T cells after virus reactivation. Cytolytic T lymphocytes (CTLs) are a major component of the host response to HIV-1. CTLs partially limit viral replication (Walker et al., 1987;

Koup et al., 1994;

Borrow et al., 1997;

Schmitz et al., 1999;

Gandhi and Walker, 2002;

Hersperger et al., 2011) Immunity 36, 491C501, March 23,

2012 ?2012 Elsevier Inc.

491 but show functional defects in patients with progressive disease that are not restored with HAART (Kalams et al., 1999;

Sa ? ez- Cirio ? n et al., 2007;

Migueles et al., 2008, 2009;

Hersperger et al., 2010). It is unknown whether CTLs can kill resting CD4+ T cells in which latent infection has been reversed. In this study, we generated latently infected cells from primary CD4+ T cells as previously reported (Yang et al., 2009). SAHA was used to reactivate latent HIV-1 in resting CD4+ T cells. We found that virus reactivation did not cause death of infected cells. CTLs from patients on HAART failed to kill autologous latently infected CD4+ T cells after latent viruses were reactivated. Antigen- speci?c stimulation of patient CTLs prior to virus reactivation led to rapid and effective killing of infected cells. Our results suggest that reactivation of latent HIV-1 will not purge the viral latent reservoir. Stimulation of HIV-1-speci?c CTL responses prior to virus reactivation may be essential for the viral eradication. RESULTS Reactivation of Latent HIV-1 In Vitro without T Cell Activation Does Not Affect the Latent Reservoir We ?rst examined the stability of HIV-1 latent reservoir in resting CD4+ T cells after treatment with agents that reactivate latent HIV-1 without causing T cell activation. If viral CPE or CTL responses cause the death of infected resting CD4+ T cells, reac- tivating latent HIV-1 should result in reduction or elimination of latent reservoir. We used the histone deacetylase inhibitor SAHA, an FDA-approved drug used to treat cutaneous T cell lymphoma. SAHA has been shown to reactivate latent HIV-1 in peripheral blood mononuclear cells (PBMCs) from HIV-1- infected individuals on HAART (Contreras et al., 2009;

Archin et al., 2009). We treated PBMCs from patients on HAART with SAHA for

6 days. After treatment with SAHA, the frequency of latently infected resting CD4+ T cells was measured by the stan- dard limiting dilution virus culture assay (Figure 1). In........