Apoptotic Activated T Cells Inhibit HIV in DCs Figure 5. APOBEC3G mRNA expression and G-to-A hypermutations are induced after co-culturing with ApoAct but not ApoRest. (A) Real-time PCR was used to determine fold changes in mRNA for APOBEC3G in DCs co-cultured with LPS, resting apoptotic allogeneic CD4+ T cells (ApoRest), or activated apoptotic allogeneic CD4+ T cells (ApoAct). mRNA levels were compared with mRNA obtained from DCs cultured in medium at different time points (2 hours, 6 hours, 24 hours, and 48 hours). Data represent the mean 6 SD from four different donors. Real-time PCR analyses of the 24 hours samples were repeated three times with similar values obtained. Significant differences were assessed by non-parametric Kruskal- Wallis test with Dunn’s Multiple Comparison Test and are indicated by *p,0.05, **p,0.01, and ***p,0.001. Non-significant, NS. (B) Intracellular p24 expression was measured by flow cytometry in DC exposed to HIV, HIV and ApoRest, HIV and ApoAct non-targeted siRNA or in DC treated with APOBEC3G siRNA (siA3G) prior to exposure to HIV and ApoAct. Data represent mean6SD from six donors and analyses were performed seven days after infection. Significant differences were assessed by the non-parametric paired Wilcoxon test and are indicated as *p,0.05. (C) G-to-A mutations in HIV-1 env clones. HIV-1 env sequences were generated from HIVBaL infected DCs co-cultured either with ApoRest or ApoAct. G-to-A mutation frequencies as compared with a HIVBaL sequence obtained from DCs cultured with the same stock of HIVBaL were measured in clones from HIVBaL infected DCs co-cultured either with ApoRest (n = 35) or ApoAct (n = 26). Significant differences were assessed by the t-test (unpaired), and significance is indicated by *p,0.03. doi:10.1371/journal.pone.0021171.g005 cells were activated before induction of apoptosis and subsequent co-culturing with DCs. There was no difference in HIV-infected or uninfected ApoAct to induce DC maturation or cytokine release and that there was no clear induction of DC maturation by the virus itself-rather it was the activated apoptotic cell component that made a difference. It should be noted that we removed cell debris from the virus preparations to avoid possible contaminating dead cells from the activated PBMCs used for viral growth. We also show that part of the anti-HIV activity is mediated by TNF-a released into the supernatant after co-culture with ApoAct. TNF-a is an example of a cytokine that can differentially inhibit or promote HIV replication depending on the target cell type, which adds to the complexity of events occurring in vivo [14]. We detected up-regulation of APOBEC3G mRNA expression in the DC co- cultures with ApoAct indicating that inhibition might occur at least in part at the transcriptional level [25,50]. Furthermore, PCR analyses showed that the amount of HIV-DNA present in DCs after co-culture with ApoAct is lower as compared to DCs co- cultured with ApoRest, which is in concordance with these findings, although it should be pointed out that there was not a PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21171