Apoptotic Activated T Cells Inhibit HIV in DCs Figure 1. ApoAct or ApoInf induce CD86 and CD83 expression in human DCs. Human in vitro–differentiated monocytes cultured for 6 days in the presence of IL-4 and GM-CSF were used as the source of human immature DCs. (A) Immature DCs were co-cultured with apoptotic allogeneic CD4+ T cells for 72 hours or 7 days and then analyzed for expression of CD86 molecules by flow cytometry. Gates were set on large CD1a+CD3– cells. LPS, which is a potent DC activator, was used as a positive control, and DCs cultured in medium only were used as a negative control. Immature DCs were cultured in medium alone or in the presence of LPS (LPS), HIVBaL (HIV), apoptotic anti-CD3– and anti-CD28–activated CD4+ T cells (ApoAct), ApoAct in the presence of exogenous HIVBaL (ApoAct+HIV), or apoptotic HIVBaL-infected activated CD4+ T cells (ApoInf). Representative flow cytometry data from one donor after 7 days of co-cultures are shown. (B) The average percentages of CD86+ DCs 6 SD for 11 donors and of CD83+ DCs 6 SD for 7 donors are depicted. Data from at least four independent experiments are included. Significant differences compared with medium control were assessed by the non-parametric Mann-Whitney U-test, and significance is indicated by *p,0.05, **p,0.01, and ***p,0.001. doi:10.1371/journal.pone.0021171.g001 are able to induce pro-inflammatory cytokine and chemokine production in DCs. Reduced percentage of HIV-infected DCs detected in co-cultures with ApoAct but not ApoRest The finding that several cytokines were released into the super- fonatants, including those with anti-HIV activity [13], prompted us to ask whether co-culturing with ApoAct could influence the efficiency of virus infection in DCs. We measured HIV infection by determining the percentage of cells expressing intracellular p24 antigen, as described (Figure 3A) [32,33]. Addition of 39-azido-39- deoxythymidine (AZT) to the DC cultures inhibited detection of p24, which showed that intracellular p24 as detected by flow cytometry was the result of productive infection in DCs [32]. Immature DCs were exposed to HIVBaL; there was large vari- ability between donors with regard to HIV infection efficiency, ranging from 4.0 to 46.4% after 7 days (Figure 3B). Co-culture of DCs with ApoAct, either uninfected or HIV-infected, resulted in a significantly reduced percentage of p24+ DCs as compared with DCs exposed only to HIVBaL (Figure 3B,C; p,0.01 and p,0.05, respectively). There was no significant reduction in p24 expression in DCs after exposure to ApoRest (Figure 3D). We detected similar induction of CD86 expression and reduction in percentage of p24 positive DCs using either autologous or allogeneic ApoAct (Figure S1). The critical step to gain the capacity to induce DC maturation and reduction in HIV-infection was the activation step (Figure 3D).We also measured HIV infection efficiency by quantitative PCR. There was an almost tenfold reduction in viral DNA copies in samples co-cultured with ApoAct but not ApoRest (Figure 3E). Alu-PCR was used to determine the relative propor- tion of integrated HIV DNA to investigate whether the inhibition occurred pre- or post-integration. The values obtained in the HIV Figure 2. Cytokine and chemokine released from DCs exposed to ApoAct or ApoInf. Immature DCs were co-cultured with activated apoptotic allogeneic CD4+ T cells for 24 hours, and culture supernatants were analyzed for the presence of TNF-a, MCP-1, MIP-1a and MIP-1b. DCs were cultured in medium alone (negative control) or in the presence of LPS, which is a potent DC activator (positive control). DCs were exposed to HIVBaL (HIV), anti-CD3– and anti-CD28–activated apoptotic CD4+ T cells (ApoAct), ApoAct in the presence of HIVBaL (ApoAct+HIV), apoptotic anti-CD3– and anti-CD28–activated CD4+ T cells infected with HIVBaL (ApoInf), or ApoInf in the presence of exogenous HIVBaL (ApoInf+HIV). Control wells included ApoAct without any DCs (no DC). The results shown are the mean 6 SD from at least seven donors except for the MCP-1 analysis with ApoInf which shows data from two donors. Significant differences compared with medium control were assessed by non-parametric Mann-Whitney U-test and Kruskal-Wallis test with Dunn’s multiple comparison test and are indicated by *(p,0.05), ** (p,0.01) and *** (p,0.001), respectively. Non- significant, NS. doi:10.1371/journal.pone.0021171.g002 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21171