primer- probe was designed to target 5- 6 exon boundary of APOBEC3G and the amplicon length was 80 bp. Cycle threshold values for APOBEC3G were normalized to the value for GAPDH or 18S in control experiments. Data are presented as fold changes in mRNA copy number in the DCs co-cultures as compared to mRNA in DCs cultured in medium only. DCs were transfected with 25 nM siRNA with the transfection reagent DF4 (Dharma- con, Thermo Fisher Scientific) and were used for experiments 36 hours after transfection [6]. The siRNA (SMARTpool; Dharma- con) specific for APOBEC3G was purchased from Dharmacon (M- 013072-00). Non-targeting siRNA (D-001206-13; Dharmacon) served a control. This protocol resulted in a transfection efficiency of nearly 100%, as determined by flow cytometry of cells transfected with siGLO RISC-Free Control siRNA (D-001600-01; Dharma- con), and 79–90%of silencing of expression of APOBEC3G was achieved as verified by real-time PCR analysis. HIV-1 env sequencing and analysis. Total cell DNA was isolated 72 h after infection with a QIAamp DNA mini kit (Qiagen). HIV-1 env V1-V5 gene region was amplified by performing high-fidelity nested PCR using an EasyA kit (Stratagene). The primers used were as follows: Round 1 (product size 1415 bp) Forward 59 TTGCAATAGAAAAATT- CTCCTC 39, Reverse (59CCTGGTGGGTGCTACTCCTA 39). Round 2 (product size 1098 pb) Forward 59 CCATGTGTA- AAGTTAACCCC 39, Reverse, 59 ATGAGGGACAATTGAG- AAGTGTCTAG 39, PCR condition as described in [44]. Amplicons were purified by using a high pure PCR product purification kit (Roche), and cloned into the TA cloning vector provided in a TOPO TA cloning kit (Invitrogen) in accordance with the manufacturer’s instructions. Plasmids were isolated using Genejet plasmid mini prep kit (Fermentas) and the plasmids were sent to Eurofins MWG Operon, Germany for sequencing. Primers M13 forward (59 GTAAAACGACGGCCAG 39) and M13 reverse (59 CAGGAAACAGCTATGAC 39) were used for sequencing. G-to-A mutations were analyzed by using the program Hypermut 2.0 that is freely available at http://www.hiv.lanl.gov/content/ sequence/HYPERMUT/hypermut.html [45]. Results Up-regulation of co-stimulatory molecules on DCs after co-culture with apoptotic activated CD4+ T cells; uninfected (ApoAct) and HIV-infected (ApoInf) We previously demonstrated that apoptotic anti-CD3/CD28– activated PBMCs, but not apoptotic resting PBMCs, can induce DC maturation [29]. Here we first investigated whether apoptotic HIV-infected activated CD4+ T cells (ApoInf) are able to induce DC maturation. Primary CD4+ T cells were activated with CD3 and CD28 mAbs for 24 hours before infection with HIV. The efficiency of T cell activation was determined by expression of CD25 and CD69. After activation, the percentage of CD25+CD4+ T cells was 49–66%, and the percentage of CD69+CD4+ T cells was 44–48%, as compared with 0.7–13% and 0.4–6.3%, respectively, before activation (data not shown). We next infected activated CD4+ T cells with HIVBaL. The infection efficiency, as determined by intracellular p24 staining, was 28–43% (data not shown). Batches of cells were frozen, and on the day of experi- ment, cells were thawed, washed, and induced to undergo apoptosis by c-irradiation as described [29,33–36]. In the majority of experiments, c-irradiated T cells were added to the DC cultures immediately after radiation exposure to allow early apoptotic events to occur in the co-cultures. We used in vitro–differentiated monocytes cultured for 6 days in the presence of IL-4 and GM-CSF as the source of human Apoptotic Activated T Cells Inhibit HIV in DCs immature DCs, which are defined by the expression of CD1a; absence of CD14; and low expression of CD40, CD80, CD86, and CD83 [29,32]. These immature DCs were co-cultured with ApoAct or ApoInf for 72 hours or 7 days and then analyzed for expression of the co-stimulatory molecule CD86 (Figure 1). Repre- sentative flow cytometric analyses are depicted in Figure 1A, and a summary of 11 donors is shown in Figure 1B. The percentage of CD86+ DCs was 91.162.5% after LPS stimulation and the background medium control was 26.965.3% after 72 hours. DCs exposed to HIVBaL viral particles did not significantly up-regulate CD86 after 72 hours or 7 days of culture as compared with the medium control (Figure 1B) although some donors had a low induction of CD86 (Figure 1A). It should be noted that the virus stock used in the present study was ultra-centrifuged to remove cell debris and possible bystander soluble factors. Co-culture with ApoAct or ApoInf, on the other hand, resulted in a robust and significant induction of CD86 as compared with medium control both after 72 hours (p,0.001) and 7 days (p,0.01 and p,0.05, respectively) of culture (Figure 1B). It is conceivable that the local milieu in vivo upon DC encounter with infected or uninfected apoptotic cells will contain viral particles. We therefore investi- gated whether this mechanism for induction of DC maturation was functional in the presence of viral particles. We found that the maturation signal provided by the ApoAct or ApoInf occurred even in the presence of free HIVBaL, and the efficiency of induced CD86 expression was comparable to that of the positive control. The expression of CD83, another molecule associated with DC maturation and functional antigen-presenting capacity, showed an expression pattern that was similar to that of CD86 but with a tendency of lower expression than CD86. These findings show that both ApoAct and ApoInf are able to provide a maturation signal to immature DCs even in the presence of exogenous HIV. Cytokines and chemokines are secreted after co-culture of DCs with ApoAct and ApoInf To address whether cytokine production was induced in DCs after exposure to ApoAct and/or ApoInf, we collected superna- tants from the co-cultures after 4, 8 and 24 hours and used a multiplexed cytokine assay for simultaneous analyses of MIP-1a, MIP-1b, TNF-a, IL-10, IL-12, and MCP-1. There was a significant induction of TNF-a from the co-cultures with apoptotic anti-CD3– and anti-CD28–activated CD4+ T cells either unin- fected (p,0.001) or HIV-infected cells (p,0.001), as compared with medium control after 24 hours of co-culture (Figure 2). TNF- a was detected in co-culture supernatants even when exogenous HIVBaL was present in the cultures and reached quantities in a similar range as LPS stimulation. We did not detect any secretion of the cytokines analyzed from the ApoAct per se (denoted as ‘‘no DC’’; Figure 2). Co-cultures with apoptotic resting CD4+ T cells (ApoRest) or apoptotic neutrophils did not result in any secretion of the mentioned cytokines (data not shown and [29]). We could not detect any significant production of IL-10 or IL-12p70 after co-culturing with ApoAct, regardless of infection status (data not shown). Production of the chemokine MCP-1, which enhances mucosal IgA secretion and cytotoxic T cell responses [46], was detected in supernatants from co-cultures of DCs and ApoAct (p,0.001 as compared with medium control; Figure 2). MCP-1 production also occurred in the presence of exogenous HIVBaL. There were significantly increased quantities of MCP-1 secreted in co-cultures with ApoAct as compared with LPS (p,0.05). We also detected significant induction of MIP-1a and MIP-1b expression in co-cultures with ApoAct, regardless of HIV infection (Figure 2). Altogether, these findings suggest that both ApoAct and ApoInf PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21171