the chemokine receptors CCR5 and CXCR4 as the main co- receptors for viral entry [12]. The chemokines MIP-1a, MIP-1b, and RANTES, which bind to CCR5, have potent anti-HIV activity [13]. Although many cytokines were initially described as having anti-HIV activities, subsequent or simultaneous studies using different experimental conditions have shown that they may also activate virus production [14,15]. TNF-a was identified as the predominant inducer of HIV production in supernatants from both monocyte and lymphocyte cultures [16,17]. The proviral activity is dependent on the activation of NF-kB present in the cytoplasm of both monocytes and lymphocytes [18–20]. The dual activity of TNF-a with regard to HIV replication was later suggested by a study showing inhibition of HIV replication before proviral DNA integration [21]. TNF-a was furthermore shown to induce DC maturation, leading to down-regulation of HIV co- receptors [22]. Hence, there are several cytokines, including TNF- a, IFN-c, TGF-b and MCP-1, that have either pro-HIV or anti- HIV effects depending on the experimental system and/or the types of cells analyzed [14,15]. In addition type I interferons were shown to induce the restriction factor APOBEC3G [23]. Members of the APOBEC family of proteins confer intrinsic immunity to retroviral infection [24]. APOBEC3G levels are increased upon DC maturation, suggesting a role for APOBEC3G as a possible candidate for viral restriction in maturing DCs exposed to HIV [23,25]. APOBEC3G is restricting HIV replication by deamina- tion of viral cDNA cytidine during reverse transcription. This will lead to a characteristic induction of G-to-A hypermutations. However, some studies suggest deaminase-independent effects by APOBEC3G proteins interfering with steps in reverse transcrip- tion or integration (reviewed in [23]). Uptake by DCs of apoptotic bodies originating from HIV-infected cells in vitro results in activation of CD4+ and CD8+ T cells [26–28]. Antigens contained in apoptotic bodies are introduced into MHC class I presentation pathways for cross-presentation and stimulation of HIV-specific CD8+ T cells. We recently demonstrated that activated, but not resting, apoptotic peripheral blood mononuclear cells (PBMCs) are able to induce maturation of DCs in terms of up- regulation of costimulatory molecules (CD80 and CD86) and induction of pro-inflammatory cytokine release [29]. In the present study, we determined whether apoptotic CD4+ T cells support DC maturation and whether HIV infection interferes with this process. We show that apoptotic HIV-infected (ApoInf) and uninfected activated CD4+ T cells (ApoAct) provide maturation signals to DCs that result in the release of TNF-a, MIP-1a, MIP-1b and MCP-1. Furthermore, upregulation of CD86 molecules in DCs was reduced in the presence of anti-TNF-a. We further found a significantly reduced percentage of HIV-infected DCs when they were co- cultured with ApoAct as compared with DCs co-cultured with apoptotic resting CD4+ T cells (ApoRest); this effect was partly blocked in the presence of anti-TNF-amAbs. We detected upregula- tion of APOBEC3G mRNA expression in DCs co-cultured with ApoAct but not with ApoRest. In addition, we demonstrated induction of G-to-A hypermutations in env-DNA isolated from HIV infected DCs co-cultured with ApoAct but not with ApoRest, as a sign of APOBEC3 activity in mature monocyte derived DCs These findings may have relevance for cell-associated HIV transmission and pathogenesis, as dying uninfected and HIV-infected cells constitute a characteristic hall mark in acute and chronic HIV infection [30,31]. Materials and Methods Human subjects and blood collection Buffy coats from healthy human blood donors were obtained from the blood bank at Karolinska University Hospital Huddinge. Apoptotic Activated T Cells Inhibit HIV in DCs Ethical approval was obtained from the medical ethics committee in Stockholm. In vitro differentiation of DCs CD14+ monocytes were enriched from buffy coats by negative selection using RosetteSep Human Monocyte Enrichment (1 ml/ 10 ml blood; Stem Cell Technologies, Vancouver, BC, Canada). Monocytes were separated using Lymphoprep density gradient (Nycomed, Oslo, Norway) and were cultured for 6 days in DC medium containing RPMI 1640 (GIBCO Life Technologies, Paisley, United Kingdom), supplemented with 1% HEPES (N-(2- hydroxyethyl)piperazine-N’-2-ethanesulfonic acid) (GIBCO), 2 mM L-glutamine (GIBCO), 1% streptomycin (GIBCO), and 1% peni- cillin (GIBCO), 10% endotoxin-free fetal bovine serum (FBS) (GIBCO), recombinant human cytokines IL-4 (6.5 ng/ml; R&D Systems, Minneapolis, MN) and granulocyte-macrophage colony- stimulating factor (GM-CSF; 250 ng/ml; Peprotech; London, United Kingdom) to obtain immature DCs [32]. Purification and activation of CD4+ T cells CD4+ T cells were enriched from buffy coats obtained from whole blood by negative selection using RosetteSep Human CD4+ T Cell Enrichment (1 ml/10 ml blood; Stem Cell Technologies). CD4+ T cells were separated using Lymphoprep density gradient (Nycomed). Cells were frozen in FBS and 10% dimethylsulfoxide (DMSO) or were added to flasks containing monoclonal anti- human CD3 (10 mg/ml; clone OKT 3; Ortho Biotech Inc., Raritan, NJ) that had been adhered to the plastic overnight at 4uC and soluble monoclonal anti-human CD28 (2 mg/ml; L293; BD Biosciences, San Diego, CA). After 24 hours of stimulation, cells were frozen in FBS/DMSO. On the day of experiment, frozen T cells were thawed, washed, and induced to undergo apoptosis by c-irradiation (150 Gy) as described [29,33-36]. HIV growth and preparation The HIV isolates HIVBaL (which uses CCR5 receptors) and HIVIIIB (which uses CXCR4 receptors) were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID, NIH, Bethesda, MD). HIV isolates were grown in PBMC cultures stimulated with phytohe- magglutinin (PHA; 2.5 mg/ml; Sigma, St Louis, MO) and IL-2 (75 IU/ml; Chiron, Emeryville, CA). Primary virus was isolated from HIV-infected patient blood by co-culture with PHA-acti- vated donor PBMCs. To concentrate the virus and to minimize the presence of bystander activation factors in the supernatant that could induce DC maturation, virus stocks were ultracentrifuged (138,0006g for 30 minutes at 4uC. (Beckman L-80 Ultra-cen- trifuge, rotor 70.1; Beckman Coulter, Fullerton, CA), and the virus pellets were resuspended in RPMI 1640 with 10% FBS to obtain a virus concentrate. An aliquot of each viral stock was thawed and titrated for infectivity using a limiting dilution culture method with PHA-activated PBMCs (mix of 3 different donors) [37]. The 50% tissue culture infectious dose (TCID50) was calculated by the method of Reed and Muench as described in [38]. The titers of virus stocks were 1.76106/ml TCID)50 for both HIVBaL and HIVIIIB. The primary isolates (207 and 208) had a TCID50 of 1.06103/ml and 4.06104/ml, respectively. HIV infection of CD4+ T cells and DCs Activated (24 hours with CD3 and CD28 monoclonal antibod- ies (mAbs) as described above) CD4+ T cells were incubated with HIVBaL (3000–6000 TCID50) in the presence of IL-2. The PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21171