Item Analyzed Total no. of clones sequenced Total no. of base pairs sequenced Total no. of clones with Fisher Exact p-value ,0.05 Total no. of G-to-A mutations No. of G-to-A mutations (GG context) No. of G-to-A mutations (GA context) Avg no. of G-to-A mutations/100 bp 21,000 35 0 39 8 11 0.090 Apoptotic Activated T Cells Inhibit HIV in DCs 26 15,600 3 201 60 40 1.288 Table 1. Summary of G-to-A mutation frequencies in HIV-1 env sequences obtained from HIVBaL infected DC co-cultures. DC co-cultured with ApoRest DC co-cultured with ApoAct doi:10.1371/journal.pone.0021171.t001 complete elimination of HIV p24+ cells in the cultures. In addition, our data suggests that the inhibition was occurring both pre- and post-integration. We showed that the anti-retroviral restriction factor APOBEC3G was important for the anti-viral effect in the present system by silencing APOBEC3G in DCs prior to addition of HIV and ApoAct. Signs of APOBEC3 activity were measured by sequence analyses of a region in env, which revealed significant induction of G-to-A hypermutations in co-cultures with ApoAct but not ApoRest. Of note is that the mutations occurred both in a GG and GA context, which implies that the mutations were induced at least in part by APOBEC3G as GG dinucleotides are target sites specific for APOBEC3G [23]. APOBEC3A was also reported to induce G-to-A mutations in a GG context but there is currently little data showing a HIV restricting role for APOBEC3A [23]. Several family members of the APOBEC3 family were reported to induce G-to-A hypermutations leading to HIV restriction in a GA context of which APOBEC3F is well studied [23]. The present system used a complete HIVBaL with capacity to produce Vif, a protein know to counteract the antiviral effect by APOBEC3G [23]. We observed the antiviral activity after a 7-10 fold increase in APOBEC3G mRNA expression, which appeared sufficient to restrict HIV-1 replication in the DCs. We previously reported that apoptotic PBMCs obtained after 24 hours of activation with either CD3/CD28 mAbs or PHA efficiently support DC maturation, while 4 days of PHA activation prior to apoptosis induction resulted in a reduced capacity (although still readily detected) to induce CD86 expression [29]. We therefore here activated CD4+ T cells with CD3/CD28 mAbs for 24 hours and induced apoptosis no more than 3–4 days after initial T cell activation in either uninfected or HIV infected CD4+ T cells. Hence, we induced apoptosis before extensive HIV- mediated apoptosis occurred in the cultures. Longer exposures to activation or activation induced cell death as well as resting apoptotic cells do not promote DC maturation and such cells are instead involved in peripheral tolerance mechanisms [29,51–53]. Cell death is a hallmark of HIV pathogenesis [31]. SIV infection is accompanied by an early depletion of CD4+ T cells in the gut of macaques, and progression to AIDS is characterized by depletion of CD4+ T cells [54]. However, it is not only HIV-infected cells that die during HIV infection. There is also a massive depletion of non-infected bystander cells. HIV-mediated bystander killing was suggested to occur through viral molecules, such as Nef, Tat, Vpr, Vpu, Vif, and Env [55]. Increased apoptotic cell death occurs during HIV infection, but the question of which type of cell death that is induced in HIV-infected cells in vivo is still somewhat controversial [31,56]. It is largely unknown whether the HIV- infected cells that undergo cell death in vivo are dying an immu- nogenic cell death. Nevertheless, the contribution of different apoptotic cells during HIV infection in vivo is likely to be complex and the integrated net effect may in part depend on whether the apoptotic cells are dying in a state when they are able to induce cytokine production and DC maturation. Hence, we propose to make a clear distinction between recently activated cells, cells that have been activated for a prolonged period before apoptosis, and resting apoptotic T cells regarding subsequent DC-induced re- sponses. The data presented here suggest that apoptotic CD4+ T cells that were efficiently activated prior to their cell death may be an important mechanism to reduce HIV infection of DCs. Aberrant immune activation is another characteristic hall mark of HIV infection [57] and it remains to be investigated whether the apoptotic lymphocytes formed during HIV infection has capacity to reduce HIV infection in DCs. Together these findings show that apoptotic activated CD4+ T cells, either uninfected or HIV-infected, have the capacity to induce DC maturation and cytokine release of TNF-a, MIP-1a, MIP-1b, and MCP-1. In addition, we detected a reduced percent- age of HIV-infected DCs after exposure to ApoAct or conditioned media from DC/ApoAct co-cultures but not with ApoRest. We revealed that TNF-a was at least partially mediating the maturation and reduced HIV infection in DCs. We could show that the anti-viral effect induced by ApoAct was largely dependent on upregulation of the restriction factor APOBEC3G in the DCs leading to induction of G-to-A hypermutations. These findings have implications for transmission of HIV and HIV pathogenesis involving interactions between HIV, apoptotic cells and DCs. Supporting Information Figure S1 Increase in the percentage of CD86+ cells and reduced percentage of HIV-infected DCs after exposure to either autologous or allogeneic ApoAct. (A) The capacity to induce CD86 expression in DCs by either autologous (AutoApoAct) or allogeneic (AlloApoAct) apoptotic anti-CD3– and anti-CD28–activated CD4+ T cells was measured by flow cytometry after 72 hours in co-culture. The mean 6 SD from nine donors is shown. (B) The capacities of AutoApoAct and AlloApoAct PLoS ONE | www.plosone.org 11 June 2011 | Volume 6 | Issue 6 | e21171