infected DC were set to one in all donors. There was a significantly reduced value obtained in DCs co-cultured with ApoAct, sug- gesting that a block occurred at least in part prior to HIV integration (Figure 3F). We next performed dose-response experiments to investigate the DC/ApoAct concentration required to obtain a reduced percentage of infection in DCs. DCs were co-cultured with serially diluted ApoAct (Figure S2). We detected an increasing percentage of p24+ DCs with decreasing numbers of ApoAct. We also performed kinetic experiments by first incubating DCs with HIVBaL for 30 minutes, 1 hour, or 2 hours followed by addition of ApoAct. We observed the same reduction in p24 expression if the DCs were exposed to the virus for 30 min, 1 hour or 2 hours prior to addition of ApoAct (Figure S3A). Conversely, DCs were first exposed to ApoAct for 30 minutes, 1 hour, or 2 hours before addition of HIVBaL. Again, the percentages of p24+ DCs were similar for all three pre-incubation time points (Figure S3A). We also performed experiments with longer kinetics by adding ApoAct 16 hours after HIVBaL infection of DCs or adding ApoAct 16 hours before infection. Again, reduced percentage of infected DCs was detected when they were co-cultured with ApoAct under any of these conditions (Figure S3B). ApoAct- mediated inhibition of HIV in DCs was also detected using three additional virus isolates (HIVIIIB and primary isolates 207 and 208) (Figure S4). TNF-a released in conditioned media from DC/ApoAct partially blocks HIV infection in DCs To assess whether conditioned medium (CM) collected from DC/ApoAct co-cultures reduces HIV infection, we collected supernatants after 24 hours of co-culture to use as CM. Different amounts of CM were added to cultures of immature DCs and HIV. There was a dose-dependent response in the reduction of p24 expression in DCs after exposure to CM (Figure 4A). To further elucidate the role of CM mediated reduction of HIV infection in DCs and to investigate possible candidate cytokines and/or chemokines for the effect, we added CM from DC/ ApoAct cultures to DCs exposed to HIVBaL (Figure 4B,C). A reduced p24 expression was detected in DCs exposed to HIV and CM as compared with DCs exposed to only HIV (p,0.05). The addition of blocking antibodies to MIP-1a, MIP-1b, IFN-c and RANTES to the CM did not restore p24 expression, whereas addition of blocking antibody to TNF-a did (Figure 4B). CM added 2 hours or 16 hours after HIV exposure did not sig- nificantly reduce p24 expression in DCs (Figure 4C). However, ApoAct added 2 hours or 16 hours after HIV exposure reduced HIV p24 expression in DCs (Figure 4D; p,0.001). The HIV inhibitory effect by ApoAct was partially lost in the presence of TNF-a mAb (Figure 4D). In addition, the TNF-a mAb blocked CM-mediated up-regulation of CD86 in DCs (Figure 4E; p,0.05). We next treated immature DCs with different concentrations recombinant TNF-a and exposed them to HIV. There was a dose- dependent inhibition of HIV infection in DC by TNF-a where the highest concentration (25 ng/ml) showed an effective inhibition (Figure 4F). The HIV inhibitory effect by TNF-a was lost in the presence of TNF-a mAb (Figure 4F). These data show that TNF-a treatment can inhibit HIV infection in these DCs and that the HIV inhibitory effect by ApoAct can be partially explained by release of TNF-a. APOBEC3G-mediated HIV inhibition in DCs exposed to ApoAct The anti-viral factor APOBEC3G is induced upon DC maturation by LPS [25]. We therefore investigated whether Apoptotic Activated T Cells Inhibit HIV in DCs APOBEC3G was induced after ApoAct-mediated DC maturation. RNA was prepared from DC co-cultures after 2, 6, 24, and 48 hours, and APOBEC3G expression was assessed by real-time PCR. We detected induction of APOBEC3G after LPS stimula- tion and after ApoAct exposure in DCs when analyzed at the time points 6, 24 and 48 hours. However, we did not detect APOBEC3G after 2 hours of incubation, which showed that the APOBEC3G detected was not derived from the ApoAct. DC co- cultures with ApoRest did not promote induction of APOBEC3G (Figure 5A). In control experiments, we used two different house- keeping genes; GAPDH and 18S RNA, to normalize the data and obtained a similar pattern of upregulation of APOBEC3G after stimulation with either LPS or ApoAct (Figure S5). To investigate whether APOBEC3G induced by ApoAct played a role in the HIV inhibitory effect in DCs, we silenced APOBEC3G using siRNA. The transfection procedure per se, neither altered the expression levels of APOBEC3G, nor induced secretion of TNF-a (Figure S6). The efficiency of silencing as revealed by real-time PCR was in the range of 79–90% in four different DCs donors co-cultured with ApoAct. The silencing of APOBEC3G resulted in a significantly increased p24 frequency (p,0.03 Wilcoxon paired test) that returned to baseline infection levels (Figure 5B). These data show induction of APOBEC3G expression in DC upon co-culture with ApoAct but not ApoRest. Moreover, siRNA analyses revealed that the HIV inhibitory effect mediated by ApoAct can largely be explained by induction of APOBEC3G in DCs. To verify that APOBEC3 proteins exerted a function to restrict HIV infection in the DC, we sequenced a region in env and measured the frequencies of G-to-A mutations using the program HYPERMUT 2.0. We sequenced 6 clones from HIVBa-L infected DC of which one was used as reference sequence, 35 clones from HIVBa-L infected DC co-cultured with ApoRest and 26 from DC co-cultured with ApoAct (Table 1). In total 21,000 base pairs were sequenced from ApoRest co-cultures and 15,600 from ApoAct. None of the clones sequenced from ApoRest co-cultures were found to have significant induction of G- to-A hypermutations according to the HYPERMUT program, while 3 clones had significant p,0.05 Fisher exact p-value in co- cultures with ApoAct. The total number of G-to-A mutations detected in ApoRest co-cultures were 39 while 201 mutations were detected in clones derived from ApoAct co-cultures. The average numbers of G-to-A mutations per 100 base pairs were 0.09 and 1.288 in the ApoRest and ApoAct co-cultures, respectively. G-to-A mutations were detected both in the GG and GA context. There was a significant difference (p,0.03 unpaired t-test) in the muta- tion frequency in clones obtained from ApoRest DC co-cultures compared with ApoAct (Figure 5C). These data demonstrate signs of APOBEC3G activity in DCs after co-culture with ApoAct. Discussion Infection of DCs is likely to be an important contributor to the spread of HIV by capturing virus at sites of viral entry followed by migration to lymphoid tissues, which constitute an optimal site for HIV transmission to T cells [30]. Production of HIV can be blocked by exposure of DCs to maturation stimuli such as for example LPS [3,10,48]. Here we have demonstrated that apo- ptotic activated CD4+ T cells (ApoAct), either uninfected or recently HIV-infected (ApoInf), induce expression of co-stimula- tory molecules, release of cytokines as well as reduce HIV infection in DCs, whereas unactivated apoptotic CD4+ T cell (ApoRest) do not. We show that induction of the restriction factor APOBEC3G was important for the antiviral activity in the DCs. However, these findings do not preclude involvement of other restriction factors PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21171