percentage of infected cells was analyzed by intracellular staining for the HIV Gag protein p24 at 3, 4, 5, 6, 7, and 10 days after infection [32,33]. The highest percentage of cells infected was detected at days 4, 5, and 6; thereafter infection percentages declined (data not shown). Batches of infected cells collected at day 3–4 were frozen in FBS/DMSO until use. For infection of DCs, 3000–6000 TCID50 of HIVBaL and HIVIIIB, and 500 TCID50 of the primary isolates were used. The percentage of infected DCs was determined by intracellular p24 staining after 7 days of infection. Quantification of HIV protein in T cells and DCs The percentage of HIVBaL-infected DCs and CD4+ T cells was determined by intracellular staining for p24 [32,33]. Cells were first stained for cell surface markers and then fixed in 2% formaldehyde (Sigma) for 10 minutes at room temperature. Cells were washed in saponin solution consisting of PBS with 2% FBS, 2% HEPES, and 0.1% saponin (Sigma), to allow permeabilization of the cell surface membrane, and then incubated for 1–2 hours at 4uC with a p24 mAb (clone KC57; Coulter, Hialeah, FL) or the corresponding isotype control antibody. Intracellular p24 expres- sion was assessed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest Software (Becton Dickinson, San Jose, CA). DC and apoptotic CD4+ T cell co-cultures Differentiated, immature DCs were co-cultured with irradiated CD4+ T cells at a ratio of 1:2 DC:T cells in DC medium. Con- ditioned medium (CM) was prepared by co-culturing DCs with ApoAct for 24 hours. In some experiments, CM was added to the DC cultures at 40% of the total volume. Supernatants were col- lected from co-cultures at 4, 8, and 24 hours. The TLR4 agonist LPS (100 ng/ml; Sigma) was used as a positive control for activation and maturation of DCs. DCs were exposed to HIVBaL, and apoptotic cells or CM was added at 0, 2, or 16 hours. For cytokine blocking experiments, CM was pre-incubated for 2 hours at 37uC with blocking antibodies alone or in combination (all obtained from R&D Systems): anti-MIP-1a (clone 14215; 2.5 mg/ ml), anti-MIP-1b (clone 24006; 1 mg/ml), anti-IFN-c (clone 25718; 4 mg/ml), anti-RANTES (clone 21445; 2.5 mg/ml), and anti-TNF-a (clone 1825; 0.5 mg/ml). In DC/ApoAct co-cultures, anti-TNF-a was added at the beginning of the co-culture. DCs were analyzed by flow cytometry for p24 and CD86 expression 7 days after infection. Phenotypic characterization of DCs and T cells DCs were stained with the following anti-human mAbs: CD1a (clone NA1/34; DAKO, Glostrup, Denmark), CD14 (clone TU¨ K4; DAKO), CD3 (clone SK7; BD Biosciences), CD83 (clone HB15e; BD Biosciences), and CD86 (clone 2331/FUN-1; BD Biosciences). T cells were stained with anti-human mAbs CD19 (clone HD37; DAKO), CD3 (clone SK7; BD Biosciences), CD4 (clone RPA-T4; BD Biosciences), CD8 (clone SK-1; BD Biosciences), CD25 (clone 2A3; BD Biosciences), and CD69 (FN50; BD Biosciences). Cell surface expression was measured by a FACSCalibur flow cytometer (Becton Dickinson). Co-culture samples were collected at 72 hours or 7 days, washed, and incu- bated with the previously mentioned CD1a, CD83, CD86, and p24 mAbs, as indicated. Cytokine and chemokine production Supernatants from apoptotic CD4+ T cells and DC co-cultures were analyzed for cytokines and chemokines with the Bio-Plex Apoptotic Activated T Cells Inhibit HIV in DCs assay (Biosource, Nivelles, Belgium). A Luminex reader (Luminex Corporation, Austin, TX) was then used to simultaneously quan- tify the concentration of IL-2, RANTES, IL-10, IL-12, TNF-a, MCP-1, MIP-1a, and MIP-1b in the supernatants. Quantification of HIV-long terminal repeat (LTR) in DCs Genomic DNA was extracted from infected DCs using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA). HIV-LTR sense and anti-sense primer sequences were 59-GCCTCAA- TAAAGCTTGCCTTGA-39 and 59-GGGCGCCACTGCTAG- AGA-39. The probe sequence was 59-6-FAM-CCAGAGTCACA- CAACAGACGGGCACA-TAMRA-39, and the resulting amplicon was 121 bp. Primer and probe design was based on published sequences [39] and were from Applied Biosystems, Foster City, CA. Real-time PCR reactions were performed in 10-ml reactions (Applied Biosystems) using the 7500 Real-Time PCR System (Applied Biosystems). For absolute quantification of HIV-LTR, a control plasmid was constructed by cloning the 121-bp HIV-LTR amplicon into pCRH4-TOPOH (Invitrogen Life Science). Plasmid copy number was quantified as described [40]. HIV-1-integration Alu-PCR assay Total cell DNA was isolated at 72 h after infection with a QIAamp blood isolation kit (Qiagen). Integrated HIV-1 DNA was measured by two-step Alu-PCR [6]. In the first round of pre- amplification PCR, Alu-LTR sequences were amplified with an HIV-1-specific primer (LTR R region) in combination with a primer that anneals to the abundant genomic Alu repeats. The HIV-1-specific primer was extended with a marker region at the 59 end, which was used for specificity in the second-round PCR. The second round was nested quantitative real-time PCR of the first- round PCR products with primers annealing to the aforemen- tioned marker region in combination with a HIV-1-specific primer (LTR U5 region). Primer sequences were as follows: first round, HIV-1 LTR R forward, 59-ATGCCACGTAAGCGAAACTG- CTGGCTAACTAGGGAACCCACTG-39 (marker sequence un- derlined); Alu reverse, 59-TCCCAGCTACTGGGGAGGCTG- AGG-39; second-round marker forward, 59-ATGCCACGTAA- GCGAAACTG-39; HIV-1 LTR U5 reverse, 59-CACACTGAC- TAAAAGGGTCTGAGG-39. Samples were assayed at two con- centrations to ensure that PCR inhibitors were absent. Dilutions were prepared with genomic DNA from uninfected cells to ensure that the number of Alu sites per reaction mixture remained constant. For monitoring of the signal contributed by non- integrated HIV-1 DNA, the first-round PCR was also done with the HIV-1-specific primer alone as a control. HIV-1 integration was normalized relative to GAPDH DNA. For relative HIV-1 integration, the value obtained for BaL-infected DCwere set to 1 for each donor and compared with DC from the same donor co-cultured with ApoAct or ApoRest. Real-Time PCR analysis and silencing of APOBEC3G RNA was extracted either from freshly isolated DCs or from DCs co-cultured with LPS or with ApoRest or ApoAct as described above. RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Amplifi- cation of APOBEC3G, 18s RNA and GAPDH cDNA was performed using the 7500 Real-Time PCR System (Applied Biosystems) and 6-carboxyfluorescein dye-labeled TaqMan MGB probes and primers (Applied Biosystems) [41-43]. We did a homology BLAST for the primer-probe, which is highly specific to APOBEC3G. The reference sequence ID is NM_021822.3, NCBI chromosome location is Chr. 22 - 39473010 – 39483748. The PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21171