. ... Table 1. List of primers designed for amplification of HIV Gag, . .. Rev, Vpr and Nef regions. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Primer Name Primer Sequence . . ... GAG F 124 ACTCTGGTAACTAGAGATCC ... GAG F 304 AATTTTGACTAGCGGAGGC . ... GAG F 334 AGATGGGTGCGAGAGCGT . ... GAG F 334.1 AGATGGGTGCGAGACCGT . ... GAG R 1881 GCTCCTGTATCTAATAGAGC ... GAG R 1881.1 GCTCCTGTATCTAATAAAGC . ... GAG R 1881.2 GCTCCTGTATCTAACAGAGC . ... GAG R 1913 TTTGGTTTCCATCTTCCTGG ... GAG R 1913.1 TTTGGTTTCCATCTTCCTGC . ... GAG R 1913.2 TTTGGCTTCCATCTCCCTGG . ... GAG R 1913.4 TTTGGTTTCCATTTCCCTGG .... GAG R 1913.5 TTTGGTTTCCATTTTCCTGG ... VPR F 4995 GCAGGACATAACAAGGTAGG . ... VPR F 4995.4 GCAGGACATAACAAAGTAGA . ... VPR F 5058 AAGATAAAGCCACCTTTGCC ... VPR R 5507 TTCTTCCTGCCATAGGAGATGC . ... VPR R 5507.1 TTCTTCCTGCCATAGGAAATGC . ... VPR R 5419 GCAGTTGTAGGCTGACTTCC .... VPR R 5419.1 GCAGTTGTAGGCTGACTCCC ... VPR R 5419.2 GCAGTTGTAGGCTGGCTTCC . ... REV F 7750 GGGATTTGGGGTTGCTCTGG .... REV F 7750.1 GGGATTTGGGGCTGCTCTGG ... REV F 7830 TGATAGTAGGAGGCTTGGTAGG . ... REV F 7830.1 TGATAGTAGGAGGCTTAATAGG . ... REV F 7830.2 TGATAGTAGGAGGCTTGATAGG ... REV F 7911 GTTAGGCAGGGATATTCACC . ... REV F 7911.1 GTTAGGCAGGGATACTCACC . ... REV R 8300 CCCTGTCTTATTCTTCTAGG .... REV R 8300.1 CCCTGTCTTATTCTTACAGG ... REV R 8300.2 CCCTGTCTTATTCTTGTAGG . ... NEF F 8235 TAGCTGAGGGGACAGATAG .... NEF F 8235.1 TAGCTGAGGGAACAGATAG ... NEF F 8235.2 TAGCTGGCTGGACAGATAG . ... NEF F 8343 ATGGGTGGCAAGTGGTCAAAAAG . ... NEF F 8343.1 ATGGGTGGCAAGTGGTCAAAACG ... NEF F 8343.2 ATGGGTGGCAAATGGTCAAAAAG . ... NEF F 8343.3 ATGGGTGGCAAGTGGTCAAAAGG . ... NEF R 9069 CCAGTACAGGCAAAAAGC .... NEF R 9069.1 CAGTACAGGCGAAAAGC ... NEF R 9069.2 CAGTACAGGCAAGAAGC . . .. . All sequences listed in 59-39 orientation. 59 primers are represented by letter F . . (forward) and 39 primers are represented by letter R (reverse). Numbers . .. correspond to relative location of each primer within sequence chosen as the .. reference. . .. doi:10.1371/journal.pone.0001489.t001 HIV RNA-transfected DCs stimulate antigen specific T cells in vitro The goal of active HIV immunotherapy is to stimulate the preferential expansion of antiviral effector T cells. To demonstrate that HIV RNAs generated by our approach can express antigens RT-PCR for Autol DC Therapy . . Table 2. Composition of primer groups. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... GAG F 124 VPR F 4995 REV F 7750 NEF F 8235 . ... GAG F 124 VPR F 4995 REV F 7750 NEF F 8235 . ... GAG F 304 VPR F 4995.4 REV F 7750.1 NEF F 8235.1 .... GAG F 304 VPR F 5058 REV F 7830 NEF F 8235.2 ... GAG F 334 VPR F 5058 REV F 7830 NEF F 8343 . ... GAG F 334 VPR R 5507 REV F 7830.1 NEF F 8343 . ... GAG F 334.1 VPR R 5507 REV F 7830.2 NEF F 8343.1 ... GAG R 1881 VPR R 5507.1 REV F 7911 NEF F 8343.2 . ... GAG R 1881 VPR R 5419 REV F 7911 NEF F 8343.3 . ... GAG R 1881.1 VPR R 5419 REV F 7911.1 NEF R 9069 . .. GAG R 1881.2 VPR R 5419.1 REV R 8300 NEF R 9069 . ... GAG R 1913 VPR R 5419.2 REV R 8300 NEF R 9069.1 . ... GAG R 1913 REV R 8300.1 NEF R 9069.2 . ... GAG R 1913.1 REV R 8300.2 ... GAG R 1913.2 . ... GAG R 1913.4 . ... GAG R 1913.5 . . .. Primers were combined according to their position in the genome. . .. Bold indicates the primer group name. Non-bold: names of the primers which ... comprise each primer groups. Sequence of all primers is given in Table 1. .. doi:10.1371/journal.pone.0001489.t002 capable of inducing CD8+ T-cell immunity, we prepared DC electroporated with all four autologous HIV antigens encoded as RNAs. 1 mg Gag RNA, 0.25 mg Nef RNA, 1 mg Rev RNA, and 1 mg Vpr RNA were electroporated along with 1 mg of CD40L RNA per 106 DC. Since cells were fully matured by overnight incubation in the presence of TNFa, INFc and PGE2 the maturation status of the DCs did not change after electroporation with the RNAs (Data not shown). These cells were co-cultured with autologous PBMCs pre-labeled with CFSE. After 6 days of co-culture, the frequency and phenotype of proliferating cells was detected by residual CFSE fluorescence within the CD8+ T cell population with effector (CD45RA+/CD282) or effector/memory (CD45RA2/ CD28+) phenotypes. After 6 days of co-culture, the CD8+ T-cell population was stimulated with either eGFP RNA-transfected DC (negative control) or HIV RNA-transfected DC. The frequency of CFSE-low cells stimulated with GFP RNA-loaded DC was 3.75% while those stimulated with HIV RNA-loaded DC had a frequency of 7.41% (Figure 4 Panel A). No proliferation above the negative control background was observed within the CD4+ T cell subset, with all DC populations inducing ,1% CD4+ CFSE-low cells within total PBMCs (data not shown). Within the proliferating CFSE-low CD8+ T cell subset stimulated with HIV RNA-loaded DC, 24.7% of cells exhibited a phenotype consistent with fully differentiated effector cells (CD8+CD282CD45RA+) versus 54.8% of cells had a phenotype indicative of effector/memory cells (CD8+CD28+CD45RA2) (Figure 4 Panel B). To assess the specificity and effector function of the T cells responding with antigen-induced proliferation, the cultures were further re-stimulated with either DC electroporated with GFP RNA (negative control), a pool of all 4 HIV RNAs, or each HIV RNA independently. After 4 hours CFSE-low CD8+ T cells were tested for IFN-c expression by intracellular staining. Induction of IFN-c expression above the GFP negative control background was observed for all HIV RNA DC-stimulated cultures (Figure 4, Panel C). Co-cultures that were originally established with GFP RNA-electroporated control DC for 6 days and then restimulated PLoS ONE | www.plosone.org 4 January 2008 | Issue 1 | e1489