Institutional Review Board of Massachusetts General Hospital and the Fenway Community Heath Care Center, Boston or via Center for HIV/AIDS Vaccine Immunology (CHAVI) clinical sites . All patients provided written informed consent for the collection of samples and subsequent analysis. Culture medium, cytokines, and reagents Culture medium RPMI 1640 (Mediatech, Herndon, VA) or Yssel’s T cell medium (Gemini Bio-Products, Sacramento, CA) was supplemented with 20ug/ml gentamicin (Gibco BRL, Gaithersburg, MD) and 1mM HEPES (Mediatech), and 1% human plasma or 5% PHS (Valley Biomedical, Winchester, VA). rhGM-CSF (Immunex, Seattle, WA) and rhIL-4 (R&D Systems, Minneapolis, MN) were obtained for the generation of DCs. rhIL- 12, rhIL-6, and rhIL-7 (R&D Systems) and rhIL-2 (Chiron Corp, Emeryville, CA) were purchased for the priming cultures. Dendritic cells Leukopacks were purchased from BRT Laboratories, Inc. (Baltimore, MD) Donors were tested prior to their initial donation and again at each donation for the presence antibodies to HBV, HCV, HTLV-I and II, HIV-1/HIV-2 and syphilis. PBMCs were separated by density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ) DCs were grown from adherent PBMCs supplemented with 300U/ml IL-4 and 100IU/ml GM-CSF. After five days, immature DCs were collected, transferred to new plates, and induced to mature by adding a cocktail consisting of 150ng/ml IL-6, 5ng/ml TNF, and 5ng/ml IL-1b (R&D Systems) and 1 mg/ml PGE2 (Sigma). Viruses and infection of cells Virions of HIV-1MN (X4-tropic), clade B were produced by infection of CL.4/CEMX174 (T1) cells and purified by sucrose gradient ultracentrifugation as described previously[61]. Samples were titrated for the presence of infectious virus using AA2CL.1 cells and HIV-1 p24 antigen capture kits (AVP, NCI). Aldrithiol-2 (AT-2)-inactivated HIV-1 (AT-2 HIV) was prepared as described previously[62]. Used in this study include infectious HIV-1MN (Lots 3807, 3966, and 3941: AVP, SAIC Frederick, Inc., NCI Frederick) and AT-2 HIV-1MN (Lots 3808, 3965, 3937, and 3934). 1500ng/106 p24 equivalents of HIV-1 was added to the cytokine- matured DCs on day six and incubated overnight (14–16 hours) at 37uC and after incubation the DCs were washed. Priming and Restimulation of T cells Na¨ıve CD4+ and CD8+ T cells were isolated by negative selection from frozen PBMCs, using magnetic beads (mb) to remove monocytes (CD14mb), B cells (CD19mb), NK cells (CD56mb), and memory T cells (CD45ROmb) (Miltenyi Biotec, Auburn, CA) and co-cultured with the HIV-pulsed MDDCs at a 1:10 or 1:15 ratio. Priming cultures were restimulated at day 7, 14, 21, and 28 with the addition of autologous HIV-pulsed MDDCs to the priming cultures. Measurement of expansion of antigen-specific CD8+ and CD4+ T cells after 7 days expansion by recall ELISPOT Mature MDDCs pulsed with microvesicles, AT-2 or infectious HIV-1 were cultured with autologous bulk T cells at 1:10–1:30 ratios for 7 days. The expanded T cells were harvested and added to IFN-c ELISPOT plates at 50,000 T cells/well and restimulated with DCs infected with vaccinia-vectors (MOI = 2) (V-ctr, V-env, V-gag, V-pol, V-nef) or pulsed with control, gp160, nef, p66, or p24 proteins (5 mg/ml; Protein Sciences Corporation, Meriden, HIV Specific T Cell Priming CT). The ratio of DCs to T cells was 1:10. The ELISPOT assays were carried out as described above. DCs infected with vaccinia- vectors were used to monitor CD8+ T cell responses[63] and HIV-protein pulsed MDDCs were used to monitor CD4+ T cell responses. Mapping of Epitopes Mapping of epitopes was done using pools of overlapping peptides based on the consensus clade B sequence spanning the entire HIV proteome (Los Alamos immunology database). The peptides were mostly 18-mers and overlapped by 10 amino acids. To facilitate the screening of the peptides, peptide pool matrices were used in ELISPOT assays as described previously[19]. Reconfirmations of positive wells were done with single peptides. Assessment of polyfunctionality of primed CD4+ and CD8+ T cells Analysis of the capacity of primed CD4+ and CD8+ T cells to secrete multiple cytokines/chemokines was done by initially stimulating them with MDDCs pulsed with 1500ng/106 p24 equivalents HIV-1 for 6 hours with 10 mg/ml of the intracellular transport inhibitor brefeldin A (BFA; Sigma) before staining for the intracellular production of cytokines/chemokines. Antibodies and flow cytometry MDDCs were stained with PE-conjugated antibodies to CD80, CD83, CD86, CD40, CD209, MHC class I and MHC class II (BD Pharmingen San Jose, CA). Primed T cells were stained with fluorochrome-conjugated antibodies to CD3, CD4, CD8, and IFN-c (BD Pharmingen). For the assessment of cytokine/ chemokine production, primed T cells were stained with fluorochrome-conjugated antibodies to IL-2, TNF, MIP-1b, and IFN-c (BD Pharmingen). Flow cytometry analysis was performed on a FACSCalibur or LSRII (BD Biosciences). Data was analyzed using Cell Quest (BD Biosciences) or FlowJo (Tree Star, Inc, Ashland, OR) software. Subjects with acute or recent HIV infection 24 individuals with acute or early HIV-infection were enrolled in this study. Acute HIV-infection (n = 9) was defined by the presence of HIV-RNA in the plasma, a negative or weakly positive HIV-antibody by HIV-1/2 ELISA, and the detection of no more than three bands in an HIV Western blot (n = 3); recent HIV infection was defined by a positive ELISA and confirmed by detuned negative ELISA or confirmed previously negative ELISA. All but one of participants had symptoms compatible with the acute retroviral syndrome and the first blood sample available for immunological studies (baseline) was drawn at a median of 12 days (range 5–27) after onset of symptoms. Nine subjects were treated with HAART after this visit whereas three remained without antiretroviral therapy. Study participants were recruited either from the Massachusetts General Hospital and the Fenway Community Heath Care Center, Boston or via Center for HIV/AIDS Vaccine Immunology (CHAVI) clinical sites. All individuals gave written consent to participate, and the study was approved by the respective institutional review boards and conducted in accordance with the human experimentation guidelines. Acknowledgments We would like to thank the CHAVI for their generous contributions of patient samples. PLoS ONE | www.plosone.org 11 January 2009 | Volume 4 | Issue 1 | e4256