HIV Specific T Cell Priming Figure 3. HIV-specific T cells are polyfunctional and recognized a broad repertoire of antigens. (A) Mapping of epitopes was done using pools of overlapping peptides. The peptides spanned the entire HIV-1 genome and were based on the consensus B sequence (http://www.hiv.lanl. gov/content/immunology/index.html). The peptide pool matrices and the HIV-1 primed T cell cultures were added to IFN-c Elispot assays as described previously [19]. The assays were developed after overnight culture and evaluated. Reconfirmations of all positive wells were done the following day with the single peptide by flow cytometry to detect intracellular IFNc production. (B) The reconfirmed HIV-1 specific T cell responses from all donors tested were analyzed to determine which HIV-1 antigens the primed T cells recognized and the percentile they constituted of total primed responses from those screened. (C) The effector functions of the HIV-1 specific polyclonal T cells were examined by stimulation with MDDCs pulsed with AT-2 HIV-1 or infectious HIV-1 and the intracellular production of the effector cytokines/chemokines; IFN-c, TNF-a, IL-2 and MIP-1b, was measured by flow cytometry. The polyfunctionality of the T cell responses were analyzed as the ability to produce the combination of one, two, three or all four of the cytokines/chemokines tested by the primed CD4+ (D) and CD8+ (E) T cells in response to HIV. doi:10.1371/journal.pone.0004256.g003 cells when pulsed with HIV-1 (Figure 1C)[25]. Alternatively, the lack of nef responses may be attributed to lack of viral replication within the priming cultures. Relatively little nef is found in virions and in the absence of robust productive infection, there may be insufficient antigen available for priming, particularly compared to more abundant proteins like gag, pol, and env. The specificity of the in vitro primed T cell responses reflect those seen in infected individuals primed in vivo Several studies have analyzed the specificity of HIV-1 memory T cell responses existing in infected subjects by ex vivo assays [19,31,32] and the CD8+ T cells are so far the most extensively studied [32,33]. Less is known about HIV-specific CD4+ T cells as these are specifically targeted by the virus as the infection progresses [34,35]. The majority (86%) of the confirmed primed responses were CD4+ T cells (Figure 4A). The basis for this CD4+ skewing could be attributed to several factors including use of whole virions as the source of antigens, and the in vitro system used for delivery of the virus. In addition, CD4+ T cell responses are the first to arise in vitro and may thus overwhelm the CD8+ T cells. However, we can rule out the possibility that our in vitro system merely selects for the development of HIV-specific CD4+ T cells. Though they made up a minority of the responses observed, we did see unequivocal development of HIV-specific CD8+ T cells. Furthermore, when we depleted CD4+ T cells from the co-culture system, we saw enhancement of priming of CD8+ T cells (Figure 4C). Therefore, it is possible to prime HIV-specific CD8+ T cells using our in vitro system. PLoS ONE | www.plosone.org 5 January 2009 | Volume 4 | Issue 1 | e4256