HIV Specific T Cell Priming Figure 2. HIV-1 pulsed MDDCs prime HIV-1 specific T cells. (A) Autologous na¨ıve bulk (CD4+ and CD8+) T cells were negatively isolated from PBMCs by magnetic beads to remove CD14, CD19, CD56 and CD45RO positive cells. (B) The autologous na¨ıve bulk T cells were co-cultured with the mock (no virus), AT-2 or infectious HIV-pulsed DCs. Cultures were restimulated weekly by the addition of DCs pulsed with mock, AT-2 HIV-1 or Inf HIV- 1 for 4 weeks. HIV-specific responses were detected by intracellular cytokine staining for IFN-c after 4 weeks of culture. (C) HIV-1 specific priming was attempted with bulk T cells from 11 HIV na¨ıve donors to establish the efficiency of in vitro MDDC priming. 8 out of 11 donors were successfully primed. (D) The priming efficiency of MDDCs pulsed with AT-2 HIV-1 vs. infectious HIV-1 of the 8 donors that was successfully primed were compared. (E) Priming cultures were analyzed for the presence of viral replication. Supernatants were collected from the cultures after the 3rd restimulation on day 21 and virus replication was measured using HIV p24 ELISA kit. Mock, AT2-HIV, and Inf HIV priming cultures were compared. doi:10.1371/journal.pone.0004256.g002 (Figure 2E). Our study demonstrates for the first time that HIV-1 pulsed DCs can prime HIV-specific T cells from na¨ıve donors and that AT-2 inactivated HIV-1 was slightly more efficient than infectious HIV-1 as an antigenic source. DCs pulsed with infectious or AT-2 HIV-1 primed polyfunctional T cell responses that target a broad repertoire of HIV-antigens The requirement for initiation and maintenance of broad and strong T cell responses capable of controlling viral load is clear when examining the HIV-infected individuals that do succeed to control the virus for many years [1]. To establish the antigenic repertoire of the primed T cells, the HIV specific polyclonal T cell populations were evaluated by IFN-c Elispot using a set of overlapping peptides spanning the complete HIV Clade B consensus sequence (Figure 3A). We found broad responses targeting epitopes located in gag MA (15%), CA (8%), pol INT (22%), RT (15%), env gp120 (10%) and gp41 (8%) (Figure 3 B, Table 1). In most cases, primed T cells from various donors recognized as few as two epitopes or as many as nine. For instance, the specific responses we detected in donor MP880 were located in env gp41, env gp120, gag MA (p17), gag CA (p24) and pol INT, thus demonstrating that within one donor a broad repertoire of HIV-specific T cell responses can be primed using MDDCs. We examined if the HIV-1 specific polyclonal cell lines, selected based on the production of IFN-c, had the ability to produce other effector cytokines such as IL-2, TNF-a or MIP-1b when stimulated with MDDCs exposed to HIV-1 (Figure 3C). The HIV-specific CD4+ T cells (Figure 3D) and CD8+ T cells (Figure 3E) in the polyclonal population were found to produce one to four cytokines upon stimulation. Taken together, these findings demonstrate that the primed CD4+ and CD8+ T cells target a broad repertoire of HIV-1 antigens and that they are polyfunctional in their ability to secrete multiple cytokines upon stimulation. The responses most frequently detected in HIV-1 infected individuals, independent of the stage of the disease, are gag p24- and nef-specific T cells[30]. Besides yielding the highest frequency of HIV-specific T cells, these two proteins also contain the highest epitope density [20,30]. Our in vitro priming induced a variety of p24-specific responses and they constituted 22% of all HIV-1 specific responses primed. However, in contrast to Addo et al [30] no nef specific responses were confirmed in our assays. The reason for this discrepancy between the in vivo and the in vitro responses is unclear. However, we can rule out the possibility that the DCs are unable to process and present the nef protein as we have previously shown that DCs can activate nef specific memory T PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4256