Thus, DCs appear to be central in the generation of protective immunity against HIV-1. Previous studies examining in vitro HIV-1 T cell priming have used MDDCs pulsed with different HIV-antigenic constructs, such as peptides, proteins, liposome complexed proteins and cDNA [11,12,13,14] to prime na¨ıve T cells. However, the capacity of DCs pulsed with whole virions; the most physiological source of HIV-antigens, to prime T cells in vitro has not been investigated. Here, we examined whether infectious and AT-2 HIV-1, with functional binding and fusion abilities, can serve as efficient sources of antigen for DCs to prime HIV-specific T cells from na¨ıve cells in vitro. The use of AT-2 HIV-1 as a source of antigen may be particularly relevant as the majority of virions found in circulation do not possess culturable infectivity [15,16,17,18]. Even if not infectious, these viruses may serve as an important source of antigens in vivo. Using both infectious and AT-2 HIV-1 also allowed us to compare the nature of responses generated with these antigen sources, findings of great interest for vaccine design. Our findings show that DCs loaded with AT-2 or infectious HIV-1 primed HIV-specific responses from na¨ıve T cells from uninfected individuals in vitro. Priming with AT-2 HIV-1 yielded slightly more frequent responses. MDDCs pulsed with HIV-1 stimulated significantly more CD4+ than CD8+ T cell responses. The specificities of the primed T cells consisted of epitopes located in env gp41 and gp120, gag p17 and p24, and pol INT and RT. For the CD4+ T cell responses, five out of ten of the in vitro primed responses matched the responses seen ex vivo in HIV- infected individuals at different stages of disease[19]. Furthermore, we identified several novel HIV-1 CD4+ T cell responses in env and pol, and we were able to detected 4 out of 5 of these novel responses in acute and recent HIV-infected individuals. The overwhelming CD4+ T cell responses seen in our in vitro priming are consistent with the strong and broad CD4+ T cell responses we have observed in individuals with acute HIV-infection and which disappeared or were greatly reduced within three months after onset of infection. Although less frequent, CD8+ T cell responses were also observed. Of note, all in vitro primed CD8+ T cell responses matched the responses seen ex vivo in HIV-infected individuals at different stages of disease [19,20]. Our study shows that DCs pulsed with HIV-1 can prime the same responses that arise naturally in vivo in HIV-infected individuals and demon- strates that AT-2 HIV-1 is a excellent source of antigens that may have the capacity in a vaccine setting, not only to restimulate existing memory responses but also prime for new T cell responses or re-establish responses lost very early on during infection. Results The aims of this study were to establish whether DCs pulsed with infectious or chemically inactivated HIV-1 could prime HIV- specific T cell responses, examine the quality of primed T cell responses, and compare them with the responses observed in vivo in primary HIV-infection. Infectious and noninfectious (AT-2 HIV-1) virions of HIV-1MN, were tested side by side to evaluate priming efficiency and qualitative characteristics of the T cell responses elicited. HIV-1 specific priming was performed using MDDCs and na¨ıve CD45RA+ CD62L+ bulk T cells from PBMCs from 11 HIV-negative individuals. Exposure of mature MDDCs to AT-2 or infectious HIV-1 does not reverse their phenotypic and functional maturation The contact between the virus and DC subpopulations likely represents a fundamental part of HIV-1 pathogenesis and may HIV Specific T Cell Priming affect the DCs phenotype and ability to stimulate T cells [21,22,23,24]. To evaluate potential deleterious effects of HIV-1 exposure on the antigen presenting properties of DCs to be used for T cell priming, we assessed the effect of HIV-1 on the viability and expression of costimulatory molecules of matured MDDC after overnight incubation with AT-2 or infectious HIV-1MN. At the dosage of virus used in the subsequent experiments, 150ng p24 equivalent/105 cells, we did not observe any negative effect on the viability (data not shown), maturation status (Figure 1A) or ability to activate HIV-specific T cell clones (Figure 1B). Furthermore, the ability of MDDCs pulsed with either infectious or AT-2 HIV-1 to activate and expand CD4+ and CD8+ HIV-specific T cells from chronic HIV-infected individuals was examined and DCs pulsed with either form of virus expanded broad HIV-1 specific memory T cell responses targeting major HIV antigens gag, pol, env, and nef (Figure 1C and Larsson et al[25]). Additionally, our previous study established that both forms of virus are processed and presented in a similar manner using, for the most part, the classical pathways for MHC class I and II presentation [26]. These findings establish that the ability of DCs to function as APCs is preserved in this system, both for infectious and AT-2 HIV-1. Both infectious and AT-2 inactivated HIV-1 can be used as antigenic sources by DCs to prime HIV-specific T cells from CD45RA+ na¨ıve T cells The ability of DCs to prime HIV-specific responses in vitro has previously been established by several studies using various antigen preparations, e.g. peptides, proteins, liposome complexed proteins and cDNA[11,12,13,14] but not whole virions, the most physio- logically relevant source of HIV-antigens. We tested whether we could prime HIV-specific T cells in vitro using an autologous system consisting of mature DC, pulsed with AT-2 or infectious HIV-1, and na¨ıve CD45RA+ CD62L+ bulk T cells (Figure 2A) from uninfected individuals. HIV-priming was usually achieved after the third or fourth restimulation, as measured by intracellular IFN-c staining after antigenic rechallenge, (Figure 2B) and considered positive when the level of responding cells, i.e. IFN-c producing T cells, was .1%–5% above the non-specific IFN-c production. Priming was achieved in 8 out of the 11 donors examined (Figure 2C), and reasons for the failure to induce priming in all donors are unknown, but may include the death of newly activated T cells, the HLA differences among donors, or even T cell impairing effects exerted by the virus on the immune cells from some individuals. Cultures using unpulsed DCs served as controls for the specificity of the HIV-priming assays and no significant HIV- specific responses were observed in these mock-primed cultures (Figure 2B). Of note, we observed a background production of cytokines when the HIV-1 primed T cells were tested with unpulsed DCs and this is attributed to T cells still active from the weekly restimulation with HIV-1 pulsed DC as the culture with mock DC did not have this production. We have shown equivalent efficiency of MDDCs to process and present antigens derived from AT-2 HIV-1 or infectious HIV-1 as measured by activating HIV-specific memory T cells from chronic HIV infected individuals[27] (Figure 1C). However, as the source of antigens for the in vitro priming of na¨ıve T cells by MDDCs, AT-2 HIV-1 was slightly more efficient, 59% of all primed responses was achieved using AT-2 HIV-1 compared to 41% by its infectious counterpart (Figure 2D). This outcome may be attributed to negative immune regulatory effects exerted by infectious HIV-1 on newly activated T cells and/or effects on the DCs in these long term cultures [24,28,29]. Of note, we observed low levels of replication in the priming cultures with infectious HIV compared to priming cultures with AT2-HIV PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4256