HIV Specific T Cell Priming Figure 4. The majority of the responses primed by MDDCs are HIV-1 specific CD4+ T cell responses. (A) The HIV-1 specific T cell responses from all donors tested were characterized for expression of CD4 or CD8 and plotted as the percentile of total confirmed primed responses. (B). Single peptide confirmation of HIV-1 specific T cells were done with the specific peptide by flow cytometry to detect intracellular IFNc production. The dot plots shows HIV-1 specific CD4+ T cell responses for gag OLP 37, gp41 OLP 377 and gp41 OLP 382. (C) HIV-1 specific priming of CD8+ T cells. CD8+ T cells were obtained by flow cytometry sorting. After the 4th restimulation, HIV-specific responses were detected by intracellular cytokine staining for IFN-c. doi:10.1371/journal.pone.0004256.g004 that HIV-specific CD4+ T cell responses at the time of acute infection target various proteins and that the overall responses declined over time. Gag-specific responses remained detectable six months after AHI whereas most subjects had lost their Pol-, Nef-, and Env-specific responses at this time point. Of note, the magnitude of Env-specific CD4+ T cell responses detectable during AHI is remarkable compared to their rarity in chronic infection [37]. We used intracellular cytokine staining for IFN-c and IL-2 to confirm this decline in HIV-specific CD4+ T cell responses after AHI with another experimental approach (Figure 5C). The strong IFNc- and IL-2 HIV-Gag-specific CD4+ T cell response present at baseline before institution of therapy declined over time, contrasting with the stability of the CMV- specific CD4+ T cell response, demonstrating that this phenomenon is HIV-specific. Thus, robust and broad HIV- specific CD4+ T cell responses can be detected during AHI however, they quickly decline. In particular, Env-specific CD4+ T cell responses, which are not commonly detected in chronic HIV- infected subjects, are readily detected during AHI. However, these responses disappear very quickly. Therefore, the CD4+ T cell responses we saw in our priming could be a reflection of what is happening during acute infection whereby broad HIV-1 specific CD4+ T cell responses are activated very early on, even before the development of HIV-specific CD8+ T cells and decline as activated CD4+ T cells are directly or indirectly destroyed by the virus. Novel epitopes primed for in vitro correlated with CD4 responses that arise and are present during acute and recent HIV-1 infection Our findings that the CD4+ T cell responses decline rapidly in acute HIV-infected subjects could explain why several of the CD4+ T cell responses we succeeded to prime for in vitro have not previously been described in the literature. The novel HIV-1 T cell responses we found in env and pol are contained within peptides ELKKIIGQVRDQAEHLK in pol IN157-173, MTKI- LEPFRKQNPDIVIY in pol RT163-181, AVLSIVNRVRQ- GYSPLSF env gp41700-717, LELDKWASLWNWFNITNW in env gp41601-678, and RPVVSTQLLLNGSLA in env gp120252-266. The ELKKIIGQVRDQAEHLK in pol IN, MTKI- LEPFRKQNPDIVIY in pol RT and AVLSIVNRVRQGYSPLSF env gp41 have so far been found in only 1 out of 36 chronic HIV infected subjects tested (data not shown). In addition, screening of acute and recent HIV-infected subjects revealed that CD4+ responses to 3 of 5 novel epitopes within pol and env are readily detected. We have been able to confirm CD4+ T cell responses to PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4256