Cell-associated HIV cross-presentation by pDC is potentiated by non-cognate CD8+ T cell pre-activation

Abstract

Antigens (Ag) from apoptotic HIV-infected cells can be cross-presented by human plasmacytoid dendritic cells (pDC) to CD8 + T cells 1 . We studied the mechanisms leading to this cross-presentation. For this, we generated HLA-A2-restricted HIV-Gag-specific CD8 + T cell clones. HLA-A2 negative, HIV-infected or uninfected, H9 cells were used as Ag donor cells. They were irradiated by UV-C to induce apoptosis. Direct viral presentation was prevented by using saquinavir. DC populations were immunomagnetically and FACS-sorted to >95% purity, from HLA-A2+ or -donor buffy coats. CD8 + T cells IFN-γ secretion or intracellular production were assessed by ELISA or flow cytometry, respectively. Apoptotic H9-HIV cell cross-presentation by pDC led to CD8 + T cells IFN-γ secretion, which was fully MHC-class I-restricted. Surprisingly, a lower level of non-MHC restricted intracellular IFN-γ production was also induced by HLA-A2-negative pDC incubated with HIV-infected cells. Both MHC-Class I-dependent and -independent IFN-γ intracellular productions were partly dependent on TLR-7 signalization, indicating a role for stimulation of TLR7 by HIV. Indeed, H9-HIV, and not H9 cells, induced maturation and production of cytokines by pDC. Among those cytokines, IFN-α or β induced intracellular IFN-γ production when added to the CD8 + T cell clones, in the absence of pDC or H9-HIV cells. To perform cognate activation of the CD8 + T cell clones without viral stimulation, we used CD3-and CD28-bound beads: IFN-γ secretion was stimulated, and was increased by adding these cytokines. Conversely, neutralizing antibodies against these cytokines abolished the non-MHC-restricted intracellular IFN-γ production triggered by pDC incubated with by HIV-infected cells. Compared to pDC, purified cDC1 or cDC2 required additional TLR-3 or -2/4 stimulation to cross-present antigens from H9-HIV cells. Indeed, H9-HIV apoptotic cells induced maturation of pDC, but not cDC. Our work demonstrates that pDC produce type I IFNs following HIV-infected cells detection, which pre-activate CD8+ T cells by making them produce, but not release, IFN-γ. Upon additional cognate interaction, IFN-γ. release is potentiated by this previous intracellular accumulation.