br Results br Discussion HIV

Results
Discussion HIV reservoirs represent a major barrier to HIV eradication (Archin et al., 2014, Kimata et al., 2016), and substantial research efforts are being directed toward the goal of identifying and eliminating this obstacle to cure (Archin and Margolis, 2014, Pitman et al., 2018). Latently infected resting CD4+ T cells represent the majority of the HIV reservoir (Bruner et al., 2015, Siliciano and Siliciano, 2015), and it has been shown that HIV latency can be established in resting CD4+ T cells directly after infection, without intervening productive infection (Chavez et al., 2015). Therapeutic strategies aimed at eliminating HIV reservoirs have been dominated by the “kick and kill” paradigm, which involves reactivating latent CD4+ T cells to induce de novo production of viral proteins, and the subsequent elimination by the immune system, notably CD8+ T cells (Archin and Margolis, 2014, Pegu et al., 2015). Another strategy could be the elimination of infected CPI-203 before the establishment of the reservoir. Here, we show that ex vivo CD8+ T cells from HIV controllers (possessing at least one HLA-B∗27 or HLA-B∗57 allele), but not HIV progressors expressing the same alleles, are able to directly recognize and kill HIV-infected non-activated CD4+ T cells through the formation of immunologic synapses and class I restricted recognition of processed viral peptides. This occurs within a few hours following viral entry, allowing an antiviral response before HIV reverse transcription and thus before the eventual establishment of HIV latency. Moreover, this recognition precedes the production of the incomplete reverse transcripts that are needed to induce pyroptosis and the release of inflammatory molecules (Doitsh et al., 2010, Doitsh et al., 2014). These data indicate that HIV-specific CD8+ T cell responses have the potential to reduce the establishment of an HIV reservoir in non-activated CD4+ T cells and reduce HIV-induced inflammation, hence contributing to HIV control. To further define the mechanism of recognition, we show that the recognition of non-productively infected resting CD4+ T cells by HIV-specific CD8+ T cells occurs through synapse formation and requires the presentation of viral peptides derived from incoming viral particles on HLA-I molecules. Moreover, we show that non-activated cells can be targeted either following exogenous infection or cell-to-cell spread, which is likely a major mode of ongoing viral replication in vivo (Chen et al., 2007, Dimitrov et al., 1993, Phillips, 1994). Our results indicate that incoming viral proteins are degraded in the cytosol of non-activated cells by the proteasome and the aminopeptidases to produce antigenic peptides for presentation by HLA class I, as blocking these enzymes or HLA class I reduced the CD8+ T cell responses. It is important to note that these results were obtained using PBMCs from HIV controllers expressing HLA-B∗27 or HLA-B∗57 and with the laboratory strain of HIV NL4.3 possessing a wild-type sequence for most of the optimal epitopes. Further studies will be required to examine the recognition of non-activated infected cells in the context of other restricting HLA-I alleles. However, our results provide support for the hypothesis that proteins from incoming particles are degraded in the cytosol and presented directly at the surface of the target cell through HLA class I. Previous studies have reported pre-integration presentation of the SIV-Gag peptides (Sacha et al., 2007) and the HIV-Gag KRWIILGLNK epitope on HLA-B∗27 in activated CD4+ T cells (Buseyne et al., 2001, Kløverpris et al., 2013). In addition, a previous study (Buckheit et al., 2013) performed with cells from HIV controllers possessing HLA-B∗57 reported the elimination of HIV+ resting CD4+ T cells by CD8+ T cells. The present study adds to these previous reports by using the Vpr-βlam assay and a GFP-expressing virus to precisely distinguish entry from protein production, by demonstrating that the observed effect is induced by the presentation of viral peptides on HLA-I molecules, by showing that functional immune synapses are required for recognition, and by demonstrating that more physiologic cell to cell transmission results in the sensitization of cells for CD8+ T cell recognition before the process of reverse transcription.