Supplementary MaterialsS1 Fig: Gating strategies for flow based functional assays. chronically HIV infected (CHI) individuals. (PDF) ppat.1007970.s008.pdf (96K) GUID:?860DF5DD-78DF-4891-96FD-999607411B4A Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFN responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of APX-115 PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted APX-115 dendritic cell (DC) maturation and CD4 T cell CD8 T cell responses can be generated in response to emerging escape mutations in chronic HIV-1 infection [15]. Our group and others have previously demonstrated that CD8 T cell cross-reactivity broadens from acute to chronic infection [16C18]. Interestingly, there has also been increasing evidence that HIV-1 adaptations may confer several viral benefits other than just classical escape, such as increasing viral fitness [19, 20], compensating for fitness costly mutations [21, 22], and acting as a decoy by drawing CD8 T APX-115 cell responses away from other epitopes [23]. Another intriguing viral advantage non-classical adaptation was put forth by cytotoxicity in chronic infection. In spite of this apparent increase in immune pressure, AE remained the dominant epitope encoded by viral quasispecies in chronic infection. We further found that AE-specific CD8 T cells promoted viral by adapted epitopes exhibit higher cytotoxicity against CD4 T cells during chronic HIV-1 infection Since we observed increased AE-specific APX-115 IFN responses in chronic infection and these responses could often be attributed to cross-reactive CD8 T cells, we evaluated the cytotoxicity of CD8 T cells recognizing AE versus NAE pulsed targets. We expanded antigen-specific cells by co-culturing the isolated CD8 T cells with peptide pulsed autologous Vegfc monocytes. Using these peptide-specific CD8 T cell lines generated against NAE or AE, we assessed cytotoxicity with a 7AAD APX-115 killing assay, in which we quantified the percentage of 7AAD+ CD4 T cell targets at various effector to target ratios as an output of CD8 T cell cytotoxicity, as described previously [14, 16]. Overall, cytotoxicity was assessed for six different NAE/AE pairs in seven CHI patients (S3 Table). A representative example of flow cytometry based gating and normalized data from CHI-6 is shown in S1A Fig, Fig 4A and 4B. Cumulative data analysis showed that the CD8 T cell lines generated against AE consistently elicited stronger cytotoxic responses to peptide-pulsed CD4 T cells (p = 0.02) than their corresponding NAE counterparts (Fig 4C) even though their IFN ELISpot response magnitude were not significantly different (S2A Fig). Whenever cell number was not limited, we also tested these CD8 T cell lines for cytokine/effector molecules production, including IFN, TNF, CD107a, perforin, and granzyme A/B production, which have been shown to be relevant to CD8 T cell cytotoxicity [26C28]. We did not detect any significant differences in the frequency of their production (either mono or polyfunctional responses) between NAE and AE specific CD8 T.