CD8 T cells cross-restricted by HLA-B*57 and HLA-E*01 recognize HIV Gag with different functional profiles [dataset 2]

Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFN?, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control. Overall design: CD8+ T cells from 8 people with HIV (PWH), specifically 4 controllers (C), 2 noncontrollers (NC), 1 elite controller (EC), and one noncontroller on antiretroviral therapy (ART+ NC) were used for this assay. Initially, indicated condition cell lines (41A3.CD4, 41A3.CD4.B57, 41A3.CD4.E01, 41A3.CD4.E03) were pulsed with KF11 peptide at 10 ug/mL for 2 hrs at 37°C before peptide was washed off with 1X serum-free media (SFM). CD8+ T cells were then isolated from PBMCs using the StemCell Easy-Sep CD8 Enrichment kit (cat. NC0050243) and coincubated with peptide-pulsed cell lines as indicated for 18 hrs in the presence of a-CD28 and -49d. Cells were then stained with Aqua LIVE/DEAD stain (Thermo Fisher Scientific), anti-CD3-PacBlue, -CD4-AlexaFluor780, -CD8-AlexaFluor700, -CD14/16/19/56-PerCP-Cy5.5 (dump channel, all separate antibodies), -CD69-APC, -CD137-PE, -CD94-PE-Cy7, and -CD107a-FITC. As per gating strategy shown in Figure S1A, single cell lymphocytes were gated on, followed by live cells (Aqua -), CD4/dump -/-, CD8+ CD3-dim, CD69/137+.. Cells which fit this gating strategy and met the criteria of having a CD3-dim activated response to the given HLA pulsed with KF11 significantly (and 3X) higher than 41A3.CD4 also pulsed with KF11 were then sorted into a 96-well plate containing the lysis buffer previously described using the Aria II FACS sorter. scTCR and scRNA-seq was performed as previously described (Bansal et al. 2021).