CD8+ T cells cross-restricted by HLA-B*57 and HLA-E*01 recognize HIV Gag with different functional profiles.

Few HIV-specific epitopes restricted by non-classical HLA-E 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 KF11 (KAFSPEVIPMF) restricted 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 HLA-E*01:03. CD8 responses were analyzed using single-cell RNA and TCR sequencing. Supernatants were also assessed for soluble protein profiling. HLA-I multimers were developed to identify CD8s restricted by HLA-B57 and/or HLA-E ex vivo. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFN-γ, whereas E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL-27, findings that were corroborated through single-cell RNA sequencing. TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01:03 as demonstrated by in vitro T cell reporter assays and ex vivo multimer screening. Ex vivo CD8s were singly restricted by HLA-B57 and HLA-E, with dual restriction only observed in PWH with lower viral load. These findings demonstrate that certain HIV-specific CD8s in PWH exhibit dual restriction by HLA-B*57 and HLA-E*01:03, leading to functionally distinct immune responses depending on the restricting allele(s).