Structural features underlying T-cell receptor sensitivity to concealed MHC class I micropolymorphisms

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.The MHC class I (MHCI) locus is described consistently as a major host factor influencing disease outcome in the setting of HIV-1 infection (1). As MHCI molecules select the repertoires of viral epitopes presented to CD8⁺ T cells, they shape the immune response against the virus. A broad variety of distinct MHCI allotypes, which can demonstrate up to 10% amino acid diversity, allows extensive sampling of epitope repertoires, and these differences also influence the efficacy of viral control, as illustrated by the strong association of individual MHCI types with prolonged AIDS-free survival (1, 2). Less understood is the role of minor polymorphisms (termed “micropolymorphisms”) that distinguish closely related MHCI subtypes, often by as few as one amino acid change. Although such minimal changes frequently allow identical epitopes to be presented, they often influence the efficacy of viral clearance (3) and disease susceptibilities in vivo (4). These effects have important implications in the context of HIV-1 infection, where the delicate interplay between CD8⁺ T-cell selection, viral evolution, and fitness cost is assumed to shape the clinical course of disease (5).MHCI subtypes separated by micropolymorphisms frequently permit the presentation of shared epitopes, however, these differences can affect the conformation of peptides in their binding grooves (6–8) and/or the positioning of MHC α1/α2 helices (9, 10), with implications for T-cell receptor (TCR) usage in vivo. The protective HLA-B*5703 and B*5702 subtypes, for example, are distinguished by a single amino acid substitution that does not notably alter their peptide-binding motifs. However, the positioning of this polymorphism within the TCR footprint is likely to affect the use of the TCR repertoire and could explain the greater association of HLA-B*5703 with lower viral set point and immune control (11). Yet for other MHCI subtype–peptide combinations, minor polymorphic differences result in minimal, if any, conformational disparities (4) and the molecular processes underlying differential T-cell selection are not well understood. This is especially true for longer epitopes, which comprise an important group of ligands (12, 13) for which the diversity of their responding TCR repertoires may be further limited by their atypical structural conformations when presented by MHCI. Notable examples are the HLA-B*57 subtypes HLA-B*5701 and B*5703, which consistently are associated with prolonged AIDS-free survival (14–16). Despite two polymorphic amino acids distinguishing these subtypes at residues 114 (D-N) and 116 (S-Y), both bind an equivalent repertoire of peptides (17) and in HIV-1 infection share similar CD8⁺ T-cell immunodominance hierarchies from acute infection through to chronic disease (18–22). The contribution of these HLA-B*57 subtypes to successful viral control is thought to relate to the epitopes selected, most notably to three p24-derived capsid epitopes targeted. Two of these, the ISPRTLNAW (IW9) and TSTLQEQIGW (TW10) epitopes, are targeted in early infection, presumably contributing to rapid viral control (22, 23). The nature of T-cell–driven escape mutations that accrue for these epitopes are conserved in the presence of both B*57 subtypes, presumably reflecting shared modes of peptide presentation and shared T-cell recognition conformations in vivo. However, the subtype-specific differences appear to impact a third epitope, KAFSPEVIPMF (KF11), which dominates the B*57-restricted immune response in chronic disease (18, 21). Targeting of this epitope is important in patients in whom circulating IW9 and TW10 mutations lead to immune escape (20, 24, 25), and its recognition is associated with lower plasma viral load (26). In HLA-B*5703⁺ patients, diverse KF11 variants circulate, which frequently associate with elevated viral loads (20, 27). However, viruses harboring these mutations are rare in carriers of HLA-B*5701 (24, 28), a finding not readily explained by factors specific to the infecting viral clades (27). We and others have analyzed the KF11-specific TCR repertoire in HLA-B*57⁺ patients and have reported common use of a conserved and frequently immunodominant Vα5/Vβ19 TCR pair sharing highly conserved CDR3α and -β motifs in unrelated HLA-B*5701⁺ individuals (21, 27, 29). This “public” TCR displays cross-reactivity against broad KF11 variants (30). However, this receptor pair does not represent a KF11-specific clonotype in carriers of HLA-B*5703 and its absence might contribute to the higher incidence of circulating KF11 variants in HLA-B*5703⁺ individuals (21, 27, 31).Although the majority of MHCI restricted epitopes are 8–10 amino acids, peptides of noncanonical length up to 13 residues, and particularly viral peptides targeted in humans, represent an important category of epitopes, (32–34). Longer peptides bind MHCI molecules either by extending beyond the peptide-binding groove (PBG) (35) or, more frequently, by forming a central bulge that arches above the cleft while maintaining standard A, B, and F pocket binding (13, 33, 34, 36). Although many arched peptides remain mobile (33), others assume a rigid conformation due to stabilizing interactions that involve hydrophobic forces (36) and water-mediated and direct peptide–MHC hydrogen bonds (9). Having previously determined the structure of KF11 in complex with HLA-B*5703, we observed a central peptide bulge, with two P residues forming the basis of the stable peptide arch (36). Here we present a structural, thermodynamic, and functional study describing the molecular features underlying TCR-mediated recognition of the atypical KF11 epitope and highlight the vital role played by germline-encoded TCR α-chain residues. We also demonstrate how minimal differences between the HLA-B*57 subtypes involving subtle alterations in the interplay of polymorphic residues and specific networks of water molecules in the PBG are paramount in facilitating optimal TCR binding, influencing the kinetics of the TCR–pMHCI interactions and cellular function. Collectively, our findings illustrate how subtle subtype-specific polymorphic differences can have important implications for T-cell use, repertoire diversity, and presumably, disease outcomes.