Uncovering structural determinants of peptide recognition by public and private T-cell receptors

Public T cell receptors (TCRs) recurrently emerge across individuals in response to common pathogens, yet the structural and biophysical basis distinguishing public from private clonotypes remains incompletely defined. Here, we combine epitope mapping, single-cell TCR sequencing, and single-particle cryo-electron microscopy to dissect CD8 ⁺ T cell responses to the immunodominant SARS-CoV-2 ORF3a(207-215) epitope presented by HLA-A*01:01. Among responding clonotypes, we identify a shared public TCR (TCRpub) and an individual-specific private TCR (TCRpriv) that use nearly identical TRBV5-1 β chains but distinct α chains. Both clonotypes exhibit comparable micromolar affinity and functional avidity, yet their structures reveal different antigen-recognition modes. We determined cryo-EM structures of the TCR _pub and TCR _priv in complex with ORF3a _(207-215) / HLA-A*01:01 at ∼3 Å resolution. Despite targeting the same epitope, the two receptors engaged the peptide-MHC complex with distinct CDR-loop orientations and contact footprints: TCRpub engages the peptide through a peptide-centric AGDL CDR3β motif and focuses interactions on the MHC α2-helix, whereas TCRpriv distributes contacts across both MHC α-helices via a canonical CDR3β configuration. These findings illustrate how near-identical β chains can yield divergent recognition strategies to recognise the same pMHC ligand through alternative α-chain pairing. More broadly, this work establishes cryo-EM as a robust approach for resolving physiological-affinity TCR/pMHC complexes, providing mechanistic insight into how public TCRs emerge and persist in antiviral immunity.