Conserved non-canonical recognition with distinct structural solutions underlies dual-HLA recognition of HIV-1 Gag...

Dual-reactive T cell receptors (TCRs) that recognize the HIV-1 Gag TL9 epitope presented by both HLA-B*81:01 and HLA-B*42:01 are associated with improved immune control. However, whether such TCRs rely on a single universal recognition blueprint or adopt distinct structural solutions remains unclear. Here, we characterize 14D7, an additional dual-reactive TL9-specific TCR, and compare its structural features with those of the previously reported T18A clonotype. The 14D7-HLA-B*81:01-TL9 structure reveals a conserved non-canonical recognition framework shared with T18A: in both TCRs, CDR3β primarily engages the HLA α2 helix rather than the peptide, while CDR2β compensates via critical peptide contacts. However, 14D7 adopts a distinct CDR3 loop conformation, likely reflecting differences in V/J gene usage and junctional rearrangement, demonstrating that dual-reactive TCRs can achieve cross-reactivity through related but non-identical molecular solutions. AlphaFold 3 modeling further suggests that, like T18A, 14D7 actively remodels the TL9 conformation in the HLA-B*42:01 context toward a state resembling that observed in the HLA-B*81:01-bound complex. Surface plasmon resonance analysis revealed differential affinity profiles across HLA backgrounds, with greater tolerance to common escape variants in the HLA-B*81:01 context. Structural comparison further suggests that polymorphic HLA residues, particularly at positions 143 and 163, reshape the peptide- and TCR-facing interaction network and thereby influence tolerance to epitope variation. Together, these findings show that dual-reactive TCRs operate within a conserved non-canonical recognition framework while retaining flexibility at the level of CDR3 loop configuration, providing a refined structural basis for HIV-specific cross-reactive T cell immunity.