11393 (to ZS and JK), Marie Curie 749010 (to DXB), NIH grants R01GM100114 (to DSL), P50GM085273 (to the New Mexico Spatiotemporal Modeling Center), and P30CA118100 (to the UNM Comprehensive Cancer Center). DF and AJRH acknowledge financial support from the NWO-funded Netherlands Proteomics Centre through the National Road Map for Large-scale Infrastructures program X-Omics (project 184.034.019). ES and LB are supported by grants from INSERM, CNRS, Université de Nantes, FRM (DEQ20170839118), and Ligue Contre le Cancer AO GO2019 (Côtes d'Armor, Association pour la Recherche contre le Cancer (PJA20191209404). This work was realized in the context of the LabEx IGO program, which is supported by the French National Research Agency Investissements d'Avenir.

Key Points We describe a novel allo-tumor–reactive and CD8α-dependent Vγ5Vδ1TCR. The molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 is an essential determinant of recognition.

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2+ T cells compared with other tumor types. By reconstructing matched Vδ2– TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.