gamma 9 delta 2T cell diversity and the receptor interface with tumor cells

gamma 9 delta 2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded gamma 9 delta 2T cells, we explored the clonal diversity of gamma 9 delta 2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded gamma 9 delta 2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected gamma 9 delta 2T cell receptors (gamma 9 delta 2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the gamma 9 delta 2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR gamma chain and modulated by the affinity of the CDR3 region of the TCR6 chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of gamma 9 delta 2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the gamma 9 delta 2TCR and required membrane flexibility of the gamma 9 delta 2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.