Deciphering the phenotypic and functional complexity of γδ T cells

Gamma delta (γδ) T cells are the prototypic members of the family of unconventional T cells. γδ T cells are defined by the expression of the γδ T cell receptor (TCR), which shows human leukocyte antigen (HLA)-unrestricted activity. Moreover, γδ T cells combine characteristics of adaptive T cells with features of innate effector cells including the expression of natural killer (NK) cell receptors, making them an interesting candidate for cancer immunotherapy. This project aimed to decipher the phenotypic and functional complexity of γδ T cells, with particular focus on a subset expressing the HLA-E/peptide-specific activating NK cell receptor CD94/NKG2C. Our data indicated an increased frequency of NKG2C+ γδ T cells in cytomegalovirus (CMV)-seropositive donors, aligning with existing findings on NKG2C+ NK cells and suggesting that CMV drives the expansion of these cell subpopulations. Additionally, multi-parameter flow cytometry together with state-of-the-art TCR sequencing revealed distinct chain usage and lower TCR diversity of NKG2C+ γδ T cells compared to the NKG2C- subset. Furthermore, NKG2C+ γδ T cells co-expressed other activating as well as inhibitory NK cell receptors and displayed a terminally differentiated phenotype with high cytotoxic potential. Further investigations on the functional capacity demonstrated that NKG2C+ γδ T cells degranulated and responded robustly upon co-culture with cancer cells presenting HLA-E/peptide ligands, while NKG2C- γδ T cells did not. This response involved directly the activating receptor CD94/NKG2C, as shown by the CD94 blocking experiments. Moreover, although NKG2C+ γδ T cells exhibit adaptive-like clonal expansion, they still retain an innate-like response to pro-inflammatory cytokines, much like the less expanded NKG2C- γδ T cells. Altogether, these results advance our fundamental understanding of γδ T cells expressing NKG2C and provide a potential basis for the future application of this cell type in cancer cell therapy.