Background Immune checkpoint blockade (ICB) set a milestone in cancer immunotherapy, but still only a fraction of patients responds. Thus, there is an urgent need for biomarkers predicting outcome, and also for understanding the responsible mechanisms. γδ T cells constitute a numerically minor subset of 1-10% of the peripheral T cell compartment in healthy people and have a major role in defense against multiple microbial and non-microbial challenges. Unlike the majority of T cells, γδ T cells bind their ligands in an MHC-independent manner. We previously studied γδ T cells, that also express checkpoint molecules, in patients in the pre-checkpoint blockade era and thereafter, and identified correlations between subset frequencies of these unconventional T cells and patients‘ overall survival (OS). Here, we present a detailed phenotyping and functional investigation of tumor-resident as well as peripheral γδ T cells.

Methods Phenotyping was performed in stage IV melanoma patients before and under PD-1+/-CTLA-4 blockade using as basis our published OMIP-20 protocol.¹ Cytokine expression patterns and proliferative capacities were determined as described according to our established protocols.² Primary flow cytometry data analysis was performed using FlowJo (BD) and correlations with clinical meta data were determined using Prism (GraphPad) and SPSS (IBM).

Results We found previously that low frequencies of peripheral Vδ1 γδ T cells were associated with prolonged OS. Here, we investigated functional aspects and abundance of γδ T cells within the tumor as well as in the blood. The peripheral Vδ1 but not the Vδ2 differentiation signature revealed significantly lower proportions of naive and effector cells as well as an accumulation of late differentiated cells in patients with high Vδ1 frequencies. The cytokine expression pattern (IFNγ, TNF and IL-17) and the degranulation marker CD107a were different in patients with high versus low peripheral Vδ1 frequencies. The proliferative capabilities of Vδ1 cells in melanoma were limited in comparison to healthy subjects. Both Vδ1 and Vδ2 cells were found in tumor tissues, and these analyses are ongoing, including analyses of replicative senescence through CD57 expression.

Conclusions Our data provide novel insights into the role of γδ T cells in cancer rejection. The previously found negative correlation of Vδ1 T cells with OS is likely due to an accumulation of mal-functioning, probably exhausted Vδ1 T cells in patients with poor outcome of ICB. Thus, we suggest that Vδ1 T cells are promising candidates for future exploitation in novel ICB-approaches.

Ethics Approval This study was approved by K. Wistuba-Hamprecht´s Ethics Committee (approval nos. 490/2014BO1 and 792/2016BO2).

References

1. Wistuba-Hamprecht K, Pawelec G, Derhovanessian E. OMIP-020: Phenotypic characterization of human gammadelta T-cells by multicolor flow cytometry. Cytometry A 2014;85:522–524. doi:10.1002/cyto.a.22470

2. Beucke N, et al. Pitfalls in the characterization of circulating and tissue-resident human gammadelta T cells. J Leukoc Biol 2020. doi:10.1002/JLB.5MA1219-296R