17-chanel FACS analysis and single cell RNA Sequencing identifies a dysfunctional spectrum of CD8⁺ cytotoxic T cell states in head and neck cancer

Introduction CD8+ T cells are a main effector cell type mediating anti-tumor immunity using direct and indirect mechanisms of cancer cell destruction (Fas-Ligand, cytotoxic granules, cytokines). Current immunotherapy approaches aim to strengthen these cells to enhance their anti-tumor effect. Here, we explore differences in protein expression and transcriptomic states of CD8+ T cells in head and neck cancer.

Methods We used 17-color FACS analysis (n=10) and single cell RNA sequencing (n=18), scRNAseq, 10x Genomics 3’ single cell (V2) platform) on blood and tumor specimen from treatment naïve head and neck cancer patients. The flow cytometry data was analyzed using FlowJo, the single cell data data was integrated using the 10x pipeline and visualized using scanpy.

Results FACS analysis of circulating CD8+ T cell subsets showed a different kinetic of divergent T cell substates (naïve to effector): while PD1 and TIGIT expression increased continuously, LAG3 and Tim-3 expression was low across all cell types and CD73, ICOS und CTLA4 showed a fluctuating kinetic. Clustering analysis of scRNAseq data allowed to identify 27,013 CD8+ T cells that were further subdivided into naïve-like T cells, cytotoxic T cells and dysfunctional/exhausted cells. Importantly, even amongst the therapeutically relevant exhausted T cell state there was a continuum between pre-dysfunctional (GZMKhigh, CXCL13low, LYAR+) and terminally dysfunctional cells (GZMKlow, CXCL13high, ENTPD1+).

Conclusions FACS and transcriptomic analyses showed a heterogenic expression of checkpoint receptors across CD8 T cell states. Here, we identified GZMK, CXCL13, LYAR and ENTPD1 as potential markers to differentiate between – clinically relevant – pre-dysfuntional and terminally dysfunctional CD8+ cells.

Publication History

Article published online:
24 May 2022