A portrait of gamma delta T cell exhaustion: Chronic stimulation models, RNA transcriptomics and CRISPR screens.

Abstract

T cell exhaustion has been identified as a major challenge for T cell therapeutics. In conventional T cells (ab T cells), the study of exhaustion has led to insights into its aetiology, and potential therapeutic approaches to overcome it. Our research focuses on a novel area of study-T cell exhaustion in human γδ T cells, particularly the Vγ9Vδ2 subtype, which is the most prevalent in peripheral blood. These cells hold therapeutic promise due to their broad cytotoxic characteristics and ability to bridge innate and adaptive immunity. However, the understanding of how exhaustion can affect the behaviour and functionality of these cells is yet to be established. Our study involved in vitro chronic stimulation protocols using combinations of zoledronic acid (ZOL) and OKT3 (anti-CD3 monoclonal antibody) or Daudi target cells, both stimulating via the Vγ9Vδ2 T cell receptor. The findings from our ZOL model, which showed a notable decrease in absolute T-cell numbers and significant upregulation of genes associated with T cell exhaustion, could have significant implications for understanding and potentially overcoming T cell exhaustion in Vγ9Vδ2 T cells. Bulk-RNA sequencing of Vγ9Vδ2 T cells identified distinct signatures for the multiple stimulated samples, and a list of highly differentiated genes was selected for further investigation by a pooled CRISPR screen. We optimised and tested a suitable protocol for a CRISPR screen in Vδ2 T cells. Two gRNA hits were enriched in the ZOL and multiple stimulated samples (targets for SLC37A3 and GORASP2 genes). In conclusion, a simple in vitro assay was developed to study hypofunctionaity in Vδ2 T cells, allowing studies to evaluate RNA profiles and regulatory mechanisms of exhaustion induction in this cell type. Finally, selected genes were screened in a CRISPR-based pooled screen and hit gRNAs from stimulated Vδ2 T cells were identified.