An In Vitro Exhaustion Model for Vγ9Vδ2 T Cells: Characterization and Reactivation Strategies for Advancing Immunotherapy Development

These cells, initially recognized for their broad activity in combating infections and tumors, are now emerging as promising candidates for targeted immunotherapeutic strategies, including T cell Engagers (TCEs). However, like other immune cells, Vγ9Vδ2 T cells can enter a state of dysfunction and exhaustion within the tumor microenvironment, driven by factors such as TCR overstimulation, immunosuppressive cytokines (e.g., TGF-β), and hypoxia.

While the mechanisms of T cell exhaustion have been extensively studied in in vivo CD8+ T cells, the lack of a murine Vγ9Vδ2 subset complicates the investigation and generation of large numbers of exhausted Vγ9Vδ2 T cells. To address this, we present a novel in vitro protocol for rapidly generating exhausted Vγ9Vδ2 T cells through co-culture with Zoledronate activated human tumor cells. We characterized the resulting cells using phenotypic, metabolomic, and transcriptomic analyses, comparing their profiles to published data on in vivo-exhausted cells.