Modeling Vγ9Vδ2 T-cell exhaustion in vitro: phenotypic and functional insights for immunotherapy

• An in vitro model induces Vγ9Vδ2 T cell exhaustion, allowing detailed phenotypic, metabolic, and transcriptomic analysis. • Exhausted Vγ9Vδ2 T cells retain partial function and can be reactivated with anti-CD3 antibodies or T cell engagers. Recent advancements in cancer immunotherapies have highlighted the potential of Vγ9Vδ2 T cells as key players in the immune response against cancer. 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. Furthermore, we measured the reactivation potential of these exhausted Vγ9Vδ2 T cells using anti-CD3 monoclonal antibodies, demonstrating that CD3 stimulation can partially reverse exhaustion and restore anti-tumor effector functions. Furthermore, exhausted Vγ9Vδ2 T cells retained some cytotoxic activity upon stimulation with T Cell Engagers, underscoring the versatility and applicability of our in vitro exhaustion model. This model offers a robust platform for the evaluation of novel immunotherapies targeting Vγ9Vδ2 T cells, facilitating preclinical assessments before transitioning to in vivo studies.