BTN3A1 expressed in cervical cancer cells promotes Vγ9Vδ2 T cells exhaustion through upregulating transcription factors NR4A2/3 downstream of TCR signaling

BackgroundClinical trials have shown that immunotherapy based on V gamma 9V delta 2 T cells (V delta 2 T cells) is safe and well-tolerated for various cancers including cervical cancer (CC), but its overall treatment efficacy remains limited. Therefore, exploring the mechanisms underlying the suboptimal efficacy of V delta 2 T cell-based cancer immunotherapy is crucial for enabling its successful clinical translation.MethodsTumor samples from CC patients and CC cell line-derived xenograft (CDX) mice were analyzed using flow cytometry to examine the exhausted phenotype of tumor-infiltrating V delta 2 T cells. The interrelationship between BTN3A1 expression and V delta 2 T cells in CC, along with their correlation with patient prognosis, was analyzed using data from The Cancer Genome Atlas (TCGA) database. CC cell lines with BTN3A1 knockout (KO) and overexpression (OE) were constructed through lentivirus transduction, which were then co-cultured with expanded V delta 2 T cells, followed by detecting the function of V delta 2 T cells using flow cytometry. The pathways and transcription factors (TFs) related to BTN3A1-induced V delta 2 T cells exhaustion and the factors affecting BTN3A1 expression were identified by RNA-seq analysis, which was confirmed by flow cytometry, Western Blot, and gene manipulation.ResultsTumor-infiltrating V delta 2 T cells exhibited an exhausted phenotype in both CC patients and CDX mice. BTN3A1 expressed in CC is highly enhancing exhaustion markers, while reducing the secretion of effector molecules in V delta 2 T cells. Blocking TCR or knocking down nuclear receptor subfamily 4 group A (NR4A) 2/3 can reverse BTN3A1-induced exhaustion in V delta 2 T cells. On the other hand, IFN-gamma secreted by V delta 2 T cells promoted the expression of BTN3A1 and PD-L1.ConclusionsThrough binding gamma delta TCRs, BTN3A1 expressed on tumor cells, which is induced by IFN-gamma, can promote V delta 2 T cells to upregulate the expression of TFs NR4A2/3, thereby affecting their activation and expression of exhaustion-related molecules in the tumor microenvironment (TME). Therefore, targeting BTN3A1 might overcome the immunosuppressive effect of the TME on V delta 2 T cells in CC.