Targeting Btn2a1 Modulates Anti-Tumor Activity Of Vg9vd2 T Cells

Background Vg9Vd2 T constitute the predominant subset among gd T cells in peripheral blood. Their infiltration into malignant tissues is associated with a favorable prognosis. Their anti-tumor activity is triggered by intracellular accumulation of organic phosphoantigens (pAgs) due to tumorigenesis. Recently, BTN2A1 was shown to bind to the Vg9TCR chain allowing immune synapse between cancer and Vg9Vd2T cells, thus initiating the anti-tumoral response. In this study, we generated monoclonal antibodies against BTN2A1 and evaluated their ability to modulate γδT cell cytotoxicity. Methods Anti-BTN2A1 mAbs were generated by mouse immunization. Their effect on Vg9Vd2 T cell degranulation, secretion of IFNγ/TNFα, and target cell killing as depicted by caspase 3/7 cleavage, were tested in co-cultures with Daudi, HL-60 cell lines and primary acute myelocytic leukemia (AML) blasts with or without zoledronate or the anti-BTN3A mAb 20.1. These readouts were measured by flow cytometry. Endometrial cancer spheroids were used to assess the ability of the anti-BTN2A1 antagonistic mAb to inhibit Vg9Vd2 T cell killing of cancers cells. Results We generated 7 anti-BTN2A1 mAbs and tested their effect on Vg9Vd2 T cell degranulation against Daudi cells with or without zoledronate. Six out of 7 anti-BTN2A1 mAbs significantly inhibited basal Vg9Vd2 T cell degranulation against Daudi up to 17-fold, and 5 of them were able to inhibit Vg9Vd2 T cell degranulation against Daudi in presence of zoledronate. Consistently, anti-BTN2A1 mAbs abrogated zoledronate and anti-BTN3A 20.1-induced apoptosis with different efficiencies. The level of apoptosis inhibition after zoledronate and 20.1 treatment were correlated. Anti-BTN2A1 7.48 mAb was the clone with the highest inhibitory potential. Increasing concentrations of 7.48 abrogated not only Vg9Vd2 T cell degranulation (IC50= 0.033±0.0003 µg/mL) but also TNFα (IC50= 0.03±0.006 µg/mL) and IFNγ (IC50= 0.015±0.004 µg/mL) secretion against Daudi cells in presence of pAgs. The ability of anti-BTN2A1 antibodies to inhibit Vg9Vd2 induced tumor cell apoptosis was also shown in 3D endometrial cancer spheroids. In co-cultures of Vg9Vd2 T cells with primary AML blasts, the anti-BTN2A1 7.48 inhibited Vg9Vd2 T cell degranulation as well as TNFα, IFNγ production and killing of AML blasts. Conclusions Antagonist antibodies to BTN2A1 highlighted its critical role in Vg9Vd2 anti-tumor responses. BTN2A1 is involved in Vg9Vd2 T cell anti-tumoral activity and can constitute an interesting therapeutic target for gdT cell response immunomodulation in cancer or immune diseases treatment. Ethics Approval The research was approved by the relevant institutional review boards (ethic committee and ANSM, HEMATO-BIO IPC 2013-015, Ref ANSM 131368B-11, Sponsor Institut Paoli Calmettes N° ID RCB 2013-A01437-38). Consent Informed consent was obtained from all donors in accordance with the 121 Declaration of Helsinki.