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The success of immunotherapy has highlighted the critical role of the immune microenvironment in acute lymphoblastic leukemia (ALL); however, the immune landscape in ALL remains incompletely understood and most studies have focused on conventional T cells or NK cells. This study investigated the prognostic impact of circulating γδ T-cell alterations using high-dimensional analysis in a cohort of newly diagnosed adult ALL patients (10 B-ALL; 9 Philadelphia+ ALL; 9 T-ALL). Our analysis revealed common alterations in CD8+ T cells and γδ T cells of relapsed patients, including accumulation of early stage differentiation and increased expression of BTLA and CD73. We demonstrated that the circulating γδ T-cell signature was the most discriminating between relapsed and disease-free groups. In addition, Vδ2 T-cell alterations strongly discriminated patients by relapse status. Taken together, these data highlight the role of ɣδ T cells in adult ALL patients, among whom Vδ2 T cells may be a pivotal contributor to T-cell immunity in ALL. Our findings provide a strong rationale for further monitoring and potentiating Vδ2 T cells in ALL, including in the autologous setting.

International audience; The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

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.

Background: Anti-tumoral response of Vg9Vd2 T cells requires sensing of phosphoantigens accumulated in malignant cells through binding of butyrophilin 3A(BTN3A). Moreover, an unknown partner located in human Chr6 was shown to be mandatory to BTN3A-mediated Vg9Vd2 T cell activation in murine models. Here, we identified butyrophilin 2A (BTN2A), which is located to Chr6, as a requirement for BTN3A-mediated Vg9Vd2 T cell cytotoxicity against cancer cells. Methods: CRISPR-Cas9-mediated inactivation of BTN2A1/2A2 isoforms was performed in Daudi, K562 and HEK-293T cells. Vg9Vd2 T cells expanded from healthy PBMCs were co-cultured with wild-type or BTN2AKO cells +/- BrHPP (1 µM), HMBPP (0.1 µM) or zoledronate (45 µM), or anti-BTN2A mAb, and Vg9Vd2 T cell degranulation (%CD106ab+ cells), and intracellular TNFa and IFNg assessed after 4h. Mouse T cell hybridoma 53/4 expressing TCRVg9Vd2-MOP were co-cultured overnight with NIH3T3 murine fibroblasts transfected with BTN3A- and/or BTN2A-encoding plasmids +/-HMBPP(10 µM), or increasing doses of HMBPP or anti-BTN3 20.1 mAb. BTN2A transcript expression in normal vs. tumoral tissue was analyzed using GEPIA tool. Anti-BTN2A mAb staining was performed on human samples of primary AML, cervical and pancreatic carcinoma and assessed by flow cytometry. Results: Degranulation and intracellular IFNg/TNFa (n=6) were abolished in Vg9Vd2 T cells co-cultured with BTN2AKO Daudi, K562 and HEK-293T cells compared to wild-type, in all conditions tested including anti-BTN3 20.1. Murine cells do not express no BTN2A1 or BTN3A orthologs and are unable to activate human Vg9Vd2 T cells. Ectopic expression of BTN2A and BTN3A combination but neither BTN2A or BTN3A alone in murine NIH3T3 cells, allows triggering of IL-2 secretion in mouse 53/4-TCRVg9Vd2-MOP reporter cells in presence of HMBPP or 20.1 mAb in dose-dependent manner. Anti-BTN2A mAb was able to suppress Vg9Vd2 T cell degranulation/cytokine secretion against cancer cell lines and activation of mouse 53/4-TCRVg9Vd2-MOP reporter by BTN2A/BTN3A-expressing NIH3T3 in a dose-dependent manner. BTN2A transcript was significantly up-regulated in pancreatic, ovarian and cervical carcinoma vs. normal tissue. Extracellular BTN2A protein was detected in primary hematological and solid tumors. Conclusion: Here, we show that BTN2A is mandatory for BTN3A-mediated function in human Vg9Vd2 T cells. Moreover, concomitant BTN2A and BTN3A expression empowers murine T cells with activation through Vg9Vd2 TCR, opening new roads for mouse models of Vg9Vd2 T cell anti-tumoral responses. We describe an anti-BTN2A able to suppress Vg9Vd2 T cell function, and we show BTN2A expression in primary tumors. These results are relevant for understanding Vg9Vd2 T cell antitumoral immunity triggered by phosphoantigens and amino-bisphosphonates. Disclosures Olive: ImCheck Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; GlaxoSmithKline: Patents & Royalties.

Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

Erratum inCorrigendum to "Socioeconomic deprivation is associated with decreased survival in patients with acute myeloid leukemia" [Cancer Epidemiol. 66 (2020) 101699].Le Floch AC, Eisinger F, D'Incan E, Rey J, Charbonnier A, Caymaris L, Stoler M, Mancini J, Boher JM, Sfumato P, Vey N.Cancer Epidemiol. 2020 Dec;69:101832. doi: 10.1016/j.canep.2020.101832. Epub 2020 Oct 14.PMID: 33067156 No abstract available.; International audience; Background: Socioeconomic deprivation is associated with poor prognosis in patients with solid tumors. However, few studies have assessed the association between socioeconomic parameters and prognosis in Acute Myeloid Leukemia (AML), and these report conflicting results. Our monocentric study assessed the impact of socioeconomic deprivation using the validated EPICES (Evaluation of Deprivation and Inequalities in Health Examination Centers) score in a prospective cohort of intensively treated AML patients.Methods: EPICES questionnaires were given to patients receiving intensive chemotherapy for newly diagnosed AML at the Paoli Calmettes Institute between July 2012 and December 2014. Study participants were categorized as non-deprived (score

Introduction: Vγ9Vδ2 T cells immunotherapy is an emerging strategy in acute myeloid leukemia (AML). Partial effects of phosphoantigens like zoledronate, prompted us to investigate other molecules. Using Vγ9Vδ2 T cells expanded from healthy volunteers, recent works of our team demonstrate that anti-BTN3A 20.1 agonist monoclonal antibody (mAb) improves Vγ9Vδ2 T cells cytotoxicity in vitro and in vivo, including in zoledronate resistant blasts. Efficacy of anti-BTN3A 20.1 agonist mAb remains undetermined using autologous Vγ9Vδ2 T cells and mechanism of its action on AML blasts is understudied. Material and methods: Expression of co-stimulatory molecules on surface of AML blasts and Vγ9Vδ2 T cells, from 37 PBMC of AML patients, was assessed by flow cytometry. Vγ9Vδ2 T cells expansion was realized from the same samples with zoledronate, and administrations of Il-2 and Il-15. Purity was assessed by flow cytometry at day 13. Degranulation assay was performed at day 14. Results: 20 degranulation assays were realized on successfully expanded Vγ9Vδ2 T cells (69% of analyzable samples). Degranulation with anti-BTN3A 20.1 agonist mAb was enhanced compared to zoledronate condition (p=0.0085) or negative control (p Conclusion: Efficacy of anti-BTN3A 20.1 agonist mAb on primary AML blasts is confirmed in an autologous setting. Degranulation seems to play a major role in enhancement of Vγ9Vδ2 T cells cytotoxicity under anti-BTN3A 20.1 agonist mAb treatment, where DNAM-1 could play a determinant role. An autologous strategy for Vγ9Vδ2 T cells may be studied and considered in AML patients, with potent extrapolation to γδ T cell therapy protocols in targeting strategies. Disclosures Vey: Novartis: Consultancy, Honoraria; Janssen: Honoraria. Olive:ImCheck Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; GlaxoSmithKline: Patents & Royalties.