Published OnlineFirst September 10, 2019; DOI: 10.1158/1078-0432.CCR-19-0375 Translational Cancer Mechanisms and Therapy Clinical Cancer Research NKG2D Controls Natural Reactivity of Vg9Vd2T Lymphocytes against Mesenchymal Glioblastoma Cells Cynthia Chauvin1,2,Noemie Joalland1,2, Jeanne Perroteau1,2, Ulrich Jarry1,2, Laura Lafrance1,2, Catherine Willem1,3, Christelle Retiere 1,3, Lisa Oliver1,4, Catherine Gratas1,4, Laetitia Gautreau-Rolland1,2, Xavier Saulquin1,2, Francois¸ M. Vallette1,2,5, Henri Vie1,2, Emmanuel Scotet1,2, and Claire Pecqueur1,2 Abstract Purpose: Cellular immunotherapies are currently being Results: We evidenced that GBM cells displaying a mes- explored to eliminate highly invasive and chemoradioresistant enchymal signature are spontaneously eliminated by allo- glioblastoma (GBM) cells involved in rapid relapse. We recent- geneic human Vg9Vd2 T lymphocytes, a reactivity process ly showed that concomitant stereotactic injections of non- being mediated by gd T-cell receptor (TCR) and tightly alloreactive allogeneic Vg9Vd2 T lymphocytes eradicate regulated by cellular stress–associated NKG2D pathway. zoledronate-primed human GBM cells. In the present study, This led to the identification of highly reactive Vg9Vd2T we investigated the spontaneous reactivity of allogeneic lymphocyte populations, independently of a specificTCR human Vg9Vd2 T lymphocytes toward primary human GBM repertoire signature. Moreover, we finally provide evidence cells, in vitro and in vivo, in the absence of any prior of immunotherapeutic efficacy in vivo, in the absence of any sensitization. prior tumor cell sensitization. Experimental Design: Through functional and transcrip- Conclusions: By identifying pathways implicated in the tomic analyses, we extensively characterized the immuno- selective natural recognition of mesenchymal GBM cell reactivity of human Vg9Vd2 T lymphocytes against various subtypes, accounting for 30% of primary diagnosed and primary GBM cultures directly derived from patient 60% of recurrent GBM, our results pave the way for novel tumors. targeted cellular immunotherapies. Introduction Current standard therapy, defined by the Stupp protocol, includes surgery followed by radiotherapy with concomitant and adjuvant Glioblastomas (GBMs) are the most frequent primary brain chemotherapy (1). Despite such aggressive treatments, the medi- tumors in adult with an incidence of five per 100,000 people. an survival does not exceed 18 months, with less than 5% of patients alive at 5 years. This dismal prognosis might be explained by deep invasive tumor growth, limited surgical efficiency, poor 1CRCINA, INSERM, CNRS, Universite d'Angers, Universite de Nantes, Nantes, drug delivery across the blood–brain barrier, and a high degree of 2 3 France. LabEx IGO "Immunotherapy, Graft, Oncology," Nantes, France. Eta- GBM tumor heterogeneity. First, GBM display intertumor hetero- ¸ 4 blissement Francais du Sang, Nantes, France. Centre Hospitalier-Universitaire geneity mostly characterized by distinct genetic alterations occur- (CHU) de Nantes, Nantes, France. 5Institut de Cancerologie de l'Ouest (ICO), St Herblain, France. ring in individual tumors and leading to various responses in patients. The genetic landscape of GBM has been performed Note: Supplementary data for this article are available at Clinical Cancer through genome-wide association studies allowing the identifi- Research Online (http://clincancerres.aacrjournals.org/). cation of up to four molecular subtypes with relative prognostic or C. Chauvin and N. Joalland contributed equally to this article. predictive significance (2, 3). There are marked differences E. Scotet and C. Pecqueur contributed equally to this article. between the mesenchymal subtype (MES) and the three others Corresponding Authors: Claire Pecqueur, CRCINA, INSERM, CNRS, Universite (defined here as CNP, referring to Classical, Neural, and Proneural d'Angers, Universite de Nantes, 8 quai Moncousu, 44007 Nantes, France. subtypes). The MES subtype is associated with poor survival, in Phone: 332-0228-080302; Fax: 332-0228-080204; E-mail: contrast to CNP subtype which is generally associated with a more [email protected]; and Emmanuel Scotet, Centre de Recherche en favorable outcome (2, 4, 5). Second, spatial heterogeneity within Cancerologie et Immunologie Nantes-Angers, INSERM UMR1232 CNRS the same tumor, including active tumor zones as well as hypoxic ERL6001, Universite de Nantes, 8 quai Moncousu, 44007 Nantes, France. and necrotic zones, is common in GBM (6, 7). Importantly, Phone: 33-2-2808-0222; Fax: 332-2808-0204; E-mail: [email protected] hypoxic zones constitute cellular niches for MES GBM cells and also cancer stem–like cells (CSCs) with singular phenotypic Clin Cancer Res 2019;XX:XX–XX properties including transient quiescence, self-renewal, resistance doi: 10.1158/1078-0432.CCR-19-0375 to radiation-induced DNA damages, and the ability to reconsti- Ó2019 American Association for Cancer Research. tute the initial tumor. Thus, new strategies targeting highly www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 10, 2019; DOI: 10.1158/1078-0432.CCR-19-0375 Chauvin et al. Vg9Vd2 T lymphocytes toward some human GBM cells. Using Translational Relevance primary GBM cells from patients, we demonstrate that alloge- Glioblastoma (GBM) representing the majority of primary neic Vg9Vd2 T lymphocytes specifically and spontaneously malignant brain tumors displays a dismal prognosis with a react against GBM cells with a mesenchymal signature. This recurrence rate of more than 90%. Therapeutic immunothera- work next showed that highly reactive Vg9Vd2Tlymphocytes pies, including the emerging use of adoptive cell therapy, have are engaged through both TCR and NKG2D pathways, but shown promising antitumor efficacy. In this work, we clearly independently of a specific TCR signature. Finally, we provided show that the more resistant subsets of GBM cells are spon- evidence of allogeneic Vg9Vd2 T-lymphocyte immunotherapy taneously killed by nonalloreactive allogeneic human Vg9Vd2 efficacy in vivo, in the absence of any prior sensitization. T-lymphocyte effectors. This natural reactivity process, medi- ated by the gd T-cell receptor and tightly regulated by cellular stress–associated NKG2D pathway, led to the identification of Materials and Methods highly reactive allogeneic Vg9Vd2 T-lymphocyte subsets. Human primary GBM cultures Importantly, we provide evidence of allogeneic Vg9Vd2 Human primary GBM cultures were obtained after mechanical T-lymphocyte immunotherapy efficacy in vivo, in the absence dissociation of surgical resection tumor samples from patients of any prior tumor cell sensitization. Our study brings new (n ¼ 17). All procedures involving human patients were per- insights into novel immunotherapeutic options for therapy formed in accordance with the ethical standards of the ethic directed at the recurrence of GBM based on adoptive transfer of national research committee and with the 1964 Helsinki decla- allogeneic Vg9Vd2 T lymphocytes at the tumor bed. ration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual patients included in this study. Primary cultures were established and stored at À180C. Cell-frozen vials were grown in defined medi- resistant cancer cells, including mesenchymal GBM cells and CSC, um [DMEM/Ham F12 (Gibco), 2 mmol/L L-glutamine (Gibco), may significantly improve patients' outcomes. N2- and B27-supplement (Gibco), 2 mg/mL heparin (Sigma In line with their spectacular effects evidenced in various solid Aldrich), 20 ng/mL EGF and 25 ng/mL bFGF (Peprotech), and and circulating cancer indications (8–11), immunotherapies have 100 IU/mL penicillin and 100 mg/mL streptomycin (Gibco)] at also been proposed for treating patients with GBM (12), including 37 C in a humidified atmosphere with 5% CO2. All experiments adoptive transfer of immune cells. Among attractive immune were performed with GBM cells in culture for less than 3 months effectors, peripheral Vg9Vd2 T lymphocytes, mostly present in (passages < 7), and cells were regularly checked for mycoplasma þ primates and representing 5% to 10% of blood CD3 lympho- contamination. There are 5 MES cultures (GBM-1, 4, 8, 11, and cytes in healthy adults, are important players in natural host 12) and 12 CNP (GBM-3, 5, 6, 7, 8, 9, 10, 13, 14, 16, 17, 18, and defenses against infection and malignancies (13). These transi- 19). For pilot studies, GBM-1 and GBM-10 were used as repre- tional T lymphocytes are selectively activated in a T-cell receptor sentative of human mesenchymal and CNP tumor cells, (TCR)–dependent, but MHC-independent, manner, by nonpep- respectively. tidic small molecules [hereafter called phosphoantigens (PAg)], such as isopentenyl pyrophosphate. Accordingly, target cells' Generation and expansion of allogeneic human Vg9Vd2T sensitization by pharmacologic aminobisphosphonate (NBP) lymphocytes compounds, such as zoledronate, inhibits a key enzyme of the After informed consent was obtained, human peripheral blood mammalian mevalonate pathway that degrades PAg and upre- mononuclear cells (PBMCs) were isolated from blood samples of gulates the reactivity of Vg9Vd2 T lymphocytes. We have described healthy adult volunteers recruited at the Etablissement Francais¸ a mandatory role played by BTN3A/CD277 butyrophilins which du Sang (EFS). For specific