Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector γδ T cells with antitumor activity

Abstract

Amino-bis-phosphonates (N-BPs) such as zoledronate (Zol) have been used in anticancer clinical trials due to their ability to upregulate pyrophosphate accumulation promoting antitumor Vg9Vd2 T cells. The butyrophilin 3A (BTN3A, CD277) family, mainly the BTN3A1 isoform, has emerged as an important structure contributing to Vg9Vd2 T cells stimulation. It has been demonstrated that the B30.2 domain of BTN3A1 can bind phosphoantigens (PAg) and drive the activation of Vg9Vd2 T cells through conformational changes of the extracellular domains. Moreover, BTN3A1 binding to the cytoskeleton, and its consequent membrane stabilization, is crucial to stimulate the PAg-induced tumor cell reactivity by human Vg9Vd2 T cells. Aim of this study was to investigate the relevance of BTN3A1 in N-BPs-induced antitumor response in colorectal cancer (CRC) and the cell types involved in the tumor microenvironment.In this paper, we show that (i) CRC, exposed to Zol, stimulates the expansion of Vd2 T lymphocytes with effector memory phenotype and antitumor cytotoxic activity, besides sensitizing cancer cells to gd T cellmediated cytotoxicity; (ii) this effect is partially related to BTN3A1 expression and in particular with its cellular re-distribution in the membrane and cytoskeleton-associated fraction; (iii) BTN3A1 is detected in CRC at the tumor site, both on epithelial cells and on tumor-associated fibroblasts (TAF), close to areas infiltrated by Vd2 T lymphocytes; (iv) Zol is effective in stimulating antitumor effector Vd2 T cells from exvivo CRC cell suspensions; and (v) both CRC cells and TAF can be primed by Zol to trigger Vd2 T cells.

BTN3A is a prognosis marker and a promising target for Vγ9Vδ2 T cells based-immunotherapy in pancreatic ductal adenocarcinoma (PDAC)

“…BTN3A expression has rarely been investigated in the primary tumor context. 24,25,27,28,29,34 Based on the analysis of human primary tumor samples and novel patient-derived xenograft, we have now established that BTN3A overexpression and a dominant expression of the BTN3A2 isoform is strongly associated with a poor prognosis, in accordance with what was recently described in primary leukemic blasts 25 and in gastric cancer. 29 In this line, the overexpression of BTN3A2 gene was associated with an increased proliferation and invasion of gastric cancer cell lines.…”

Zoledronate Triggers Vδ2 T Cells to Destroy and Kill Spheroids of Colon Carcinoma: Quantitative Image Analysis of Three-Dimensional Cultures

“…Immunofluorescence assay was performed as described ( 29 ) with the anti-ICAM1 monoclonal antibody (mAb) (14D12D2, IgG1) ( 29 ), or anti-CD133-specific mAb (W6B3C1, IgG1, Miltenyi Biotec, Bergish Gladbach, Germany) on CRC cell lines or with anti-Vδ2 mAb (BB3, IgG1) ( 30 ) or anti-CD3 mAb (289/10/F11, IgG2a) or anti-CD16 mAb (VD4, IgG1) ( 29 ) on Zol-stimulated lymphocytes, followed by Alexafluor647 anti-IgG2a or PE-anti-IgG1 goat anti-mouse antiserum (GAM) (Life Technologies, Milan, Italy). The Fc chimeras (soluble receptors fused with the Fc of human immunoglobulins): Fc-NKG2D and Fc-DNAM1 were purchased from R&D System (Minneapolis, MN, USA) and used on CRC cell lines in immunofluorescence assay followed by Alexafluor647 goat anti-human antiserum (Life Technologies).…”

“…Peripheral blood mononuclear cells (PBMC) were obtained from healthy adult donor’s buffy coat (institutional informed consent signed at the time of donation and EC approval PR163REG2014) by density gradient centrifugation using Lymphocyte Separating Medium (Pancoll human, Density: 1.077 g/ml, PAN-Biotech, Munich, Germany) as described ( 29 ). To obtain Vδ2 T lymphocyte populations, 10 5 PBMC were cultured in 96 W U-bottomed plates in 200 µl of RPMI-1640 (Gibco Life Technologies) medium supplemented with 10% FBS (Gibco™), penicillin/streptomycin and l -glutamine (BioWhittaker ® Reagents) and with 1.0 µM zoledronic acid as zoledronate (Zol, Selleckem, Munich, Germany) at 37°C humidified cell incubator with 5% of CO 2 .…”

How to Hit Mesenchymal Stromal Cells and Make the Tumor Microenvironment Immunostimulant Rather Than Immunosuppressive

“…Another approach to downregulate the inhibitory effect of MSC on immune system is to convert their behavior from immunosuppressive to immunostimulant. Recently, it has been demonstrated, both in NHL and CRC, that priming of MSC, derived from lymph nodes or colon mucosa, with the aminobisphosphonate (N-BP) zoledronic acid can trigger Vδ2 T cell proliferation ( 25 , 234 , 235 ). In NHL, zoledronate-pulsed MSC are impaired in the secretion of TGF-β, whereas there is an increment in the production of IL-15 ( 234 ) (N-BPs in Figure 3 ).…”