Supplementary Figure S2. BTN2A1 is broadly expressed in normal and tumor cells.

Supplementary Figure S8. Segmentation of live cells using Refractive Index (RI).

Supplementary Figure S10. The caspase-3-specific inhibitor zDEVD-FMK inhibits the cleavage of GSDME.

Supplementary Figure S6. Anti-BTN2A1 107G3B5 increases Vɣ9Vδ2 T cell effectors functions against hematological cell lines.

Supplementary Figure S5. Cytotoxicity assays of 4 cancer cell lines (PC3, KARPAS, 697 and SUP-T1) and primary ALL blasts (n=4) using Annexin and 7-AAD labelling.

Supplementary Figure S4. Anti-BTN2A1 107G3B5 mAb characterization.

Supplementary Figure S9. Anti-BTN2A1 sensitizes Vɣ9Vδ2 T cell cytotoxicity against PC3 cell line.

Visualization of Vγ9Vδ2 T cells (E) and SUP-T1 (T) coculture using a label-free method within 20 hours. Ratio T/E = 1/2. Cells treated with 107G3B5 antibody (10ug/ml).

Supplementary Figure S7. 107G3B5 primes Vγ9Vδ2T cells by mechanisms that are dependent on BTN3A and BTN2Abut independent of the pAg-binding B30.2 intracellular domain.

Supplementary Figure 12. The caspase-3-specific inhibitor zDEVD-FMK inhibits the cleavage of GSDME.

Supplementary figure S11. Anti-BTN2A1 107G3B5 induces a colocalization of BTN3A1 and BTN2A1.

Supplementary Raw Data Excel File S1

Abstract Vγ9Vδ2 T cells are potent but elusive cytotoxic effectors. Butyrophilin subfamily 2 member A1 (BTN2A1) is a surface protein that has recently been shown to bind the Vγ9 chain of the γδ T-cell receptor, but its precise role in modulating Vγ9Vδ2 T-cell functions remains unknown. Here, we show that 107G3B5, a monoclonal BTN2A1 agonist antibody, was able to significantly enhance Vγ9Vδ2 T-cell functions against hematologic or solid cell lines and against primary cells from patients with adult acute lymphoblastic leukemia. New computer vision strategies applied to holotomographic microscopy videos showed that 107G3B5 enhanced the interaction between Vγ9Vδ2 T cells and target cells in a quantitative and qualitative manner. In addition, we found that Vγ9Vδ2 T cells activated by 107G3B5 induced caspase 3/7 activation in tumor cells, thereby triggering tumor cell death by pyroptosis. Together, these data demonstrate that targeting BTN2A1 with 107G3B5 enhances the Vγ9Vδ2 T-cell antitumor response by triggering pyroptosis-induced immunogenic cell death. These new pyroptosis-based therapies have great potential to stimulate the immune system to fight cancer, especially “cold” tumors. See related Spotlight by Kabelit, p. 1662

Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAbs), and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift toward an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T cell proliferation and interferon-gamma (IFNγ) secretion. Mechanistically, BTN2A1 engagement induced spleen tyrosine kinase (SYK) recruitment, leading to sequential SYK and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of SYK or ERK phosphorylation abolished M2 reprogramming upon BTN2A1 engagement. Our findings, derived from an analysis of macrophages from healthy donors and human tumors, underscore the pivotal role of BTN2A1 in immunosuppressive macrophage differentiation and function, offering potential applications in cancer immunotherapy.

ABSTRACTVγ9Vδ2 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of Vγ9Vδ2 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of Vγ9Vδ2 T cells to an anti-BTN3A antibody. Blood Vγ9Vδ2 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated Vγ9Vδ2 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNFα, IFNγ and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and Vγ9Vδ2 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro, SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 Vγ9Vδ2 T cells cytotoxicity and IFN-γ and TNFα in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that Vγ9Vδ2 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response.KEY POINTSSARS-CoV-2 mediates upregulation of the key receptor of Vγ9Vδ2 T cells BTN3A on lung tissues and cell lines as well as monocytesDuring SARS-CoV-2 infection, Vγ9Vδ2 are differentiated and efficiently degranulate and secrete cytokines upon activation with BTN3A mAb

Supplementary Figures 1-6 from Tumor Protein 53–Induced Nuclear Protein 1 Is a Major Mediator of p53 Antioxidant Function

PDF file, 232K, Predicted pathways regulated by Nupr1.

PDF file, 355K, mRNA expression in glucose starvation- and hypoxia-treated pancreatic cancer cells expressing or not Nupr1.

PDF file, 137K, Protective role of Nupr1 and AURKA in BxPC-3 cells.

PDF file, 57K, siRNA sequences used for silencing Nupr1, Beclin1, ATG5 and AURKA mRNA and Primer sequences used to amplify human genes.