Journal: Nature

Article Title: CRISPR screens decode cancer cell pathways that trigger γδ T cell detection

doi: 10.1038/s41586-023-06482-x

Figure Lengend Snippet: (a) Schematic of OXPHOS, inhibitor targets, and genetic KOs. (b) Surface BTN3A MFI in Daudi-Cas9 KOs cultured in different glucose concentrations for 3 days in RPMI (no glucose, no pyruvate). Normalized to cells grown without glucose (0 g/L). (c, d) Surface BTN3A MFI in WT Daudi-Cas9 cells cultured with vehicles (DMSO, ethanol) or OXPHOS inhibitors of (c) Complex I (rotenone), Complex V (oligomycin A), mitochondrial membrane potential (FCCP), and (d) Complex III (antimycin A) for 72 hours in complete RPMI. (e-g) Surface BTN3A MFI in WT Daudi-Cas9 cells cultured with (e) 2-DG, (f) AICAR, and (g) Compound 991 (C991) or equivalent amount of DMSO (vehicle) for 72 hours in complete RPMI. (h) Surface BTN3A MFI in patient-derived breast cancer organoids and Daudi cells cultured for 3 days with pamidronate and AICAR, C991, or DMSO. (i) Surface BTN3A MFI in WT Daudi-Cas9 cells co-treated with an OXPHOS/glycolysis inhibitor and increasing amounts of Compound C (CC, AMPK inhibitor). (b) n=4 per condition, data combined from two independent experiments, each individually normalized. (c) n=4 per condition, data combined from two independent experiments. (d) n=3 per condition, representative data from one of two experiments. (e, f) n=3 per condition, representative data from one of three independent experiments. (g) n=3 per condition, representative data from one of two independent experiments. (h) n=5, data combined from two independent experiments. (i) n=3 per condition, representative data from one of three independent experiments. (b, e, f, h) One-way ANOVA comparison to the zero or control treatment condition with Dunnett’s multiple comparisons test. (c) Two-tailed unpaired Student’s t test with FDR adjustment for the tested concentrations (1.25-20 μM). (d, g) Two-tailed unpaired Student’s t test with Bonferroni correction. (b-i) Mean ± SD. p<0.0001 (****), p<0.001 (***), p<0.01 (**), p<0.05 (*).

Article Snippet: For 4 days prior to the co-culture, Daudi cells were cultured in complete RPMI and PDOs were cultured in BME for 4 days in the presence of 10 μM pamidronate and either 500 μM AICAR (Merck) or 40 μM Compound 991 (TargetMol).

Techniques: Cell Culture, Membrane, Derivative Assay, Comparison, Control, Two Tailed Test

Journal: Nature

Article Title: CRISPR screens decode cancer cell pathways that trigger γδ T cell detection

doi: 10.1038/s41586-023-06482-x

Figure Lengend Snippet: (a) G115 clone Vγ9Vδ2 TCR tetramer staining MFI of WT Daudi-Cas9 cells treated with 80 μM C991 (DMSO), DMSO (vehicle), 0.5 mM AICAR (aqueous), or without treatment for 72 hours. Two-tailed unpaired Student’s t test. (b) Vγ4Vδ1 TCR (clone DP10.7) tetramer staining fluorescence (MFI) of Daudi-Cas9 KO cells treated with 80 μM C991 (DMSO), DMSO (vehicle), 0.5 mM AICAR (aqueous), or water for 72 hours. This staining with a tetramer of an irrelevant γδTCR clone defines the background for Vγ9Vδ2 TCR tetramer staining in Figure 4a. (c) qPCR data for BTN2A1, BTN3A1, and BTN3A2 transcripts in Daudi-Cas9 cells treated with C991, internally normalized to ACTB transcripts and normalized to DMSO (vehicle)-treated cells. Two-tailed unpaired Student’s t test. (d) IgG1κ isotype control staining in Daudi-Cas9 KO cells treated with 80 μM Compound 991 (DMSO), DMSO (vehicle), 0.5 mM AICAR (aqueous), or water (vehicle) treatment for 72 hours. (e) Survival of eGFP+ Daudi cells treated for 3 days with AICAR or water prior to co-culture (E:T 2:1) with primary Vγ9Vδ2 T cells in the presence of an anti-BTN3A antibody (clone 103.2). Cells were quantified using real-time quantitative live-cell imaging (Incucyte). Survival was normalized to Daudi cells cultured without T cells. (a) n=4 per condition, representative data from one of two independent experiments. (b) n=3 per condition, representative data from one of two independent experiments. (c) n=4 per condition, representative data from one of three independent experiments. (d) n=3, representative data from one of two independent experiments. (e) n=4 per condition. (a-e) Mean ± SD. p<0.0001 (****). Source Data files provided for Figures 2, ​,3,3, ​,4,4, and Extended Data Figures 1, ​,4,4, ​,7,7, ​,8,8, ​,9,9, and ​and1010.

Article Snippet: For 4 days prior to the co-culture, Daudi cells were cultured in complete RPMI and PDOs were cultured in BME for 4 days in the presence of 10 μM pamidronate and either 500 μM AICAR (Merck) or 40 μM Compound 991 (TargetMol).

Techniques: Staining, Two Tailed Test, Fluorescence, Control, Co-Culture Assay, Live Cell Imaging, Cell Culture