Fig 2: IFN-? response is required for efficient killing by redirected lymphocytes.a Left, cocultures of PBMCs with BT474 or BT-R cells were treated with different concentrations of HER2-TCB in presence of an IgG control or an IFN-? blocking antibody for 72 h. Then, viable cells were quantified by flow cytometry using EpCAM as a marker. Right, BT474 cells were grown in 3D and treated with HER2-TCB in presence of an IgG control (-) or an IFN-? blocking antibody (+) for 72 h. Viable BT474 cells were quantified by flow cytometry using EpCAM as a marker. Results were normalized to untreated cells. b Parental BT474 cells were cocultured with different ratios of CAR T cells for 48 h in the presence of an IgG control or an IFN-? blocking antibody. Then, cell numbers were calculated and expressed as in (a). c Parental BT474 or resistant BT-R cells were treated with different concentrations of IFN-? for 5 days. Cell numbers were estimated with the crystal violet staining assay. d Parental BT474 or resistant BT-R cells were treated with 1 µg/ml of IFN-? and the percentages of apoptotic cells were determined by means of Annexin V+ cells measured by flow cytometry. e Cells were stained with anti-IFNGR1 or isotype antibody and analyzed by flow cytometry. IFR1 stands for IFNGR1. f Sensitivity of the indicated cells to IFN-? was analyzed as in (c). g The indicated cells were treated with different concentrations of HER2-TCB and analyzed as in (a). h The indicated cell lines were cocultured with CAR Ts and analyzed as in (b). i Totally, 6.5 × 107 BT474 cells or the same cells knock-out for IFNGR1 were injected orthotopically into NSG mice. Mice were treated as described in Fig. 1c. Tumor volumes are represented as averages ± SD (empty, n = 8; IFR1 KO, n = 4). a Left, **p = 0.001, *p = 0.05; right, *p = 0.01. b ***p < 0.001, **p = 0.002. c ***p < 0.001. d **p = 0.007. f Left, **p = 0.002, ***p < 0.001, **p = 0.003, **p = 0.0013; right, ***p < 0.001, **p = 0.002 (shIFNGR1 #31); **p = 0.009, **p = 0.006, ***p < 0.001, **p = 0.003 (shIFNGR1 #92); *p = 0.02, **p = 0.004, ***p < 0.001 (shIFNGR1 #97). g Left, ***p < 0.001; right, ***p < 0.001, **p = 0.004, *p = 0.02 (shIFNGR1 #31); *p = 0.046, *p = 0.047, *p = 0.03 (shIFNGR1 #92); *p = 0.05, *p = 0.02, *p = 0.02 (shIFNGR1 #97). h Left, ***p < 0.001; right *p = 0.02, ***p < 0.001 (shIFNGR1 #31); ***p < 0.001, *p = 0.02 (shIFNGR1 #92); ***p < 0.001, **p = 0.005, **p = 0.005 (shIFNGR1 #97). Two-tailed t test. i **p < 0.01, ***p < 0.001, two-way ANOVA and Bonferroni correction. Data are presented as mean ± SD of three (a–c, f, g right, h), four (d), or six (g left) independent experiments. Source data are provided as a Source Data file.

Fig 3: GB cells upregulate TSPO expression upon response to T cell-derived IFN? and TNFa. a-j Induction of TSPO expression in (a-e) BTIC13 and in (f-j) BTIC129 upon response to TNFa and IFN? signaling. (a, b, f, g) BTICs were treated with 50 ng/ml TNFa or IFN?. 24 and 48 h following treatment TSPO mRNA and protein expression were analyzed. a, f RT-qPCR analysis of TSPO mRNA expression in BTICs. Results are presented as fold change compared to untreated condition after RPL13A mRNA normalization. b, g Western blot analysis of TSPO protein expression in BTICs. c-e, h-j Impact of TNF-R1/2 and IFNG-R1 blockade on TSPO expression in BTICs upon response to activated T cell supernatant. BTICs were pre-incubated with anti-TNF-R1/2 and/or anti-IFNG-R1 blocking antibodies and treated with supernatant of activated (c-e) FluTC or (h-j) TIL129. Control medium (CM) treatment was used to monitor basal TSPO expression. 24 h following treatment TSPO mRNA and protein expression were analyzed. c, h RT-qPCR analysis of TSPO mRNA expression in BTICs. Results are presented as fold change compared to isotype control after ß-actin mRNA normalization. d, e, i, j Western blot analysis of TSPO protein expression in BTICs. d, i Protein quantification is presented as fold change compared to isotype control after GAPDH protein normalization. e, j Representative blot of two independent experiments. k-n Single-cell RNA-Seq data from 4700 cells sorted from 20 adult GB patients were analyzed for TSPO expression and its correlation with IFN?-induced genes [65]. k UMAP (Uniform Manifold Approximation and Projection) plot of all malignant cells. Cells were clustered using the Harmony algorithm and colored on the bases of assigned meta-modules as described in Neftel et al. [65]. (AC: Astrocyte-like, NPC: neural-progenitor-like, OPC: oligodendrocyte-progenitor-like, MES: mesenchymal-like, G1/S, G2/M: cycling cells). l UMAP plot of TSPO expression in malignant cells. m Violin plot of TSPO expression in all identified clusters. n Correlation between the expression of TSPO and IFNGR1, IFNGR2 as well as IFN?-induced genes CD274, IRF1, SOCS3, B2M, IRF7, and IFITM3 at single-cell level. R indicates Pearson`s correlation coefficient. o Comparison of TSPO expression in recurrent GB patients who underwent neoadjuvant or adjuvant Pembrolizumab therapy. RNA-Seq data (GSE121810) from clinical trial NCT02852655 were analyzed to compare TSPO expression in patients with high or low IFN? scores [18]. (CPM: Counts per million) (a, b, d, e, f, g, i, j) Representative data of at least two independent experiments are shown. c, h Cumulative data of three independent experiments. Values represent the mean ± SD. P-value was calculated using (a, c, f, h) two-tailed Student`s t-test and (o) Mann–Whitney test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001, ns: non-significant

Fig 4: Targeted epigenetic silencing (CRISPRoff) with the T.gonfio library.a, Alignment of the T.gonfio sgRNA sequences to the CRISPRoff targeting window. b, An example of flow cytometry measurement of CD151 in HEK293T cells after exposure (10 days) to pools of three separated sgRNAs (3-sgRNA, used in the CRISPRoff study) or the respective T.gonfio qgRNA. ‘Off’ represents the CRISPRoff plasmid; ‘off-mut’, CRISPRoff mutant carrying a catalytically inactive version of the DNA methyltransferase. c, Quantification of the percentage of cells with ITGB1, CD81 and CD151 silencing 10 days after co-transfection of the CRISPRoff or CRISPRoff-mutant plasmids with pools of three single sgRNAs (3-sg) or the qgRNA plasmid from the T.gonfio library in HEK293T cells. Each dot represents an independent biological repeat of the assay. Data are presented as mean ± s.e.m. d, An example of a flow cytometry measurement of the percentage of cells with IFNGR1 silencing 10 days post-CRISPR knockout with the qgRNA plasmid from the T.spiezzo library or CRISPRoff with the qgRNA plasmid from the T.gonfio library in HEK293T cells. e, Quantification of the percentage of cells with CD47, IFNGR1 and MCAM silencing 10 days post-CRISPR knockout with qgRNA plasmids from the T.spiezzo library or CRISPRoff with qgRNA plasmids from the T.gonfio library in HEK293T cells. Each dot represents a biological repeat of the assay. Data are presented as mean ± s.e.m. f, A schematic of pairwise pooled genome-wide CRISPRoff screens to determine the effectiveness of T.gonfio for gene silencing at the genome scale in HEK293T cells. Created with BioRender.com. g, Mean phenotype scores (γ) for essential and non-essential genes from screens with the CRISPRoff or the T.gonfio library. N = 2 biological repeats. The dashed lines indicate the median and interquartile range. h, SSMD* value measuring the separation between essential and non-essential genes for each repeat of the screens performed with the CRISPRoff or T.gonfio pool libraries. i, Plots of true and false positive rates for the distinction between essential and non-essential genes for screens with the CRISPRoff or the T.gonfio library. AUC, area under the curve. j, Lentiviral template switching between sgRNA2 and sgRNA3 in pre-pooled versus post-pooled T.gonfio libraries. T0 and end: baseline and endpoint as defined in the text. k, Mean phenotype scores (γ) for essential versus non-essential genes in screens with pre-pooled versus post-pooled T.gonfio libraries (2 biological repeats); IQR, interquartile range. l, Mean phenotype scores (γ) for essential genes in post-pooled versus pre-pooled screens. IQRs for each screen were depicted. m, SSMD* between essential and non-essential genes for screens performed with the pre-pooled versus post-pooled library. In g, k and l, values above 0.2 or below −0.6 were set to 0.2 and −0.6, respectively.Source data