Fig. 2

GB cells upregulate TSPO expression upon response to T cell-derived IFNγ and TNFα. a-j Induction of TSPO expression in (a-e) BTIC13 and in (f-j) BTIC129 upon response to TNFα and IFNγ signaling. (a, b, f, g) BTICs were treated with 50 ng/ml TNFα or IFNγ. 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 β-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 IFNγ-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 IFNγ-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 IFNγ 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