Fig 2: Endometrial stromal cells license to an anti-inflammatory phenotype without upregulation of human leukocyte antigen II. Representative histograms (colored white) of eSC cell surface expression of CD119 at a baseline, b 3 days, and c 7 days of interferon (IFN)γ and tumor necrosis factor (TNF)α exposure. Gray histograms indicate respective immunoglobulin controls. Representative scatterplots of eSC cell surface expression of CD120a and CD120b at d baseline, e 3 days, and f 7 days of IFNγ and TNFα exposure. Box and whisker plots showing the minimum and maximum distribution of the median fluorescence intensity (MFI) of g CD119—phycoerythrin (PE; n = 5), h CD120a—allophycocyanin (APC; n = 5), and i CD120b-PE (n = 5). Representative histograms (colored white) of eSC cell surface expression of human leukocyte antigen (HLA) I at j baseline, k 3 days, and l 7 days of IFNγ and TNFα exposure. Gray histograms indicate respective immunoglobulin controls. m Box and whisker plots showing the minimum and maximum distribution of the MFI of HLA I—Alexa Fluor (AF) 488 (n = 5). Representative histograms (colored white) of eSC cell surface expression of HLA II at n baseline, o 3 days, and p 7 days of IFNγ and TNFα exposure (n = 5). Gray histograms indicate respective immunoglobulin controls. Box and whisker plots showing the minimum and maximum distribution of baseline and post-licensing concentrations of q L-kynurenine (as a marker of indoleamine-2,3 dioxygenase activity), r interleukin (IL)-6, and s prostaglandin (PG) E2 in conditioned cell culture media (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 3: The reverse signaling of tmTNF-α enhances the sensitivity of T cells to TNF-α-induced AICD through upregulating TNFR(A, B, C, D) Jurkat cells were stimulated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by αCD3 (10 μg/ml) with or without TNF-α pAb (1:100) for 24 h. The expression of tmTNF-α (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 μg/ml) with or without TNF-α pAb were incubated for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 μg/ml) with TNF-α pAb for 3 h, followed by incubation for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-α, the effector cells were treated with TNF-α pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. *p<0.05, ** p<0.01, *** p<0.001.

Fig 4: IL-33 Promotes TNF-a Secretion and TNFR2 Expression on ILC2s(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33 or PBS.(B) On day 4, BAL fluid was collected, and supernatant TNF-a was measured by ELISA.(C) Number of TNF-a-producing CD45+ lung cells on day 4, cultured for 4 h with GolgiPlug.(D–F) Representative flow cytometry plots in naive (D) and activated (E) lungs of CD45+ TNF-a+ cells backgated for AMs (CD45+ SiglecF+ CD11b-, CD11c+, and F4/80+) and SiglecF-CD11b+ and SiglecF-CD11b- populations and (F) corresponding quantitation presented as mean frequency ± SEM.(G) AMs were FACS sorted from PBS- or rmIL-33-challenged mice on day 4 and cultured for 24 h in vitro (106 AMs/well), and supernatant TNF-a was measured by ELISA.(H and I) Gating strategy for murine ILC2s showing (H) lineage- staining and (I) lineage-CD45+ CD127+ ST2+ GATA-3hi ILC2s.(J) nILC2s were FACS sorted from naive BALB/cByJ mice and cultured (50 × 104/mL) ex vivo in the presence of rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 ng/mL) for the indicated times. nILC2s were gated as lineage- CD45+ ST2+ CD127+.(K and L) Representative flow cytometry plot of TNFR1 (K) and TNFR2 (L) expression and corresponding quantitation, presented as Mean Fluorescence Intensity (MFI) ± SEM.(M) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33 or PBS.(N and O) Representative flow cytometry plot of lung ILC2 expression of TNFR1 (N) and TNFR2 (O) expression on day 4 and corresponding quantitation, presented as MFI ± SEM.Data are representative of 4 individual experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. See also Figure S1.

Fig 5: 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