Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: a , In vitro infection of primary human glioblastoma line BT145 with HSV-1(anti-TRAIL) at the indicated multiplicity of infection (m.o.i.). GFP fluorescence detection was indicative of virus-infected cells 72 h after infection. Interferon-defective Vero cells shown as positive control. Scale bar, 100 μm. b , Experimental design (top) of GL261–mNectin1-implanted mice randomized by tumour bioluminescence signal and treated with 3 × 10 6 plaque-forming units (p.f.u.) HSV-1 intratumourally 7 days after implantation. Tumour bioluminescence (bottom) 1 day before and 9 days after treatment with HSV-1 ( n = 8 each). c , Survival of GL261–mNectin1-implanted mice treated with HSV-1 engineered to produce TRAIL-blocking single-chain antibody (scFv); mice treated with PBS, empty HSV-1 or HSV-1 expressing a non-targeting scFv were included as controls ( n = 15 for PBS, n = 30 HSV-1(empty), n = 15 HSV-1(anti-TRAIL), n = 11 HSV-1(control scFv)). Body weight fold change from initiation of study in surviving mice, HSV-1(anti-TRAIL) 0.9079 ± 0.049 g, HSV-1(control scFv) 0.9344 ± 0 g, HSV-1(empty) 0.9887 ± 0.027 g (HSV-1(anti-TRAIL) versus HSV-1(empty), P = 0.2114; HSV-1(anti-TRAIL) versus HSV-1(control scFv), P = 0.9561 by two-sided ANOVA). d , Tumour-infiltrating cleaved caspase-3/7 + CD4 + or CD8 + T cells in HSV-1-treated mice 17 days after GL261 implantation ( n = 8 each). e–h , Frequency ( e ) and absolute count ( f ) of HSV-1-specific or of GARC-1(77–85)-specific ( g , h ) CD8 + T cells infiltrating the GL261 TME in HSV-1-treated mice 17 days after implantation ( n = 8 each). i , Frequency of activated GARC-1(77–85)-specific PD-1 + CD8 + T cells. j–m , Heatmap (left) and pathway analysis (right) of bulk RNA-seq data analysis from FACS-sorted tumour-infiltrating CD8 + T cells ( j ), CD4 + T cells ( k ), microglia-derived TAMs ( l ) and monocyte-derived TAMs ( m ) in HSV-1-treated mice 17 days after tumour implantation ( n = 5 HSV-1(empty); n = 4 except monocytes, for which n = 3 for HSV-1(anti-TRAIL)); all pathways shown are P < 0.05. Data are the mean ± s.e.m. Paired two-tailed t -test ( b ). Survival analysis by log-rank (Mantel-Cox) test ( c ). Unpaired two-tailed t -test elsewhere. n indicates biologically independent samples.

Article Snippet: For Il11 overexpression, parental GL261–Luc cells were transfected with a cDNA construct encoding mouse IL-11 and GFP (Sino Biological, MG50117-ACG) as described above and selected with hygromycin B (Cayman Chemical Company, 14291) at 500 μg ml −1 .

Techniques: In Vitro, Infection, Fluorescence, Virus, Positive Control, Blocking Assay, Expressing, Control, RNA Sequencing, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: ( a,b ) Flow cytometry gating strategy for analysis of TdTomato + TRAIL + cells in Aldh1l1 Cre-ERT2/TdTomato reporter mice (a) and analysis of TRAIL expression in TdTomato +/− fractions (b). Paired two-tailed t-test. ( c ) Immunofluorescence analysis of the tumour border in GL261 tumour-bearing Aldh1l1 eGFP reporter mice at day 15 (left). The fluorescence signals of DAPI, TRAIL, GFP astrocyte reporter are shown along with a quantification of TRAIL+ cells within GFP+ or GFP− fractions (right). ( d ) Immunofluorescence analysis and quantification of TRAIL+ astrocytes in naïve or sham-injured mice after 15 days. Data shown as mean ± SEM. Unpaired two-tailed t-test used for statistical analysis. n indicates biologically independent samples.

Article Snippet: For Il11 overexpression, parental GL261–Luc cells were transfected with a cDNA construct encoding mouse IL-11 and GFP (Sino Biological, MG50117-ACG) as described above and selected with hygromycin B (Cayman Chemical Company, 14291) at 500 μg ml −1 .

Techniques: In Vivo, Flow Cytometry, Expressing, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: ( a ) Reanalysis of spatial transcriptomics data in GBM 86 . Spatially-weighted correlations of TRAIL imputation score from Fig. 2f and non-malignant reactive astrocyte signature 32 . ( b ) Spatially annotated bulk RNA-seq from Ivy Glioblastoma Atlas showing signature score of death receptor signalling (R-HSA-73887) split by anatomical niche (left). Pearson correlation analysis of TNFSF10 and TNFRSF10B expression split by anatomical niche. ( c ) Validation by flow cytometry of Tnfsf10 -targeting lentivirus-driven genetic perturbation in TdTomato + Aldh1l1 Cre-ERT2/TdTomato reporter cells isolated from naïve mice (n = 8 NTsgRNA, n = 10 sg Tnfsf10 ). ( d ) Control measurement of TRAIL in a GFP + GL261 line by flow cytometry 15 days after implantation following the lentivirus-driven genetic perturbation in astrocytes (n = 3 NTsgRNA, n = 2 sg Tnfsf10 ). ( e ) Analysis by qPCR of off-target Tnfsf10 deletion effects in sorted T cells and myeloid cells effects following lentivirus administration as shown in Fig. 3f . ( f ) Survival analysis following implantation of CRISPR control-edited or Tnfsf10 -deleted GL261 lines (n = 4 GL261-NTsgRNA, n = 6 GL261-sg Tnfsf10 ), ( g ) Survival analysis of mice implanted with genetically engineered glioma model MK007 following TRAIL inactivation in astrocytes as shown in Fig. 3f (n = 10 each). ( h ) Survival analysis of GL261-implanted Rag2 −/− mice following TRAIL inactivation in astrocytes (n = 5 each). ( i,j ) Flow cytometry quantification related to Fig. 3h of cleaved caspase-3/7 + single-positive (i) and caspase-3/7 + SYTOX + double-positive (j) CD4 + or CD8 + T cell populations. Data shown as mean ± SEM. Unpaired two-tailed t-test used for statistical analysis. Survival analysis by Log-rank (Mantel-Cox) test for (f-h). n indicates biologically independent samples.

Article Snippet: For Il11 overexpression, parental GL261–Luc cells were transfected with a cDNA construct encoding mouse IL-11 and GFP (Sino Biological, MG50117-ACG) as described above and selected with hygromycin B (Cayman Chemical Company, 14291) at 500 μg ml −1 .

Techniques: Knockdown, RNA Sequencing, Expressing, Biomarker Discovery, Flow Cytometry, Isolation, Control, CRISPR, Two Tailed Test

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p < 0.05; ** p < 0.01. For data on other tested cytokines, see Supplementary Fig. . w.p.i. weeks post injection

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Multiplex Assay, Two Tailed Test, Injection

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: IL-21 or IL-33 treatments induce clearance of established BPS persistence. Plasmids expressing murine IL-21 or IL-33 and empty vector control were injected into BPS HDI mice at 4 and 7 weeks post injection (w.p.i.) (arrows). Sera were collected at indicated time points and levels of IL-21 and IL-33 ( a ), HBsAg ( b ), HBsAb ( c ) and HBeAg ( d ) were analysed using ELISA. Group means and s.e.m. within group are presented with group sizes ( n ) indicated. Group positivity percentage data are also presented for HBsAg ( b , right panel), HBsAb ( c , right panel) and HBeAg ( d , right panel). HBV DNA for each group were analysed using pooled serum in commercial quantitative assay ( e ). Dotted lines represent cut-off thresholds ( a , b – d , right panels) and lower limit of quantification ( e ) respectively. geq genome equivalents

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Expressing, Plasmid Preparation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: IL-21-induced or IL-33-induced clearance of BPS persistence engenders protection from re-challenge. HDI mice harbouring BPS persistence were treated with injections of plasmids expressing IL-21 ( a ) or IL-33 ( b ) at 4 and 7 w.p.i. and those displaying at least 4 weeks of sustained HBsAg negativity (see Fig. ) were re-challenged in the same way as initial BPS HDI. BPS HDI mice treated with vector control were used as control and re-challenged similarly ( c ). Sera were collected at indicated time points and levels of HBsAg (left), HBsAb (middle) and HBeAg (right) were analysed using ELISA. Dotted lines represent cut-off thresholds. w.p.i. weeks post injection of BPS re-challenge

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p < 0.05; ** p < 0.01. For data on other tested cytokines, see Supplementary Fig. . w.p.i. weeks post injection

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Multiplex Assay, Two Tailed Test, Injection

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: IL-21 or IL-33 treatments induce clearance of established BPS persistence. Plasmids expressing murine IL-21 or IL-33 and empty vector control were injected into BPS HDI mice at 4 and 7 weeks post injection (w.p.i.) (arrows). Sera were collected at indicated time points and levels of IL-21 and IL-33 ( a ), HBsAg ( b ), HBsAb ( c ) and HBeAg ( d ) were analysed using ELISA. Group means and s.e.m. within group are presented with group sizes ( n ) indicated. Group positivity percentage data are also presented for HBsAg ( b , right panel), HBsAb ( c , right panel) and HBeAg ( d , right panel). HBV DNA for each group were analysed using pooled serum in commercial quantitative assay ( e ). Dotted lines represent cut-off thresholds ( a , b – d , right panels) and lower limit of quantification ( e ) respectively. geq genome equivalents

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Expressing, Plasmid Preparation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

doi: 10.1038/s41467-017-02304-7

Figure Lengend Snippet: IL-21-induced or IL-33-induced clearance of BPS persistence engenders protection from re-challenge. HDI mice harbouring BPS persistence were treated with injections of plasmids expressing IL-21 ( a ) or IL-33 ( b ) at 4 and 7 w.p.i. and those displaying at least 4 weeks of sustained HBsAg negativity (see Fig. ) were re-challenged in the same way as initial BPS HDI. BPS HDI mice treated with vector control were used as control and re-challenged similarly ( c ). Sera were collected at indicated time points and levels of HBsAg (left), HBsAb (middle) and HBeAg (right) were analysed using ELISA. Dotted lines represent cut-off thresholds. w.p.i. weeks post injection of BPS re-challenge

Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

Techniques: Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection