Journal: Journal for Immunotherapy of Cancer

Article Title: Novel post-translational modification learning signature reveals B4GALT2 as an immune exclusion regulator in lung adenocarcinoma

doi: 10.1136/jitc-2024-010787

Figure Lengend Snippet: B4GALT2-mediated regulation of immune microenvironment and neoplastic growth. ( A ) The correlation matrix revealed inverse associations between B4GALT2 levels and immune-related transcripts, with prominent immune gene clusters (highlighted by black margins) exhibiting the strongest negative correlations. ( B ) The spatial distribution of CD8 and CD20 lymphocytes was visualized via multiplexed immunofluorescence in regions displaying differential B4GALT2 abundance. ( C, D ) Successful B4GALT2 suppression in LUAD cell lines was validated through immunoblotting and quantitative PCR analyses. ( E–G ) Cellular viability and colony formation capacity following B4GALT2 manipulation were assessed via CCK8 and clonogenic methodologies. ( H ) Experimental design involved subcutaneous inoculation of C57BL/6 mice with 1×10 6 sh-B4galt2 or control LLC cells, followed by administration of either PD-1 specific antibody or corresponding IgG2a control. ( I ) Temporal monitoring of tumor progression across treatment groups demonstrated optimal therapeutic efficacy in the shB4galt2+anti-PD1 cohort, manifesting as significantly attenuated tumor expansion. B4GALT2, beta-1,4-galactosyltransferase 2; LUAD, lung adenocarcinoma; PD-1, programmed cell death protein 1.

Article Snippet: The study groups were administered either anti-programmed cell death protein 1 (PD-1) monoclonal antibody (BioXcell, BE0146) or isotype-matched IgG control (BioXcell, BE0089) via intraperitoneal route.

Techniques: Immunofluorescence, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Novel post-translational modification learning signature reveals B4GALT2 as an immune exclusion regulator in lung adenocarcinoma

doi: 10.1136/jitc-2024-010787

Figure Lengend Snippet: Synergistic enhancement of anti-PD-1 response via B4GALT2 suppression: modulation of CD8+ T cell phenotypes. ( A ) Flow cytometric analysis demonstrated augmented CD8+ T lymphocyte infiltration following both B4GALT2 suppression and PD-1 blockade, with maximal infiltration observed in the combination therapy cohort. ( B ) Representative cytometric profiles validated that dual intervention (shB4galt2+anti-PD1) substantially elevated activated CD8+ T cell populations while diminishing naive subset abundance. Technical annotation: GZMB serves as a cytolytic activity indicator, CD44/CD69 function as activation status markers, and CD62L identifies naive populations within CD8+ T lymphocytes; statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. B4GALT2, beta-1,4-galactosyltransferase 2; LUAD, lung adenocarcinoma; PD-1, programmed cell death protein 1.

Article Snippet: The study groups were administered either anti-programmed cell death protein 1 (PD-1) monoclonal antibody (BioXcell, BE0146) or isotype-matched IgG control (BioXcell, BE0089) via intraperitoneal route.

Techniques: Activity Assay, Activation Assay

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative IHC images of tumour samples from patients with low and high α V expression in tumour cells. Objective: 20×. b Kaplan−Meier curve of OS for stage I treatment-naïve lung cancer patients according to the α V expression by IHC analysis of FFPE tumours. c Kaplan−Meier curve of PFS of PD-1 blockade-treated patients with tumours harbouring low and high expression of α V integrin. d Percentages of anti-PD-(L)1-treated patients displaying α V high tumours among long-responders (LR: PFS > 6 months and OS > 12 months) or fast progressors (FP: defined by “early death” occurring within 12 weeks of treatment initiation). e Representative digital mark-up image of fluorescent IHC of CD8 (green), cytokeratin (turquoise), and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + cell density. Left, the density of CD8 + TIL in α V low and α V high tumours. The numbers of tumours in each group are indicated (* p = 0.046). Scale bar, 2 cm. f Representative digital mark-up image of CD8 + CD103 neg (green), CD8 + CD103 + (orange), CD8 - CD103 + (red), cytokeratin (turquoise) and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + CD103 + cell density. Left, the density of CD8 + CD103 + (* p = 0.016) and CD8 + CD103 neg ( p = 0.120) cells in tumour regions of α V low and α V high tumours. Scale bar, 2 cm. Each symbol represents an individual cell type from tumour samples; horizontal lines correspond to mean ± standard error of the mean (SEM) ( e , f ). Data were calculated with the log-rank test ( b , c ) and Welch’s two-sided t -test ( e , f ). Source data are provided as a Source Data file.

Article Snippet: 25 μg/mouse of anti-TGF-β (Bio-X-Cell BE0057; clone 1D11.16.8) and/or 200 μg/mouse of anti-PD-1 (Bio-X-Cell BE0146; clone RMP1-14) mAb or isotype control (Bio-X-Cell BE0089: IgG2a, clone 2A3; Bio-X-Cell BE0090: IgG2b, clone LTF-2; Bio-X-Cell BE0083: IgG1, clone MOPC-21).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Left, B16F10E and B16F10E-KO tumour growth kinetics ( p = 0.800). Right, tumour weights of B16F10E and B16F10E-KO tumours recovered at day 14 after engraftment ( p = 0.846). Tumour volumes and tumour weights are given as means ± SEM of eight mice/group. Data represent one independent experiment out of three. b The ratio of active versus total TGF-β from B16F10E and B16F10E-KO tumours ( n = 8) was measured ex vivo on day 8 by ELISA (* p = 0.038). c Absolute cell counts of CD3 + (* p = 0.040) and CD8 + T cells (* p = 0.034) infiltrating B16F10E and B16F10E-KO tumours ( n = 8). Data are from one independent experiment out of three. d Representative flow cytometry profiles (bi-exponential scale) of the expression of CD62L and CD44 in CD8 + T cells from B16F10E and B16F10E-KO tumours ( n = 8). Right, percentage of CD44 + CD62L neg cells among CD8 + T cells in B16F10E and B16F10E-KO (*** p = 0.0006). e KLRG1 ( n = 18, * p = 0.014), CD69 ( n = 13, * p = 0.025) and CD103 ( n = 17, *** p = 0.0003) expression in CD8 + T cells infiltrating B16F10E and B16F10E-KO tumours. Data are from two independent experiments out of three. f Representative flow cytometry profiles of PD-1 expression on CD8 + TIL from B16F10E and B16F10E-KO. Right, expression of PD-1 on CD8 + T cells infiltrating B16F10E and B16F10E-KO tumours ( n = 12, * p = 0.016). g Representative profiles of Ki-67 expression in CD8 + T cells from B16F10E and B16F10E-KO. Right, expression of Ki-67 in CD8 + T cells from B16F10E and B16F10E-KO tumours ( n = 11, * p = 0.028). Data are from two independent experiments out of three. Each symbol represents an individual tumour ( a − g .). Horizontal lines correspond to mean ± SEM ( a , right− g .). Data were calculated with unpaired Student t -tests ( a , right− g .) and two-way ANOVA ( a , left). Source data are provided as a Source Data file.

Article Snippet: 25 μg/mouse of anti-TGF-β (Bio-X-Cell BE0057; clone 1D11.16.8) and/or 200 μg/mouse of anti-PD-1 (Bio-X-Cell BE0146; clone RMP1-14) mAb or isotype control (Bio-X-Cell BE0089: IgG2a, clone 2A3; Bio-X-Cell BE0090: IgG2b, clone LTF-2; Bio-X-Cell BE0083: IgG1, clone MOPC-21).

Techniques: Ex Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Growth of B16F10E and B16F10E-KO tumours (**** p < 0.0001). Right, weights of tumours recovered at day 14, B16F10E+anti-PD-1 vs. B16F10E-KO+anti-PD-1 (* p = 0.023), B16F10E-KO+isotype vs. B16F10E-KO+ anti-PD-1 (* p = 0.049). Tumour volumes and weights are given as means± SEM ( n = 6). Data represent one independent experiment out of five. b Absolute number of CD8 + T cells from B16F10E ( n = 16) and B16F10E-KO ( n = 18) tumours treated with anti-PD-1 or isotype control ( n = 17, B16F10E; n = 19, B16F10E-KO). Data are from five independent experiments (* p = 0.037). c Growth of B16F10E-KO tumours ( n = 6). Tumour volumes are given as means ± SEM. Data are from one independent experiment out of two. B16F10E-KO+iso vs. B16F10E-KO+anti-PD-1 (* p = 0.043); B16F10E-KO+anti-PD-1 vs. B16F10E-KO+anti-CD8 (* p = 0.020). d Percentages of KLRG1 + cells in CD8 + T cells from tumours ( n = 6) treated with anti-PD-1 or isotype control (* p = 0.043, **** p < 0.0001). Data are from one independent experiment out of three. e Percentages of Ki-67 + T cells among CD8 + (* p = 0.012) and CD69 + CD103 + CD8 + TIL from B16F10E-KO ( n = 6) and B16F10E tumours treated with anti-PD-1 ( n = 6) or isotype control ( n = 5). Data are from one independent experiment out of three. f Percentages of granzyme B (GzmB) + (left, ** p = 0.0043, **** p < 0.0001) and CD69 + CD103 + (middle, * p = 0.025, ** p = 0.008) in CD8 + T cells from tumours ( n = 6) treated with anti-PD-1 or isotype control. One independent experiment out of three is shown. Right, percentages of granzyme B + cells in CD69 + CD103 + CD8 + T cells from B16F10E ( n = 11) and B16F10E-KO ( n = 12) treated with anti-PD-1 or isotype control ( n = 11), B16F10E+iso vs. B16F10E-KO+isotype (* p = 0.046); B16F10E-KO+isotype vs. B16F10E-KO+anti-PD-1 (* p = 0.040). Two independent experiments out of three are included. g . Cytotoxic activity of CD8 + TIL isolated from B16F10E and B16F10E-KO tumours treated with anti-PD-1 or isotype control (* p = 0.037). Indicated are the effector-to-target (E:T) ratios. Data are means of three independent experiments. Each symbol represents an individual tumour; horizontal lines correspond to mean ± SEM ( a right, b , d − f .). Data were calculated with one-way ANOVA with Tukey’s correction ( a right, d , e , f left, middle), unpaired t -test ( b , f right, g .), two-way ANOVA for tumour growth ( a , c ). Source data are provided as a Source Data file.

Article Snippet: 25 μg/mouse of anti-TGF-β (Bio-X-Cell BE0057; clone 1D11.16.8) and/or 200 μg/mouse of anti-PD-1 (Bio-X-Cell BE0146; clone RMP1-14) mAb or isotype control (Bio-X-Cell BE0089: IgG2a, clone 2A3; Bio-X-Cell BE0090: IgG2b, clone LTF-2; Bio-X-Cell BE0083: IgG1, clone MOPC-21).

Techniques: Activity Assay, Isolation

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Neutralizing anti-CD103 mAb inhibits the effect of anti-PD-1. Growth of B16F10E-KO tumours, treated with anti-PD-1 and then receiving anti-CD103 blocking mAb (i.t.) or isotype control. Tumour volumes are given as means ± SEM of six mice/group (* p = 0.043). Data are from two independent experiments out of two. b Cytotoxic activity of CD8 + TIL isolated from B16F10E-KO tumours ( n = 3) treated with anti-PD-1 (i.p.) plus isotype control (i.t.) or anti-PD-1 plus anti-CD103 (i.t.). Cytotoxicity against B16F10E-KO tumour cells was determined by a conventional 51 Cr release assay at indicated E:T ratios. Data are from one independent experiment out of two (* p = 0.030). c Effects of anti-TGF-β and anti-CD103 neutralizing mAb on tumour growth. Mice were engrafted with B16F10E-KO and then treated with anti-TGF-β, anti-CD103, or isotype control (i.t.). Tumour volumes are given as means ± SEM of six mice/group. Right, tumour weights of B16F10E-KO ( n = 6) recovered at day 17 (* p = 0.024). d Left, absolute cell counts of total TIL and right, CD8 + T cells infiltrating B16F10E-KO tumours treated with anti-TGF-β, anti-CD103, or isotype control ( n = 6). e Effects of anti-TGF-β neutralizing mAb on PD-1 blockade. Growth of B16F10E-KO tumours, treated with anti-PD-1 and then received anti-TGF-β blocking mAb (i.t.) or isotype control. Tumour volumes are given as means ± SEM of six mice/group ( p = 0.196). f Left, absolute counts of total TIL in B16F10E-KO tumours treated with anti-PD-1 plus anti-TGF-β or anti-PD-1 plus isotype control ( p = 0.418). Middle panel, absolute counts of CD8 + T cells ( p = 0.343) and right, percentages of CD69 + CD103 + (* p = 0.020) among CD8 + T cells from B16F10E-KO tumours treated with anti-PD-1 mAb (i.p.) plus anti-TGF-β (i.t.) or anti-PD-1 plus isotype control ( n = 6). Each symbol represents an individual tumour; horizontal lines correspond to mean ± SEM ( c right, d , f ). Data were calculated with unpaired t -test ( a − c , e , f ) and one-way ANOVA with Tukey’s correction ( c right, d .). Source data are provided as a Source Data file.

Article Snippet: 25 μg/mouse of anti-TGF-β (Bio-X-Cell BE0057; clone 1D11.16.8) and/or 200 μg/mouse of anti-PD-1 (Bio-X-Cell BE0146; clone RMP1-14) mAb or isotype control (Bio-X-Cell BE0089: IgG2a, clone 2A3; Bio-X-Cell BE0090: IgG2b, clone LTF-2; Bio-X-Cell BE0083: IgG1, clone MOPC-21).

Techniques: Blocking Assay, Activity Assay, Isolation, Release Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting SNORA38B attenuates tumorigenesis and sensitizes immune checkpoint blockade in non-small cell lung cancer by remodeling the tumor microenvironment via regulation of GAB2/AKT/mTOR signaling pathway

doi: 10.1136/jitc-2021-004113

Figure Lengend Snippet: SNORA38B LNAs sensitized NSCLC to ICBs treatment. (A) Transfection efficiency of LNAs in LLC cells. (B) Tumor volume examined in LLC derived tumors in tumor burden C57BL/6J mice according to formula 0.5×L×W 2 . n=5 per group. (C–D) Representative images and quantification of BLI in the luciferase reporter gene labeled LLC tumor regions in C57BL/6J mice treated with PBS, LNA, ICB or LNA+ICB. Signals were recorded every 4 days before reaching the observation endpoint. n=5 per group. (E–F) Flow cytometry detection of CD3 + CD8 + T cells, CD4 + FOXP3 + Tregs and their percentage of CD45 + cells isolated from LLC cells injected C57BL/6J mice treated with PBS, LNA, ICB or LNA+ICB. (G) IHC staining of AKT/mTOR/GAB2 signing pathway with the isolated burden tumors from C57BL/6J mice treated with PBS, LNA, ICB or LNA+ICB. Bar=100 µm. (H–K) Quantification of IHC staining of AKT/mTOR/GAB2 signing pathway. (L) Basic summary of the carcinogenic mechanism of SNORA38B in NSCLC. SNORA38B directly bound to the transcription factor E2F1, then accelerated the transcription of downstream GAB2 gene, activated AKT/mTOR pathway and its downstream effectors to perform cancer-promoting effects. Besides, SNORA38B recruited Tregs to mediate immunosuppression by promoting the secretion of IL-10. Knockdown of SNORA38B using LNAs could sensitize NSCLC to ICB treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Assays were conducted in triple. Means±SD was shown. Statistical analysis was subjected to one-way analysis of variance. Comparison between-group using LSD method. BLI, bioluminescence imaging; CTLA-4, cytotoxic T-lymphocytes-associated protein 4; E2F, E2F transcription factor; GAB2, GRB2-associated-binding protein 2; ICB, immune checkpoint blockade; IHC, immunohistochemistry; IL, interleukin; LLC, murine Lewis lung carcinoma; LNA, locked nucleic acid; LSD, the Fisher's least significant difference; NSCLC, non-small cell lung cancer; p-AKT, phosphorylation of protein kinase B; PBS, phosphate-buffered saline; PD-1, programmed cell death protein-1; p-mTOR, phosphorylation of mammalian target of rapamycin; Scr, scramble; SNORNA, small nucleolar RNA; TME, tumor microenvironment; Treg, regulatory T cells;.

Article Snippet: For in vivo combinatorial treatment of ICB plus LNA assay, 4–6 weeks old male C57BL/6J mice (weight ~20 g) were purchased from Hubei Research Center of Laboratory Animal and divided randomly into four groups: PBS (Scr LNA+αIgG (Clone: TNP6A7, BE0089, BioXcell; 10 mg/kg.bw)), LNA (LNA+αIgG), immune-checkpoint blockade (ICB) (Scr LNA+ICB (anti-programmed cell death protein-1 (PD-1) (Clone: J43, BE0146, BioXcell; 10 mg/kg.bw)+anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) (Clone: 9D9, BE0164, BioXcell; 10 mg/kg.bw)), LNA+ICB (LNA (5 mg/kg.bw)+ICB (n=5 for each group)).

Techniques: Transfection, Derivative Assay, Luciferase, Labeling, Flow Cytometry, Isolation, Injection, Immunohistochemistry, Imaging, Binding Assay