Journal: Frontiers in Immunology

Article Title: Orthotopic and Heterotopic Murine Models of Pancreatic Cancer Exhibit Different Immunological Microenvironments and Different Responses to Immunotherapy

doi: 10.3389/fimmu.2022.863346

Figure Lengend Snippet: NKG2D CAR-T rescue impaired CAR-T cell activity via eliminating MDSCs. (A) Schematic diagram of NKG2D-CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (B) Cytotoxicity assay of NKG2D CAR-T to Rae1 positive CHO cell at indicated E:T ratio. E:T, effector-to-target ratio. (n=6 compared with Control CAR-T group, ***P<0.001). (C) Cytotoxicity assay of NKG2D CAR-T to murine MDSCs at E:T=1:1. MDSCs were pre-labeled by CSFE. CSFE + 7AAD + cells indicate specific killed MDSCs. (D) Survival of orthotopic tumor recipients after NKG2D CAR-T transfer. The green arrows indicate NKG2D CAR-T cells administration. (E) Representative IHC images of Gr1 staining in orthotopic tumor 2 days after NKG2D CAR-T transfer. Scale bar=50μm. (F) Schematic outline of experimental approaches and analyses. (G) Survival of orthotopic tumor recipients after combination of NKG2D CAR-T and αMUC1 CAR-T. The green arrows indicate NKG2D CAR-T cells administration; the orange arrows indicate αMUC1 CAR-T cells administration. (***P<0.001). (H) Schematic diagram of bicistronic CAR (Upper) and flow cytometry assay of transfection efficiency (Down). (I) Survival of orthotopic tumor recipients after bicistronic CAR-T transfer. The orange arrow indicates CAR-T cells administration (***P<0.001).

Article Snippet: The extracellular (EC) and transmembrane (TM) regions of mice NKG2D (NM_001083322.2) were cloned form commercial plasmid (SinoBiological, MG57340). scFv sequence of CD19 was from patent US20210395364A1 in VL-(G 4 S) 3 -VH form. scFv sequence of MUC1 was from patent US10239950B2 in VL-(G 4 S) 3 -VH form.

Techniques: Activity Assay, Flow Cytometry, Transfection, Cytotoxicity Assay, Labeling, Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A The t-Distributed stochastic neighbour embedding (t-SNE) plot showing clustering information from GSE163558 and GSE206785 . B Venn diagram showing the overlap between mRNA microarray, GSE163558 and GSE206785 , upregulated genes associated with ECM, and Immunology Database and Analysis Portal (ImmPort) database. C Gene Ontology (GO) analysis showing enriched pathway terms for genes in GSE163558 and GSE206785 . D ULBP2 expression in fibroblasts revealed a correlation with collagen biosynthesis, collagen fibril organisation, ECM-receptor interaction, and TGF-β signalling. E ULBP2 mRNA expression of GC and corresponding adjacent normal tissues analyzed by mRNA microarrays (n = 16). F Immunoblotting of ULBP2 in the tumors and corresponding adjacent normal tissues from GC patients (n = 6). G Quantification of ULBP2 expression by immunohistochemistry (IHC) in human tissue microarrays (TMAs) from 62 gastric patients. H Kaplan–Meier survival analysis of overall survival (OS). I Representative images of gastric tissues with high ULBP2 expression compared to low-expression tissues, stained by IHC, Masson, and Sirius Red stains. J Scatter plots of ULBP2 expression versus collagen deposition in the human gastric TMAs. Data are expressed as mean ± SEM (***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Microarray, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A ULBP2 knockout efficiency in SNU-216 cells and MKN-45 cells assessed by flow cytometry (FCM). B Growth curves of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). C Colony formation assay and the statistical results of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). D Transwell invasion assay of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). E Representative images and growth kinetics of ctrl organoids and ULBP2 −/− organoids (n = 3). F Tumor growth curves of NSG mice carrying wild-type, ULBP2 −/− MKN-45 cell line-derived xenografts (CDX) (n = 5). G Representative images of tumors, H&E staining, and Ki-67 IHC staining (n = 3). Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Knock-Out, Flow Cytometry, Colony Assay, Transwell Invasion Assay, Derivative Assay, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Heat map showing differentially expressed genes (DEGs) in ULBP2 −/− MKN-45 cells compared to wild-type cells. B Transcriptome KEGG analysis revealed 20 pathways that ULBP2 knockout had a significant effect on MKN-45 cells. C ULBP2 knockout exhibited the most pronounced impact on ECM-receptor interaction and TGF-β signalling pathway of GC. D – F Transcriptome GSEA analysis revealed that the differentially expressed genes were significantly enriched in the TGF-β signalling pathway ( D ), ECM-receptor interaction ( E ), and collagen formation ( F ). G mRNA expression levels of TGF-β1 in ctrl cells and ULBP2 −/− MKN-45 and SNU-216 cells (n = 3). H Western blotting of TGF-β1, phosphorylated SMAD2 ( p -SMAD2), and total SMAD2/3 (T-SMAD2/3) in ctrl cells and ULBP2 −/− MKN-45 and SNU-216 cells. Data are represented as mean ± SEM (*p < 0.05, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Knock-Out, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B Growth curves of CAFs cultured with supernatants from ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). C The diagram of co-culture system between CAFs and GC cells. D EdU assay showing that the CAF proliferation was significantly reduced in the co-culture system of ULBP2 knockout MKN-45 cells compared with wild-type cells (n = 3). E Frequency of Ki-67 positive cells in the co-culture system, as determined by FCM (n = 3). F , G Relative levels of TGF-β1 ( F ) and collagen ( G ) content in the co-culture system (n = 6). H Diagram of the co-culture system between CAFs and GC organoids. I Representative bright field, H&E, and α-SMA staining images of the CAFs and organoid co-culture system. Data are represented as mean ± SEM (ns, nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Cell Culture, Co-Culture Assay, EdU Assay, Knock-Out, Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Schematic diagram of ULBP2 CAR. B FCM showing the transduction efficiency of CAR-T cells. C IF analysis of the binding of ULBP2 CAR-T cells (green) to MKN-45-mCherry cells (red). D Killing efficiency of MKN-45 cells by ULBP2 CAR-T cells, determined by LDH-based cytotoxicity assay (n = 3). E Protein levels of IFN-γ and granzyme B in the culture supernatant (n = 3). F Killing efficiency of ULBP2 −/− MKN-45 cells by ULBP2 CAR-T cells (n = 3). G Representative images of GC organoids and killing efficiency of organoids by ULBP2 CAR-T cells (n = 3). H Representative bright field, H&E, and α-SMA staining images of ULBP2 CAR-T cells, CAFs, and organoid co-culture system, along with quantification of α-SMA positive area (n = 3). Data are represented as mean ± SEM (ns, nonsignificant; ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Transduction, Binding Assay, LDH Cytotoxicity Assay, Staining, Co-Culture Assay

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B , C Tumor growth ( B ) and survival ( C ) curves of the CDX mice treated with ULBP2 CAR-T, anti-PD-1, or ULBP2 CAR-T cells plus anti-PD-1 (n = 5). D Bioluminescent imaging of the CDX mice at different days post tumor inoculation. E Protein levels of IFN-γ and granzyme B in the serum of CDX mice at different weeks post tumor inoculation detected by ELISA (n = 3). F Protein levels of PD-1, LAG-3, and TIM-3 in tumor-infiltrating lymphocytes at day 21 post tumor inoculation in the CDX mice that received different treatments, as shown by FCM (n = 3). G Representative images of tumors, H&E staining, Ki-67 IHC staining, and CD8 IF staining in the CDX mice that received different treatments. H Quantification of Ki-67 (n = 3) and CD8 + T (n = 6) cells in the CDX at day 21 post tumor inoculation. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Imaging, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B – D Tumor growth B and survival curves ( C , D ) of mice bearing the PDX from a patient treated with ULBP2 CAR-T or ctrl cells alone, or ULBP2 CAR-T cells plus anti-PD-1 (n = 5). E Representative images of H&E, Ki-67 IHC, CD8 IHC, Masson, and α-SMA IF staining in the PDX mice that received different treatments. F – I Quantification of Ki-67 F (n = 3), CD8 + T cells G (n = 6), collagen area H (n = 6), and α-SMA area (n = 3) in the PDX at day 21 post tumor inoculation. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: ULBP2 overexpression promotes TGF-β signalling, driving CAF activation, leading to a dense stromal microenvironment. Moreover, targeting ULBP2 effectively reduces stromal deposition, stimulates CAR-T cell infiltration, and augments the antitumor efficacy of PD-1 blockade therapy. This graphical abstract was created with BioRender.com (License #NP28FJISA5, 2025).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Over Expression, Activation Assay

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD314 , REA1228 , 50 , 130-124-341 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Scientific Reports

Article Title: Epigenetic regulation of NKG2D ligands is involved in exacerbated atherosclerosis development in Sirt6 heterozygous mice

doi: 10.1038/srep23912

Figure Lengend Snippet: ( A ) Schematic diagram of the experiments. Peritoneal macrophage, isolated from ApoE −/− and Sirt6 +/− ApoE −/− mice, were incubated to adhere to culture plate. Cells were treated with 30 μg/ml ox-LDL for 24 hours, and cocultured with NK cells, at 1:10 ratio for 6 hours. The NK cells were collected and used for determination of cytokine expression by realtime PCR. ( B,C ) Macrophages were transfected with Si-Ctrl or Si-H60b to knockdown H60b, cultured for 24 hours, treated with 30 μg/ml ox-LDL for 24 hours, and then co-incubated with NK cells at 1:10 ratio for 6 hours. ( B ) Macrophages were collected to determinate Sirt6 and H60b expression at the mRNA level and protein level. ( C ) The NK cells were collected and used for determination of cytokine expression by realtime PCR. ( D ) To block ligand-receptor interaction, isotype IgG antibody or NKG2D antibody (R&D, BAM1547, 3 μg/ml) or H60b antibody (R&D, MAB1155, 3 μg/ml) were added for 2 hours before co-incubation with NK cells. The NK cells were collected and used for determination of cytokine expression by realtime PCR. P value was obtained by two-way analysis of variance (ANOVA) plus a post hoc analysis using the Bonferroni test. (*P < 0.05, **P < 0.01, ***P < 0.005).

Article Snippet: To block ligand-receptor interaction, isotype IgG antibody or NKG2D antibody (R&D, BAM1547, 3 μg/ml) or H60b antibody (R&D, MAB1155, 3 μg/ml) were added for 2 hours before co-incubation with NK cells.

Techniques: Isolation, Incubation, Expressing, Transfection, Cell Culture, Blocking Assay