Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Targeting PDXK could promote the formation of TLSs and enhance the efficacy of immunotherapy in gastric cancer. ( A ) Heatmap displaying the metabolic feature for T-cell clusters using scMetabolism package. ( B ) Dot plot showing the expression of the genes encoding rate-limiting enzymes of vitamin B 6 metabolism in different types of T cells. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( C ) Quantification of CXCL13 in PDTFs in the presence of different enzyme inhibitors measured by ELISA. ( D ) Representative H&E staining and PDXK immunohistochemistry of gastric cancer tissues with different responses following immunotherapy. Scale bar, 500 µm. ( E ) The schematic diagram of the animal experiments. ( F–G ) Images of tumors and tumor volume curves of 615 mice treated with various agents (n=6, each group). ( H ) Paraffin sections of mouse subcutaneous graft tumor tissue stained with H&E and IHC detection for CD8, CD20 and CXCL13. Scale bar, 100 µm. ( I ) The number (left panel) and area (right panel) of TLS per tumor area were compared between groups (n=6, each group). Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AOX1, aldehyde oxidase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CR, complete response; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; PDTFs, patient-derived tumor fragments; PHOSPHO2, phosphatase orphan 2; PNPO, pyridoxamine 5'-phosphate oxidase; PR, partial response; PSAT1, phosphoserine aminotransferase 1; s.c, subcutaneous injections; SD, stable disease; TLS, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Two Tailed Test, Saline, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could promote the expression and secretion of CXCL13 in CD160 + CD8 + T cells. ( A ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in PDTFs in the presence of different drugs measured by ELISA. ( B ) The schematic diagram of orthotopic transplanted tumor model with diets containing various amounts of vitamin B 6 (n=6, each group). ( C ) Mouse orthotopic stomach xenograft tumor tissues stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( D ) The schematic diagram of orthotopic transplanted tumor model fed with different drugs or diets (n=6, each group). ( E ) The representative images of mouse bioluminescence imaging at week 3 (left panel) and the corresponding quantification analysis (right panel). ( F ) Representative micrographs of xenografts stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( G–H ) Density of TLSs (left panel) and ratio of tumor area occupied by TLSs (right panel). ( I ) Gating strategy for CD160 + CD8 + T cells. ( J ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in supernatants from CD160 + CD8 + T-cell cultures in the presence or absence of PL measured by ELISA. ( K ) Flow cytometric analysis and corresponding quantification of CXCL13 + CD160 + CD8 + T cells with or without PL treatment. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Student’s t-test. ICIs, immune checkpoint inhibitors; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PL, pyridoxol; PD-1, programmed cell death protein 1; s.c, subcutaneous injections; TLS, tertiary lymphoid structures; TNF, tumor necrosis factor.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Two Tailed Test, Immunohistochemistry, Saline

Journal: Bioactive materials

Article Title: Engineered extracellular vesicles for concurrent Anti-PDL1 immunotherapy and chemotherapy.

doi: 10.1016/j.bioactmat.2021.07.012

Figure Lengend Snippet: Fig. 5. Functional activity of havPD-1 EVs in cell-based assays. (a) Neutralization of PD-L1 in a cell assay by havPD-1 EVs in comparison with a clinical control, Atezolizumab. (b) Mixed lymphocyte reaction assay. Supernatants were measured for IFN-γ production by ELISA (n = 8). (c) Tetanus toxoids recall assay. Super natants were measured for IFN-γ production by ELISA (n = 8) (d) Antigen-dependent cell-mediated cytotoxicity assay (n = 8). (e) Complement dependent cyto toxicity assay (n = 8).

Article Snippet: After incubation for 3 days at 37 ◦C and 5% CO2, supernatants were harvested and subjected to detection of IFNγ with ELISA (R&D Systems, DIF50).

Techniques: Functional Assay, Activity Assay, Neutralization, Comparison, Control, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay

Journal: Cell Host & Microbe

Article Title: Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure

doi: 10.1016/j.chom.2020.04.009

Figure Lengend Snippet:

Article Snippet: Human IFN-γ ELISA , Diaclone , 950.000.192.

Techniques: Control, Recombinant, Saline, Enzyme-linked Immunosorbent Assay, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Immunological synapse formation as a key mechanism in T cell-dependent bispecific antibody-mediated immune activation and cytotoxicity

doi: 10.1007/s00262-025-04036-w

Figure Lengend Snippet: Cytotoxic activity and inflammatory cytokine release in co-culture systems. a–c After co-culturing WT or KO, T cells, and TDB, the color reaction with WST-8 reagent in an hour was measured. The co-culture times were 24 h, 48 h, and 72 h of co-culture, respectively. Red: WT + T cell + TDB, blue: KO + T cell + TDB. Error bars represent the mean ± SD ( n = 6), ** p < 0.005, *** p < 0.0005, n.s. not significant. d Measurement of IFN-γ release by ELISA after 24 h of co-culture time. Co-culture supernatants were obtained and used for assay using the ELISA Kit. Red: WT + T cell + TDB, blue: KO + T cell + TDB, gray: negative control (T cell + TDB). Red letters are the results of the WT and N/C statistical tests, and blue letters are the results of the KO and N/C statistical tests. Error bars represent the mean ± SD ( n = 3), *** p < 0.0005, n.s. not significant. e IFN-γ release at TDB: 10 ng/mL for each co-culture time. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. f Cancer cell death when the IFN-γ release measured in e is added to the standard. From left to right, IFN-γ release at 6 h, 24 h, 48 h, and 72 h was added. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. g-i TNF-α version of the same experiment as d – f

Article Snippet: T cells (E:T = 5:1, 1.0 × 10 6 cells/50 μL/well) were added and co-cultured. hEx3 was added at a final concentration of 0.1–100 ng/mL in 100 μL of medium and cultured for 24 h. The culture supernatant was then collected and assayed using human IFN-gamma ELISA Kit, human TNF-alpha ELISA Kit, human CXCL10 ELISA Kit (Proteintech, Rosemont, IL), and human HMGB1 ELISA Kit (Chondrex, Woodinville, WA) according to the protocols provided.

Techniques: Activity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Immunology

Article Title: Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma

doi: 10.1111/imm.13624

Figure Lengend Snippet: Glypican 3 (GPC3) chimeric antigen receptor (CAR)-natural killer (NK) cells showed enhanced antitumor activity against GPC3-positive tumour cell lines. (a) Cytolytic analysis of untransduced NK cells and CAR-NK cells against different tumour cells. The effector cells were cocultured with target cell at multiple E: T ratios for 4 h, the supernatants were collected to determine the cytotoxicity of CAR-NK cells. (b) Representative dot plots showing CD107a expression on untransduced NK cells or CAR-NK cells cocultured with target cells for 4 h. Cells were collected and stained with anti-human CD56-APC and anti-human CD107a-fluorescein isothiocyanate. The plots are gated on CD56+ NK cells, and the upper-right quadrant demonstrated the percentage of positive cells. A representative experiment was shown. (c) interferon (IFN)-γ and tumour necrosis factor (TNF)-α release of CAR-NK cells after antigen-specific stimulation. Untransduced NK cells or GPC3 CAR-NK cells were cocultured with GPC3-positive tumour cell lines for 4 h. The supernatants were collected to determine IFN-γ and TNF-α production by using enzyme-linked immunosorbent assay (ELISA) assay.

Article Snippet: The cytokine release was detected by using IFN-γ ELISA kit (Cat: RK00015, ABclonal) and TNF-α ELISA kit (Cat: RK00030, ABclonal) according to the instructions provided in the manual.

Techniques: Activity Assay, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma

doi: 10.1111/imm.13624

Figure Lengend Snippet: Increased soluble programmed death-ligand 1 (sPD-L1) release in hepatocellular carcinoma (HCC) patients. (a) The level of sPD-L1 secreted by Huh7-membrane programmed death-ligand 1 (mPD-L1) cells. The culture supernatants of Huh7 and Huh7-mPD-L1 were collected to determine the level of sPD-L1 by using enzyme-linked immunosorbent assay (ELISA). Positive control: standard PD-L1 in the ELISA kit. (b) The statistical graph was presented for comparing the level of sPD-L1 in HCC patients with healthy individuals (n = 25).

Article Snippet: The cytokine release was detected by using IFN-γ ELISA kit (Cat: RK00015, ABclonal) and TNF-α ELISA kit (Cat: RK00030, ABclonal) according to the instructions provided in the manual.

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Immunology

Article Title: Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma

doi: 10.1111/imm.13624

Figure Lengend Snippet: Expression and characterization of soluble programmed death-ligand 1 (sPD-L1). (a) Coomassie-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified sPD-L1. The purified sPD-L1 was analysed in non-reducing and reducing SDS-PAGEs. Lane 1: non-reducing SDS-PAGE, Lane 2: reducing SDS-PAGE, Lane M: protein marker. (b) Western blotting was used to detect the his-tag expression of the purified sPD-L1. Lane 1: purified sPD-L1, Lane M: protein marker. (c) The purified sPD-L1 was characterized by using PD-L1 enzyme-linked immunosorbent assay (ELISA) kit. Positive control: standard PD-L1 in the ELISA kit. (d) FACS analysis of proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with 50 ng/ml anti-CD3 and 15 ng/ml anti-CD28 in the presence or absence of purified sPD-L1. (e) Purified sPD-L1 inhibited T cell function. Cytotoxicity assays were performed by incubating alpha fetoprotein (AFP) T-cell receptor (TCR)-T cells with HepG2 cells in the presence or the absence of 10 μg/ml sPD-L1 protein at an E:T ratio of 2:1 for 24 h. LDH, lactate dehydrogenase

Article Snippet: The cytokine release was detected by using IFN-γ ELISA kit (Cat: RK00015, ABclonal) and TNF-α ELISA kit (Cat: RK00030, ABclonal) according to the instructions provided in the manual.

Techniques: Expressing, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Cell Function Assay

Journal: Immunology

Article Title: Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma

doi: 10.1111/imm.13624

Figure Lengend Snippet: High levels of soluble programmed death-ligand 1 (sPD-L1) impaired anti-tumour activity of chimeric antigen receptor (CAR)-natural killer (NK) cells. (a) Cytotoxicity assays were performed by incubating CAR-NK cells with Huh7 cells in the presence or the absence of 10 μg/ml sPD-L1 protein at an E:T ratio of 2:1 for 4 h. (b) The representative fluorescence-activated cell sorter (FACS) analysis of CD107a expression on the membrane surface of CAR-NK cells. CAR-NK cells were cocultured with Huh7 cells combined with sPD-L1 for 4 h. (c) and (d) Cytokine production detection by enzyme-linked immunosorbent assay (ELISA) was performed by incubating CAR-NK cells with Huh7 cells in the presence of sPD-L1 at an E:T ratio of 2:1. LDH, lactate dehydrogenase

Article Snippet: The cytokine release was detected by using IFN-γ ELISA kit (Cat: RK00015, ABclonal) and TNF-α ELISA kit (Cat: RK00030, ABclonal) according to the instructions provided in the manual.

Techniques: Activity Assay, Fluorescence, Expressing, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma

doi: 10.1111/imm.13624

Figure Lengend Snippet: L3C7c-Fc promoted the anti-tumour efficacy of chimeric antigen receptor (CAR)-natural killer (NK) cells. (a) L3C7c-Fc reversed the suppression effect of soluble programmed death-ligand 1 (sPD-L1) on the killing of CAR-NK cells. CAR-NK cells were cocultured with Huh7 cells at an E:T ratio of 2:1 in the presence of sPD-L1, and L3C7c-Fc was added or not. Lactate dehydrogenase (LDH) release was used to detect cytotoxicity. (b) The representative fluorescence-activated cell sorter (FACS) analysis of CD107a expression on CAR-NK cells when targeting Huh7 cells in combination of sPD-L1 and L3C7c-Fc at an E:T ratio of 2:1 after 4 h. (c) Cytokine production detection by enzyme-linked immunosorbent assay (ELISA) was performed by incubating CAR-NK cells with Huh7 cells in the presence of sPD-L1 and L3C7c-Fc at an E:T ratio of 2:1 for 24 h.

Article Snippet: The cytokine release was detected by using IFN-γ ELISA kit (Cat: RK00015, ABclonal) and TNF-α ELISA kit (Cat: RK00030, ABclonal) according to the instructions provided in the manual.

Techniques: Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay