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: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV blocks IDO activity and IDO-mediated antimicrobial effects in human MSC. (a) PBL, stimulated with OKT3, were cocultured with MSC in the presence or absence of CMV. As controls UV-inactivated CMV (uvCMV), the IDO-specific inhibitor 1-L-methyl-tryptophan (1-MT; 1.5 mM), or a neutralising anti-IFN- γ antibody ( α IFN- γ ; 10 ng/mL) was used. After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) PBL (1 × 10 5 /well), stimulated with CD3-directed mAB OKT3, were cocultured with MSC (3 × 10 4 /well) in the absence or presence of CMV (MOI 5). After three days cultures were infected with S. aureus (10–100 cfu/well) and bacterial growth was determined photometrically. As a control, cultures were supplemented with L-tryptophan (Trp; 0.6 mM) at the time point of bacterial infection. Data are given as mean OD (620 nm) ± SEM of three experiments, each done in triplicate. Significant differences ( P < 0.05) as compared to the positive control are marked by asterisks.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Activity Assay, Infection, Control, Positive Control

Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV inhibits IDO induction by recombinant IFN- γ . (a) MSC (2 × 10 4 /well) which were infected with various amounts of CMV (MOI 0.1–10) were stimulated with IFN- γ (300 U/mL). After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) MSC (1.5 × 10 6 /flask) were stimulated with IFN- γ (600 U/mL) in the absence or presence of CMV (MOI 5). Cells were harvested after 24 h and IDO protein was detected in Western blot analysis. β -Actin was utilized as a protein loading control, while the viral pp72 protein served as an infection control.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Infection, Activity Assay, Western Blot, Control

Journal: Cells

Article Title: TRPA1 as a Key Regulator of Keratinocyte Homeostasis and Inflammation in Human Skin

doi: 10.3390/cells15020192

Figure Lengend Snippet: TRPA1 silencing modestly enhances keratinocyte expression of multiple chemokines. Keratinocytes obtained from three different donors were cultured for 18 h in the presence or absence of IFN-γ and TNF-α, in both control cells and silencing-treated cells, and then analyzed by real-time PCR, as described in the Methods section.

Article Snippet: Cytokine stimulations with recombinant human (rh) IFN-γ (200 U/mL), TNF-α (50 ng/mL), and IL-17A (50 ng/mL) (R&D Systems, Minneapolis, MN, USA), as well as treatments with TRPA1 agonists, were conducted on keratinocyte cultures kept in keratinocyte basal medium (KBM-GOLD, Clonetics, Lonza, Basel, Switzerland).

Techniques: Expressing, Cell Culture, Control, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

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