Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 1. CD40L expression on SV40 TAg–specific CD8+ T cells during a protective immune response. (A) Splenocytes of WT mice challenged with 16.113 TAg+ cancer cells were stimulated with peptide IV at indicated time points. d, day. The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−-gated lymphocytes from one representative mouse (n = 4 mice). (B) The diagram summarizes the frequencies of CD40L+IFN-γ+ and CD40L−IFN-γ+CD8+ T cells and (C) the frequency of IL-2– producing cells among CD40L+ and CD40L− tumor-specific CD8+ T cells at the different time points (both means ± SD).

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 2. Prevention of tumor outgrowth is dependent on CD40L expression on CD8+ T cells. (A) RAG1−/− mice were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells and treated in parallel with intravenously injected CD8+ T cells from WT or CD40L−/− mice and/or with WT CD4+ T cells. The means ± SD of the tumor size of four or five mice per group are shown from one representative of five experiments. (B) The tumor sizes of individual mice in different groups are shown at day 26. (C) Sum- mary of the tumor formation data obtained from five independent experiments. Tumor formation was defined as an established tumor when its volume reached >500 mm3. Statistical analysis: Analysis of variance (ANOVA) with Bonferroni multiple comparisons posttest: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 3. Impaired tumor rejection in nonlymphopenic CD8+ T cell–specific CD40L KO mice. (A) Strategy for the generation of CD40Lfl/fl mice. UTR, untranslated region; FRT, flippase recognition target. (B) E8I-Cre × CD40Lfl/fl, E8I-Cre, and CD40L fl/fl mice as WT control were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells. Sum- mary of the tumor formation data obtained from three individual experiments, each with 5 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. (C and D) WT and E8I-Cre × CD40Lfl/fl mice were subcutaneously injected with 1 × 106 9.27 TAg+ cancer cells, and 7 days later, splenocytes and cells from the draining lymph nodes (dLNs) were isolated and stimulated with peptide IV. (C) The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−- gated splenocytes from one representative mouse of five mice. (D) The diagram summarizes the frequencies of TAg-specific IFN-γ+CD8+ T cells measured among spleno- cytes and lymph node cells. Statistical analysis: Log-rank test: **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Control, Injection, Isolation, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 4. Role of CD40 expression on host and 9.27 cancer cells for tumor rejection. (A) CD40L−/− and CD40−/- mice were injected subcutaneously with 1 × 106 9.27 cancer cells. Summary of the tumor formation data obtained from two individual experiments, each with 6 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. FSC-A, forward-scatter-area. (B) Cell surface expression of CD40 was analyzed after culturing 9.27 cancer cells, supplemented with or without TGFβ for 24 hours in three individual experiments. (C and D) The 9.27 cancer cells were treated for 24 hours with TGFβ and subsequently stimulated for further 24 hours with multimeric mouse CD40L. Thereafter, caspase-8 activity was determined with the fluorescence marker FITC-IETD-FMK and costained with annexin V. (C) The representative dot plots show the gating of annexin V and FITC-IETD-FMK–stained cancer cells after triggering CD40. (D) The diagrams summarize the frequencies of ac- tive caspase-8+ cancer cells, measured as triplicates of one of three representative experiments.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection, Activity Assay, Fluorescence, Marker, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 5. CD8+ T cell–mediated CD40 signaling in cancer cells prevents tumor formation. (A) Cell surface expression of CD40 was analyzed after culturing TRAMP-C1 and CD40tg TRAMP-C1 cells with or without TGFβ for 24 hours. (B) Both TRAMP-C1 cancer cell lines were stimulated for 24 hours with multimeric CD40L. Thereafter, cas- pase-8 activity was determined with the fluorescence marker FITC-IETD-FMK. The representative dot plots show the gating of 4′,6-diamidino-2-phenylindole (DAPI) and FITC-IETD-FMK–stained cancer cells after triggering CD40. The diagrams summarize the frequencies of active caspase-8+ cancer cells. (C and D) E8I-Cre × CD40Lfl/fl and E8I-Cre as WT control mice were injected subcutaneously with 5 × 106 TRAMP-C1 (C) or CD40tg TRAMP-C1 (D) cancer cells. In the diagrams [(C) and (D)], the data from one of two representative experiments are shown. Per group, five or six mice were challenged. Tumor was defined as established when its volume reached >500 mm3. Statis- tical analysis: (B) Mann-Whitney U test: **P < 0.01 and [(C) and (D)] log-rank test: *P < 0.05 and **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Activity Assay, Fluorescence, Marker, Staining, Control, Injection, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 6. CD40L+CD8+ T cells mediate cell death in human CD40+ carcinoma cell lines by caspase-8 activation. (A) Histograms represent the CD40 expression on EJ138 or A704 transfected with nontargeting single guide RNA (black, sgNon) or cells depleted for CD40 by CRISPR-Cas9 and sgRNA against human CD40 (red). Gray-filled his- tograms show isotype staining. (B) sgNon (black) or caspase-8 sgRNA–treated (red) EJ138 and A704 cells were treated with multimeric CD40L for 24 hours, and caspase-8 activation was monitored by FITC-IETD-FMK staining. Gray-filled histograms represent the basal caspase-8 activation of unstimulated WT cells. (C and D) sgNon, CD40 sgRNA-, or caspase-8 (Casp8) sgRNA–treated variants of EJ138 (C) or A704 (D) were cocultivated with CD40L-enriched CD8+ T cells for 24 hours, and apoptosis was de- tected by annexin V and DAPI staining. Shown are representative dot plots with frequencies of technical triplicates (left) and bar graphs summarizing the data from ex- periments with eight different T cell donors. Statistical analysis: [(C) and (D)] Repeated measures ANOVA, followed by Dunnett’s test: **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Activation Assay, Expressing, Transfection, CRISPR, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 7. Resistance pattern for CD40-mediated cell death and correlations between CD8 and CD40L in different RCC cohorts. (A) In the histograms, the dark gray areas display the CD40 expression, and the light gray areas display the corresponding isotype control on eight different RCC lines. The included numbers represent the mean fluorescence intensity fold change between CD40 and isotype control staining. (B) Percentages of CD40L-induced lysis per RCC line after stimulation for 48 hours are plotted. Representative lysis values after backgorund substraction of each cell line of at least three individual experiments are depicted. (C) The heatmap represents the top 10 differentially expressed genes between CD40-resistant and CD40-sensitive RCC cell lines. (D) In the Kaplan-Meier survival plot, the resistance score of low and high groups of the TCGA-KIRC patient cohort is compared with each other. (E) Partial correlation networks of different patient groups are displayed, and the numbers are the partial correlation coefficients. (F) In the Kaplan-Meier survival plots, the resistance score of low and high groups of the cohorts is compared with all patients treated within the checkmate-009, checkmate-010, and checkmate-025 studies (left) or divided into two arms of the checkmate-025 study (middle nivolumab arm and right everolimus arm). Statistical analysis: (C) Described in Materials and Methods; [(D) and (F)] log-rank test.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Control, Fluorescence, Staining, Lysis

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Functional analysis of cells stimulated with CD154. (A) Viability assay. No significant changes were observed in all four cell lines. (B) Migration assay. (C) Invasion assay. Migration and invasion were enhanced in TE-5 and TE-10 cells, which highly expressed CD40, after rsCD154 stimulation. No significant changes were observed in low CD40 expression cells (TE-4 and −11 cells). The Mann-Whitney U test was performed using GraphPad Prism 9 software. ns, not significant; OD, optical density; rsCD154, recombinant soluble CD154.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Functional Assay, Viability Assay, Migration, Invasion Assay, Expressing, MANN-WHITNEY, Software, Recombinant

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Changes in gene expression caused by the CD40-CD154 interaction in TE-10 cells. Screening of 40 genes involved in extracellular matrix remodeling. The expression ratios of target genes relative to GAPDH are shown on the left. On the right, CD154-induced changes in gene expression are displayed in descending order. Upregulation of gene expression in the order MMP-9 > PLG > LAMC2 was confirmed following rsCD154 stimulation.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Gene Expression, Expressing

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Changes in MMP-9 expression caused by the CD40-CD154 interaction in TE cells. (A) Qualitative analysis of MMP-9 secretion from TE cells using gelatin zymography. Increased MMP-9 secretion was observed in high CD40 expression cells (TE-5 and −10), regardless of the baseline MMP-9 secretion level without CD154 stimulation. (B) Quantitative analysis of MMP-9 secretion from TE cells using an ELISA. Increased MMP-9 secretion was observed in TE-5 and −10 cells. (C) Quantitative analysis of MMP-9 mRNA expression in TE cells via reverse transcription-quantitative PCR. MMP-9 mRNA expression upregulation was observed in high CD40 expression cells (TE-5 and −10). The Mann-Whitney U test was performed using GraphPad Prism 9 software. n.d., not detected; rsCD154, recombinant soluble CD154.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Expressing, Zymography, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Software, Recombinant

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Experiments using CD40-knockdown TE-10 cells using siRNA. (A) Transfection with CD40 siRNA resulted in a reduction in CD40 mRNA expression. The average relative CD40 mRNA expression was compared using GraphPad Prism 9 software. After one-way ANOVA, Bonferroni post hoc analysis was performed. (B) CD154 stimulation of CD40 knockdown TE-10 cells was performed, and MMP-9 upregulation was suppressed. (C) Migration assay. CD40 knockdown reduced cell migration, and no increase in migration was observed following CD154 stimulation. The images on the right present a visualization of migrating cells under a 1.25× optical microscope, with the cellular regions indicated by green labelling. (D) Invasion assay. Similar to the migration assay, a decrease in invasion was observed following CD40 knockdown. The images on the right present a visualization of migrating cells under a 1.25× optical microscope, with the cellular regions indicated by green labelling. siRNA, small interfering RNA.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Knockdown, Transfection, Expressing, Software, Migration, Microscopy, Invasion Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Survival analysis of patients who underwent esophagectomy was performed using the Kaplan-Meier method. The log-rank (Mantel-Cox) test was conducted using GraphPad Prism 9 software. (A) Association between DFS and MMP-9 levels. (B) Association between DFS and CD154 levels. (C) Association between OS and MMP-9 levels. (D) Association between OS and CD154 levels. DFS, disease-free survival; MST, median survival time; OS, overall survival.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Software

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Survival analysis of patients with esophageal cancer stratified by disease stage. (A) Association between DFS and MMP-9 levels in patients with pathological stage I. (B) Association between DFS and CD154 levels in patients with pathological stage I. (C) Association between OS and MMP-9 levels in patients with pathological stage I. (D) Association between OS and CD154 levels in patients with pathological stage I. (E) Association between DFS and MMP-9 levels in patients with pathological stage II–IV. (F) Association between DFS and CD154 levels in patients with pathological stage II–IV. (G) Association between OS and MMP-9 levels in patients with pathological stage II–IV. (H) Association between OS and CD154 levels in patients with pathological stage II–IV. Survival analysis was performed using the Kaplan-Meier method, and the log-rank (Mantel-Cox) test was conducted using GraphPad Prism 9 software. DFS, disease-free survival; MST, median survival time; OS, overall survival.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Software

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Schema of the CD40-CD154 interaction in the tumor microenvironment of esophageal cancer. In tumor cells, the CD40-CD154 interaction induces the secretion of molecules involved in ECM remodeling, including MMP-9, contributing to cell invasion, EMT and metastasis. In immune cells, the CD40-CD154 interaction is involved in antitumor immunity, leading to cancer cell death. ECM, extracellular matrix; EMT, epithelial-mesenchymal transition.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: