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. 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: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. ( A ) Representative images of IHC staining for RRBP1 and CD8 + T cells in BC samples. ( B ) The correlation between RRBP1 expression and CD8 + T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. ( C ) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. ( D ) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. ( E ) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. ( F ) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. ( G ) Representative images of IHC and mIHC staining for RRBP1 and CD8 + T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. ( H, I ) Flow cytometry showed the percentages of CD8 + T cells in CD3 + cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis ( B ), unpaired two-tailed t-test ( I ). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T-cell subgroup. The UMAP plot of CD8 + T cells subpopulation, color-coded by cell cluster and cell type. ( A ) The expression of markers in each CD8 + T cells subpopulation. ( B ) Bar plot showed the proportion of CD8 + T cells subpopulation in the shNC and shRRBP1 groups. ( C ) The percentage of each CD8 + T-cell clusters in shNC and shRRBP1 groups. ( D ) Heatmap showed the differentially activated pathway among all the CD8 + T-cell clusters. ( E ) The differentially expressed genes in CD8 + T cells between shNC and shRRBP1 groups. ( F ) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. ( G, H ) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. ( I–K ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes ( I ), volumes ( J ), and weight ( K ) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( H, K ) and two-way ANOVA with Tukey’s multiple comparison test ( J ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. ( A ) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. ( B ) The correlation between CXCR3 expression and CXCL10 expression or activated CD8 + T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. ( C ) MB49 cells were co-cultured with CD8 + T cells, and tumor cells were stained with crystal violet. ( D ) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8 + T cells in vitro conditioned culture model. ( E ) Schematic diagram of in vitro CD8 + T-cell migration assays. ( F ) The number of CD8 + T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. ( G–I ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( G ), volumes ( H ), and weights ( I ) were measured. ( J ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( K ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( D, F, I, K ) and two-way ANOVA with Tukey’s multiple comparison test ( H ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. ( A–D ) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. ( E–G ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( E ), volumes ( F ), and weights ( G ) were measured. ( H ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( I ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( B, D, G, I ) and two-way ANOVA with Tukey’s multiple comparison test ( F ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: Nature communications

Article Title: Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

doi: 10.1038/s41467-018-04999-8

Figure Lengend Snippet: Fig. 2 Macrophage MGLL inhibits tumor progression via CD8+T cells. a Six-week-old WT and TgMGLL mice were subcutaneously inoculated with MC-38 cells, and tumor volumes were measured dynamically. This test was performed four times. (n = 10) b The survival time of the MC-38 tumor-bearing mice described in a. The data represent the means ± s.e.ms. (n = 10, ***P < 0.005, Gehan-Breslow-Wilcoxon test). c The growth curves of the subcutaneous MC-38 tumors in Myeloid-mgll-KO mice and the control littermates (fl/fl). (n = 6). d The survival time of the MC-38 tumor-bearing mice described in c. The data represent the means ± s.e.ms. (n = 6, **P < 0.01, Gehan-Breslow-Wilcoxon test). e Incidence of metastasis in lungs and livers of the intravenous tumor model. The 6-week-old C57BL/6 WT or transgenic mice were intravenously injected with MC-38 colorectal cancer cells (5 × 106 cells per mouse) via the tail vein. The mice were sacrificed 2 weeks after tumor inoculation. The lungs and livers were dissected for pathological observation of tumor lesions. Data are means ± s.e.ms. and polled from four individual experiments. Each symbol represents an experimental replicate (n = 8~11). f–h The MC-38 tumor lesions in lungs of WT and TgMGLL mice, as described in e. Representative images of gross anatomy (f) and H&E staining (g) are displayed. The blue dotted lines indicate the tumor lesions. The relative tumor lesions were calculated (h). Scale bars, 200 μm. (n = 8~9). i Macrophage MGLL inhibited tumor growth in a Rag-1 dependent mannaer. MC-38 tumors were inoculated subcutaneously in WT, TgMGLL, Rag1KO or TgMGLL + Rag1KO mice and the tumor volumes were measured dynamically. (n = 5) j mRNA levels of IFNγ in CD8+ or CD4+ T cells that were isolated from the MC-38 tumors inoculated subcutaneously in WT or TgMGLL mice for 3 weeks. (n = 5). k Six-week-old WT or TgMGLL mice were subcutaneously inoculated with MC-38 cells and treated with anti-CD8 antibody or IgG as a control. The tumor volume was measured dynamically. (n = 10). l The survival time of the MC-38 tumor- bearing mice treated as described in k. The data represent the means ± s.e.ms. (n = 10, *P < 0.05, Gehan-Breslow-Wilcoxon test; ns, not significant). Data in a, c and e–k represent the means ± s.e.ms. (*P < 0.05, **P < 0.01, ***P < 0.005; student’s t-test; ns not significant)

Article Snippet: The MC-38 tumor-bearing mice were treated with monoclonal antibodies anti-CD8 (MAB116, Novus Biologicals) or IgG (MAB002, Novus Biologicals) as control.

Techniques: Control, Transgenic Assay, Injection, Staining, Isolation

Journal: Nature communications

Article Title: Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

doi: 10.1038/s41467-018-04999-8

Figure Lengend Snippet: Fig. 6 MGLL-CB2 axis regulates tumor progression in mice. a Relative mRNA levels of TNFα, IL-6 and IFNγ in the CD8+ T cells in the MC-38 tumors inoculated subcutaneously in WT, TgMGLL, TgCB2, and TgMGLL+CB2 mice. The data represent the means ± s.e.ms. (n = 5, ***P < 0.005, Student’s t-test; ns not significant). b Six-week-old WT, TgMGLL, TgCB2, and TgMGLL+CB2 mice were subcutaneously inoculated with MC-38 cells, and the tumor volume was measured dynamically. This experiment was repeated three times. The data represent the means ± s.e.ms. (n = 10, **P < 0.01, ***P < 0.005, Student’s t-test). c The survival time of the MC-38 tumor-bearing mice. The mice were described in b. The data represent the means ± s.e.ms. (n = 10, **P < 0.01, ***P < 0.005, Gehan-Breslow-Wilcoxon test). d The incidence of tumor metastasis in MC-38 tumor model. Six-week-old WT, TgMGLL, TgCB2, and TgMGLL

Article Snippet: The MC-38 tumor-bearing mice were treated with monoclonal antibodies anti-CD8 (MAB116, Novus Biologicals) or IgG (MAB002, Novus Biologicals) as control.

Techniques:

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing balb/c nude mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). B Double staining of CD8 and αSMA in AB1-HA tumors from balb/c mice treated with or without KL001 (studied in Fig. ). Scale bar, 200 μm. C Representative images and D quantitative evaluation of sections from KL001-treated AB1-HA tumors stained for CD8, CD4, Foxp3, and F4/80 ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Quantitative evaluation of sections from CLK8-treated AB1-HA tumors stained for CD8, CD4, Foxp3 ( n = 15 fields from five mice per group). * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Injection, Double Staining, Staining, MANN-WHITNEY

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: Total CD8 + T cells from individuals without HIV were treated with medium, vehicle (Veh.) control, or the GSK3 inhibitor (inh), followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A and B ) Analysis of CD8 + T cell subpopulations in unstimulated cells ( n = 4). ( C ) Fold change of CD8 + T cell subpopulations upon vehicle control or GSK3 inhibitor treatment, relative to the medium alone condition ( n = 4). ( D ) Expression of TCF-1 in CD8 + T cell subsets ( n = 4). ( E ) Fold change in the expression of the indicated markers induced by anti-CD3/anti-CD28 antibody stimulation relative to unstimulated cells ( n = 5). ( F ) Analysis of dead cells by Aqua LIVE/DEAD + staining (Aqua L/D) among total and T-bet + CD8 + T cells, and fold change in dead CD8 + T cells induced by anti-CD3/anti-CD28 stimulation relative to the unstimulated (Unstim.) condition ( n = 5). ( G ) Frequencies of granzyme B + (GZMB + ), IL-2 + , IFN-γ + , and TNF-α + CD8 + T cells after anti-CD3/anti-CD28 stimulation. ( H ) Expression of 1 to 4 functions in CD8 + T cells ( n = 5). * P < 0.05, by Dunn’s test ( B ) and Wilcoxon test ( C , D , and F – H ). Data obtained from 2 ( A – F ) or 3 ( G and H ) independent experiments are shown.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A and B ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A ) Analysis of the expression of 2-NBDG, BODIPY, MitoTracker Green, and CellROX among CD8 + T cell subsets. ( B ) Frequencies of 2-NBDG + , BODIPY + , MitoTracker Green + , and CellROX + cells among CD8 + T cell subpopulations after stimulation ( n = 5). ( C and D ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( C ) Flow cytometric analysis of the expression of p-S6 and p-AKT in total CD8 + T cells, and frequencies of p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – subsets in total CD8 + T cells ( n = 7). ( D ) Flow cytometric analysis of IL-2 and IFN-γ expression among TNF-α + CD8 + T cells, after anti-CD3/anti-CD28 stimulation. Histograms show the expression of p-S6 in the indicated cell subsets, in reprogrammed and nonreprogrammed cells. Frequency of p-S6 + cells among TNF-α + IFN-γ + IL-2 + or TNF-α + IFN-γ – IL-2 – subsets ( n = 6 individuals without HIV). * P < 0.05 and ** P < 0.01, by Wilcoxon test ( B and C ) and Šidák’s multiple-comparison test ( D ). Data are from 2 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: After treatment with the GSK3 inhibitor, CD8 + T cells from people with HIV were stained with HLA-matched HIV dextramers for analysis of the phenotype of HIV dextramer + cells. ( A ) UMAP plots generated from HIV dextramer + CD8 + T cells after data concatenation ( n = 4). Vehicle control and GSK3 inhibitor treatments as well as CD8 + T cell subpopulations were identified by manual gating and projected into the UMAP space. ( B ) Analysis of the expression of CCR7, CD27, and TCF-1 expression in HIV dextramer + CD8 + T cells ( n = 7). ( C ) Frequencies of CD8 + T cell subpopulations among HIV dextramer + CD8 + T cells ( n = 7). ( D – G ) After reprogramming, cells from people with HIV were stimulated for 6 hours with Gag peptides for analysis of the total frequency (IFN-γ + or CD107a + or IL-2 + or TNF-α + ) of antigen-specific CD8 + T cells ( n = 5) ( D ), the proportion of memory T cell subpopulations ( n = 5) ( E ), the expression of CD107a, IFN-γ, granzyme B, IL-2, and TNF-α on a per-cell basis ( n = 5–11) ( F ), and the frequency of polyfunctional cells ( n = 6) ( G ). ( H and I ) Cells from people with HIV were stimulated for 6 days with Gag peptides and restimulated with the same peptides for another 12 hours, followed by analysis of the viability of proliferating HIV Gag–specific CD8 + T cells ( n = 6) ( H ) and frequencies of the total (live IFN-γ + , IL-2 + , or TNF-α + ) HIV Gag–specific response ( n = 8) ( I ). ( J and K ) After reprogramming, cells from people with HIV ( n = 6) were stimulated for 6 hours with Gag peptides, followed by analysis of p-S6 + p-AKT – , p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – cell subsets ( J ) and the intensity of expression of p-S6 and p-AKT ( K ) in HIV Gag–specific (IFN-γ + and/or IL-2 + ) CD8 + T cells. * P < 0.05 and *** P < 0.001, by Wilcoxon test. Data are from 3 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Staining, Generated, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: HIV-1 BaL–superinfected CD4 + T cells from people with HIV were cultured alone or in the presence of autologous nonreprogrammed or reprogrammed CD8 + T cells. After 7 days, the levels of infection were measured by flow cytometry (KC57 anti-Gag antibody) or ELISA (p24 in culture supernatant). ( A ) Representative flow cytometric analysis of the frequency of infected CD4 + T cells (from a total of 4 donors). ( B ) HIV-suppressive capacity of nonreprogrammed and reprogrammed CD8 + T cells (log 10 decrease of p24 levels in culture supernatant; n = 5 individuals, with the median of triplicates for each experiment). ( C and D ) The frequency of IFN-γ + HIV-specific CD8 + T cells ( C ) and expression of CCR7, PD-1, and LAG-3 in HIV-specific CD8 + T cells ( D ) were measured after 7 days of coculturing. * P < 0.05 and ** P < 0.01, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Cell Culture, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A ) Total CD8 + T cells from people without HIV were treated with vehicle control or the GSK3 inhibitor, followed by evaluation of Eomes and CD122 expression in CD8 + T cell subsets ( n = 5). ( B ) After vehicle control or GSK3 inhibitor treatment, CD8 + T cells from people without HIV were left unstimulated or stimulated with IL-7 or IL-15 for 6 days, followed by analysis of cell proliferation ( n = 5; data are from 2 independent experiments). ( C and D ) CD8 + T cells from people with HIV were treated with vehicle control or the GSK3 inhibitor, followed by stimulation with IL-15 for 6 days, for analysis of the proliferation of HIV dextramer + CD8 + T cells ( n = 4; data from 3 independent experiments). * P < 0.05, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Expressing

Journal: Translational Oncology

Article Title: Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

doi: 10.1016/j.tranon.2021.101205

Figure Lengend Snippet: cEGFR p527 immunization mediates CD8 infiltration and antibody deposition into canine tumors. Postmortem bladder cancer tissue from a cEGFR p527 immune dog and tissue from healthy, non-cancerous bladder was examined for infiltration of CD8 T cells and endogenous antibody. A. CD8 isotype control. B. CD8 T cell staining (green). Arrows illustrate presence of CD8+ cells within the tumor tissue. C. & D. Antibody deposition in canine bladder tumor tissue from a cEGFR p527 immunized dog (two different fields). E. & F. Canine IgG deposition in normal canine bladder tissue (two different fields). Magnification is 40x (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: CD8 T cells were stained in tissues using mouse anti-dog CD8 (MAB6709; R&D Systems, Minneapolis, MN), followed by goat anti-mouse IgG ( H + L ) Alexa Fluor 488 (A11017; Life Technologies, Eugene, OR) and counterstaining with DAPI (Molecular Probes; Eugene, OR).

Techniques: Control, Staining