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: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Phenotpyes of primed CD8 + Foxp3 + T cells. (A) Surface staining of CD8, CD25, GITR, CTLA4, and PD-1 on the splenic cells taken from Foxp3-GFPtg mice on day 6 after infection with H5N1 virus. The dot plots represent one of four independent experiments with similar results ( n = 5 mice). (B) Splenic CD8 + CD25 + T cells from Foxp3-GFPtg mice on day 6 after H5N1 viral infection were purified for quantitative RT-PCR. Naïve CD8 + T cells were purified from naïve Foxp3-GFPtg mice as control. Data are shown as mean + SEM and are pooled from three independent experiments. ** p < 0.01. n.s., p > 0.05, unpaired two-tailed t -test. (C) Splenic cells were isolated on day 6 from Foxp3-GFPtg mice infected by H5N1 virus and were stimulated with 5 μg/mL NP peptide (NP366–374) or no peptide. Cells were incubated for 6 h with 2 μM monensin before intracellular staining. CD8 + T cells were gated to analyze the expression of Foxp3 and IL-10. The dot plots represent one of three independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Staining, Infection, Virus, Purification, Quantitative RT-PCR, Control, Two Tailed Test, Isolation, Incubation, Expressing

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: Primed CD8 + CD25 + T cells expressed IL-10. Splenic cells were isolated on day 6 from IL-10-GFPtg mice infected with H5N1 virus and surface stained for CD8 and CD25. CD8 + T cells were separated into CD25 positive and negative cells. These cells were further divided into GFP positive or negative, with GFP serving as a marker for IL-10 positivity. The percentages of CD8 + CD25 + T cells and CD8 + CD25 − T cells that were IL-10 + or IL-10 − are shown together with results of statistical analysis. The dot plots represent one of five independent experiments with similar results ( n = 5 mice).

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Isolation, Infection, Virus, Staining, Marker

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + Treg cells resulted in enhanced mortality and increased virus load in the lung. (A) Splenic CD8 + CD25 + T cells were isolated on day 6 from Foxp3-GFPtg mice infected with H5N1 virus. The cells were transferred into BALB/c mice (1 × 10 5 or 5 × 10 5 CD8 + CD25 + T cells per mouse) and the mice ( n = 10 mice per group) were then immediately challenged with H5N1 virus. (B) Survival of mice was monitored from day 7 to 16 after virus infection. Log-rank test for comparisons of survival curves between control mice and CD8 + Treg cells recipient mice; ** p < 0.01. *** p < 0.001, log-rank test. (C) Lung viral load was assayed on day 6 after virus infection ( n = 5 mice per group). (D) Total RNA was extracted from lung for real-time PCR for IFN-β and Mx-1. Data are shown as mean + SEM and are pooled from three independent experiments. n.s., p > 0.05. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Virus, Isolation, Infection, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + T-cell immunity was inhibited by CD8 Treg cells in vivo. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, total CD8 + T cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus and were transferred to CD8 KO mice. 5 × 10 5 CD8 + CD25 + T cells, 1 × 10 7 CD8 + CD25 − T cells, or 1 × 10 7 CD8 + T cells were transferred into each mouse ( n = 10 mice per group). (B) Mouse survival was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between CD8 + T-cell recipient mice and CD8 + CD25 − T-cell recipient mice; ** p < 0.01 ( n = 10 mice per group), log-rank test. (C) Lung viral loads were assayed on day 6 after virus infection ( n = 5 mice per group). (D). Lung cells taken on day 8 of infection were stimulated with PMA and ionomycin for 5 h in the presence of monensin and then stained to detect intracellular IFN-γ expression in the CD4 + and CD8 + T cells ( n = 4 mice per group). (E) Statistical analysis of IFN-γ + cells among CD4 + and CD8 + T cells (%). (F) Serum total H5N1 virus-specific IgG titers, assayed by ELISA on day 8 post virus infection ( n = 4 mice per group). Mice receiving CD8 + CD25 − T cells served as CD8nonTreg-cell controls. Data are presented as means ± SEM and are representative of three independent experiments. * p < 0.05. ** p < 0.01. *** p < 0.001, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vivo, Isolation, Infection, Virus, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: CD8 + CD25 − T-cell proliferation was inhibited by CD8 + Treg cells through IL-10 in vitro. (A) CD8 + CD25 + T cells, CD8 + CD25 − T cells, CD11c + cells were isolated on day 6 from spleens of H5N1-infected C57BL/6 mice. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD8 + Treg cells for 5 days. (B) CD4 + CD25 + T cells, CD8 + CD25 + T cells, CD8 + CD25 − T cells, and CD11c + cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus. CFSE-stained CD8 + CD25 − T cells were stimulated to proliferate in vitro; 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD4 + or CD8 + Treg cells for 5 days. (C) 2 × 10 5 CD8 + CD25 − T cells and 5 × 10 4 CD11c + cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of 1 × 10 5 CD8 + Treg cells in transwell plates for 5 days. (D) T-cell proliferation was done with 50 μg/mL anti-IL-10 mAb or isotype control antibodies in the wells. The number of CD8 + Treg cells used in the system (C and D) was 1 × 10 5 . (E) CD8 + CD25 + T cells and CD11c + cells from C57BL/6 mice and CD8 + CD25 − T cells from DNIL-10R mice were isolated 6 days after infection with H5N1 virus and T-cell proliferation assays were performed. (A–E) Data shown are representative of at least three independent experiments with four mice per group.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: In Vitro, Isolation, Infection, Staining, Virus, Control

Journal: European Journal of Immunology

Article Title: CD8 + Treg cells suppress CD8 + T cell-responses by IL-10-dependent mechanism during H5N1 influenza virus infection

doi: 10.1002/eji.201343583

Figure Lengend Snippet: The antiviral activity of CD8 + CD25 − T cells was inhibited by CD8 + Treg cells in vivo through IL-10. (A) CD8 + CD25 + T cells and CD8 + CD25 − T cells from the spleens of WT mice and CD8 + CD25 − T cells from the spleens of DNIL-10R mice were isolated on day 6 after H5N1 infection and transferred into CD8 KO mice (5 × 10 5 CD8 + CD25 + T cells and 1 × 10 7 CD8 + CD25 − T cells per mouse) that were then immediately infected with H5N1 virus (day 0 of challenge). (B) Survival of mice ( n = 10 per group) was monitored from day 4 to 16 after virus infection. Log-rank test for comparisons of survival curves between DNIL-10R CD8 + CD25 − T cells recipient mice and DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice, or between DNIL-10R CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice and WT CD8 + CD25 − T cells plus WT CD8 + Treg cells recipient mice; n.s., p > 0.05. **, p < 0.01, log-rank test. (C) Viral load in mouse lungs were assayed on day 6 after virus infection ( n = 5 mice per group). Data are presented as means ± SEM and are representative of four independent experiments. n.s., p > 0.05. * p < 0.05, unpaired two-tailed t -test.

Article Snippet: MagCellect Mouse CD4 + CD25 + T-cell Isolation Kit (R&D Systems, Minneapolis, MN, USA), MagCellect Mouse CD8 + T-cell Isolation Kit (R&D Systems), EasySep® Mouse Naïve CD8 + T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) and EasySep® CD11c Positive Selection Kit (STEMCELL Technologies) were purchased.

Techniques: Activity Assay, In Vivo, Isolation, Infection, Virus, Two Tailed Test