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: Vaccines

Article Title: Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

doi: 10.3390/vaccines11020245

Figure Lengend Snippet: Vaccination of C57Bl/6 mice with rMVA-HBc±CD70. ( A ) C57Bl/6 mice were immunized subcutaneously with HBcAg adjuvanted with CpG and PCEP at day 0 (group wt received adjuvants only). At day 21, mice were boosted with MVAwt (group wt), rMVA-HBc (group HBc) or rMVA-HBc-CD70 (group HBc-CD70) by intraperitoneal injection. At day 35, splenocytes of mice were isolated and stained with PE-labelled C 93 multimers to determine the amount of HBc-specific CD8 T cells ( B ) showing representative dot plots and ( C ) showing the group analysis. The table ( C ) shows the proportion of all CD8+ T cells. At day 35, splenocytes of vaccinated mice were also stimulated ex vivo with C 93 or B8R peptides for intracellular cytokine staining (ICS) to evaluate C 93 - or B8R- specific IFNγ-, TNFα- and IL2 production of CD8 T cells ( D ) showing representative dot plots and ( E ) showing the group analysis. ( F ) Simultaneous detection of IFNγ, TNFα and IL2 production of CD8 cells upon HBc stimulation ex vivo: dot plots show one representative mouse of each group. The table shows the mean values (% of CD8+ cells) of 4 mice/group. ( G ) The gain of function of CD8 T cells after CD70 co-stimulation during vaccination: the proportion of IFNγ+ CD8 T cells upon ex vivo C 93 stimulation was set in relation to the proportion of C 93 multimer-positive CD8 T cells in the same animals. Proportions were calculated for each mouse separately. C 93 : HBcAg CD8 T cell epitope; B8R: MVA-derived CD8 T cell epitope. Crosses and triangles represent individual mice; horizontal lines indicate mean values; error bars indicate standard deviation. Statistics: one-way ANOVA and subsequent unpaired t-test, n.s.: not statistically significant.

Article Snippet: Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA).

Techniques: Injection, Isolation, Staining, Ex Vivo, Derivative Assay, Standard Deviation

Journal: Vaccines

Article Title: Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

doi: 10.3390/vaccines11020245

Figure Lengend Snippet: Amount and functionality of CD8 T cells in the livers and spleens of vaccinated HBV1.3tg mice with and without CD70 co-expression. HBV1.3tg mice were immunized with protein-prime (day 0, HBc + CpG + PCEP, group wt only CpG and PCEP) and MVA boost (day 21, with MVAwt (group wt), rMVA-HBc (group HBc) or rMVA-HBc-CD70 (group HBc-CD70)). Mice were divided into groups with low to moderate HBeAg levels (1–8 S/CO, “low viremia”) and high antigen levels (8–23 S/CO, “high viremia”) before the experiment. At day 35, splenocytes and liver-associated lymphocytes of mice were isolated. ( A ) Representative dot blot analysis of liver-associated lymphocytes (LAL) for single mice of each vaccination group, utilizing C 93 multimer staining. ( B ) Representative dot blot analysis of intracellular cytokine staining (IFNγ) after ex vivo C 93 stimulation of LALs. ( C ) Percentages of C 93 multimer-positive CD8 T cells in vaccinated mice. ( D ) HBV core-specific CD8 T cell response for the groups of mice with low and high viremia represented by the portion of CD8 T cells expressing IFNγ after ex vivo stimulation with C 93 peptide. ( E ) MVA-specific CD8 T cell response represented by the portion of CD8 T cells expressing IFNγ after ex vivo stimulation with B8R peptide. Mice with low and high viremia are presented together. C 93 : HBV core-derived CD8 T cell epitope; B8R: MVA-derived CD8 T cell epitope; Crosses and triangles represent individual mice; Horizontal lines indicate mean values; Error bars indicate standard deviation. Differences between groups HBc and HBc-CD70 (if >2 mice/group) were not statistically significant (one-way ANOVA and unpaired t-test).

Article Snippet: Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA).

Techniques: Expressing, Isolation, Dot Blot, Staining, Ex Vivo, Derivative Assay, Standard Deviation