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: 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