anti mouse cd8 α ab  (Bio X Cell)

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: Immunostaining was performed on ice in the dark for 40 min using the following antibody: fixable viability dye (BD Pharmingen, catalog no. 565388), PerCP/Cyanine 5.5-conjugated anti-mouse CD45 (Elabscience, E-AB-F1136J), APC-conjugated anti-mouse CD3 (Elabscience, E-AB-F1013E), or PE-conjugated anti-mouse CD8 (Elabscience, E-AB-F1104D), PE-conjugated anti-mouse Granzyme B (GZMB) (Thermo Fisher Scientific, 12-8898-82), PE-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101D), FITC-conjugated anti-mouse GZMB (Thermo Fisher Scientific, 11-8898-82), FITC-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101C), and the results were analyzed using CytExpert software or FlowJo.

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: Immunostaining was performed on ice in the dark for 40 min using the following antibody: fixable viability dye (BD Pharmingen, catalog no. 565388), PerCP/Cyanine 5.5-conjugated anti-mouse CD45 (Elabscience, E-AB-F1136J), APC-conjugated anti-mouse CD3 (Elabscience, E-AB-F1013E), or PE-conjugated anti-mouse CD8 (Elabscience, E-AB-F1104D), PE-conjugated anti-mouse Granzyme B (GZMB) (Thermo Fisher Scientific, 12-8898-82), PE-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101D), FITC-conjugated anti-mouse GZMB (Thermo Fisher Scientific, 11-8898-82), FITC-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101C), and the results were analyzed using CytExpert software or FlowJo.

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: Immunostaining was performed on ice in the dark for 40 min using the following antibody: fixable viability dye (BD Pharmingen, catalog no. 565388), PerCP/Cyanine 5.5-conjugated anti-mouse CD45 (Elabscience, E-AB-F1136J), APC-conjugated anti-mouse CD3 (Elabscience, E-AB-F1013E), or PE-conjugated anti-mouse CD8 (Elabscience, E-AB-F1104D), PE-conjugated anti-mouse Granzyme B (GZMB) (Thermo Fisher Scientific, 12-8898-82), PE-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101D), FITC-conjugated anti-mouse GZMB (Thermo Fisher Scientific, 11-8898-82), FITC-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101C), and the results were analyzed using CytExpert software or FlowJo.

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: Immunostaining was performed on ice in the dark for 40 min using the following antibody: fixable viability dye (BD Pharmingen, catalog no. 565388), PerCP/Cyanine 5.5-conjugated anti-mouse CD45 (Elabscience, E-AB-F1136J), APC-conjugated anti-mouse CD3 (Elabscience, E-AB-F1013E), or PE-conjugated anti-mouse CD8 (Elabscience, E-AB-F1104D), PE-conjugated anti-mouse Granzyme B (GZMB) (Thermo Fisher Scientific, 12-8898-82), PE-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101D), FITC-conjugated anti-mouse GZMB (Thermo Fisher Scientific, 11-8898-82), FITC-conjugated anti-mouse IFN-γ (Elabscience, E-AB-F1101C), and the results were analyzed using CytExpert software or FlowJo.

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

Journal: CytoJournal

Article Title: The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer

doi: 10.25259/Cytojournal_129_2025

Figure Lengend Snippet: PGE2 upregulates PD-L1 expression in NSCLC and promotes immune escape response. (a-c) PD-L1 expression detected after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (d and e) Cytotoxicity tested by LDH kit assay after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (f and g) CD8 + T cell viability tested after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (h and i) CD8 + T cell apoptosis examined after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (j-m) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). n = 6; ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, LDH: Lactate dehydrogenase, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: First, a single-cell suspension was prepared and incubated with CD3 (E-AB-F1013E, Elabscience, Wuhan, China) and CD8 (E-AB-F1104Q, Elabscience, Wuhan, China) antibodies in the dark.

Techniques: Expressing, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: CytoJournal

Article Title: The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer

doi: 10.25259/Cytojournal_129_2025

Figure Lengend Snippet: PGE2 promotes immune escape in NSCLC in vivo by upregulating PD-L1 expression. (a) Isolated tumor images after PTGES overexpression and knockout. (b and c) Changes in tumor weight and volume after PTGES overexpression and knockout (significant difference markers marked with ✶ represent OE-NC versus OE-PTGES, and those marked with # represent sh-NC vs. sh-PTGES). (d-f) WB analysis of PTGES and PD-L1 after PTGES overexpression and knockout in vivo . (g and h) IHC analysis of CD8 after PTGES overexpression and knockout in vivo (scale bar: 20 μm, magnification, 400×). (i-l) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression and knockout in vivo . n = 5; ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ## P < 0.01. OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IHC: Immunohistochemistry, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: First, a single-cell suspension was prepared and incubated with CD3 (E-AB-F1013E, Elabscience, Wuhan, China) and CD8 (E-AB-F1104Q, Elabscience, Wuhan, China) antibodies in the dark.

Techniques: In Vivo, Expressing, Isolation, Over Expression, Knock-Out, Enzyme-linked Immunosorbent Assay, Negative Control, Immunohistochemistry