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: bioRxiv

Article Title: Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

doi: 10.1101/2020.03.11.987412

Figure Lengend Snippet: (A) Study design using the IL-10 knock-out mouse model. CPZ group dams were treated with CPZ [(0.5 mg/ml) administration in drinking water] during pregnancy from day 14 of gestation until the end of the weaning period (3 weeks of age of offspring). (B) The number of 16S rRNA gene per gram of feces in male offspring from non-treated (NT) and CPZ-exposed dams at 3 and 7 weeks of age ( n = 5 per group). (C) Shannon diversity index in male offspring at 3 and 7 weeks of age. (D) PCoA plots of both unweighted and weighted UniFrac distances of 16S rRNA gene amplicon sequences in NT and CPZ male pups at 3 and 7 weeks of age, respectively. (E) Flow cytometric analyses of live CD4 + T cells expressing Foxp3 (Treg), T-bet (Th1) or RORγt (Th17) in spleens (SPLNs) and mesenteric lymph nodes (MLNs) of NT versus CPZ males at 7 weeks of age ( n = 4-5 per group). Data represent the percentage of live TCRβ + CD4 + cells. (F) IL-12p40 production of dendritic cells obtained from 7-week-old male NT (blue) and CPZ (red) pups with stimulation with fecal slurry ( n = 5 per group). * p < 0.05 and ** p < 0.01. Data represent mean ± SEM for (B), (C), (E), and (F). Female data are shown in the supplementary figure. See also Figure S1.

Article Snippet: DCs and naïve CD4 + T cells were harvested from SPLNs of mice at 7 weeks of age using a CD11c positive selection kit II (STEMCELL Technologies) and a mouse naïve CD4 + T cell Isolation kit (STEMCELL Technologies).

Techniques: Knock-Out, Amplification, Expressing

Journal: bioRxiv

Article Title: Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

doi: 10.1101/2020.03.11.987412

Figure Lengend Snippet: (A) Flow cytometric analyses of live CD4 + T cells expressing IFNγ and IL-17A stimulated via PMA and ionomycin. Cells in spleens (SPLNs) and mesenteric lymph nodes (MLNs) were obtained from NT and CPZ males at 7 weeks of age. Data represent the percentage of live TCRβ + CD4 + cells ( n = 5 per group). (B) IFNγ and IL-17A production of CD4 + T cells obtained from SPLN and MLN of 7-week-old NT and CPZ male pups with co-stimulation via anti-CD3 and anti-CD28 antibodies ( n = 5 per group). (C) The number of 16S rRNA gene per gram of feces of male recipients before CD4 + T cell transfer (NT recipients: n = 12, CPZ recipients: n = 12, Control group: n = 9). (D) Shannon diversity index of male recipients before CD4 + T cell transfer. (E) PCoA plots of both unweighted and weighted UniFrac distances of 16S rRNA gene amplicon sequences in male recipients before CD4 + T cell transfer. (F) Percent weight change (expressed as % of starting weight) of male recipients after CD4 + T cell transfer. (G) Survival rate of male recipients during the 8-week observation period. (H) Fecal lipocalin-2 (LCN-2) levels before CD4 + T cell transfer (week 0) and at 2 weeks after post-transfer (week 2) in male recipients. (I) Histological assessment of colons from the surviving male recipients at week 8 (NT group: n = 10, and CPZ group: n = 9). Representative H&E histological sections of colon are presented for NT recipients, CPZ recipients, and the control group. * p < 0.05, ** p < 0.01, and *** p < 0.001. Data represent mean ± SEM for (A)-(D), (F), (H), and (I). Female data are shown in the supplementary figure. See also Figure S2.

Article Snippet: DCs and naïve CD4 + T cells were harvested from SPLNs of mice at 7 weeks of age using a CD11c positive selection kit II (STEMCELL Technologies) and a mouse naïve CD4 + T cell Isolation kit (STEMCELL Technologies).

Techniques: Expressing, Amplification

Journal: Science Advances

Article Title: Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy

doi: 10.1126/sciadv.abq4659

Figure Lengend Snippet: ( A ) Scheme of CD8 + T cells mediated pore formation by secreting PRF to promote the perfusion of proteins into cancer cells. ( B ) CLSM observation of the RNase A–FITC uptake in the presence or absence of PRF1. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( C and D ) Flow cytometry (FCM) analysis of RNase A–FITC or GrB uptake in the presence or absence of PRF1. Data are means ± SD ( n = 3). MFI, mean fluorescence intensity. ( E ) Cell viability detection of 4T1 cells after incubation with RNase A under PRF1(−) or PRF1(+). Data are means ± SD ( n = 3). ( F ) Cell viability detection of 4T1 cells after incubation with GrB under PRF1(−) or PRF1(+). Data are means ± SD ( n = 3).

Article Snippet: After 5 days, the 4T1-specific CD8 + T cells were isolated from the spleen or LNs of the mice by MACS (Mouse CD8 + T Cell Isolation Kit, BD Biosciences) and were cultured in DMEM containing interleukin-2 (100 U/ml) with activation by the anti-CD3/CD28 beads for further use.

Techniques: Flow Cytometry, Fluorescence, Incubation

Journal: Science Advances

Article Title: Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy

doi: 10.1126/sciadv.abq4659

Figure Lengend Snippet: ( A ) Schematic illustration of the synergistic tumor cell killing effect by CD8 + T cells and RNase A/GrB. ( B ) The tumor cell killing ability of CD8 + T cells under different CD8 + T/4T1-Luc cell ratios. Data are means ± SD ( n = 3). ( C ) The cell viability assay of 4T1 cells after incubation with proteins in the presence or absence of CD8 + T cells. Data are means ± SD ( n = 3). ( D and E ) FCM analysis of RNase A–FITC + –positive cells after incubation with RNase A–FITC in the presence or absence of CD8 + T cells. Data are means ± SD ( n = 3). ( F and G ) FCM analysis of GrB + positive cells after incubation with GrB in the presence or absence of CD8 + T cells. Data are means ± SD ( n = 3).

Article Snippet: After 5 days, the 4T1-specific CD8 + T cells were isolated from the spleen or LNs of the mice by MACS (Mouse CD8 + T Cell Isolation Kit, BD Biosciences) and were cultured in DMEM containing interleukin-2 (100 U/ml) with activation by the anti-CD3/CD28 beads for further use.

Techniques: Viability Assay, Incubation

Journal: Science Advances

Article Title: Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy

doi: 10.1126/sciadv.abq4659

Figure Lengend Snippet: ( A and B ) Tumor cell apoptosis after incubation with CD8 + T cells in the presence or absence of proteins by annexin V/PI double staining. Data are means ± SD ( n = 3). ( C ) F-GSDME, N-GSDME, Cas 3 and C-Cas 3 expression after CD8 + T cells + RNase A/GrB treatment by Western blot. ( D ) Lactate dehydrogenase (LDH) release, ( E ) HMGB1 release, and ( F ) adenosine triphosphate (ATP) release after CD8 + T cells + RNase A/GrB treatment. Data are means ± SD ( n = 3). ( G ) DC maturation after incubation with the supernatant of 4T1 cells treated by CD8 + T cells + RNase A/GrB.

Article Snippet: After 5 days, the 4T1-specific CD8 + T cells were isolated from the spleen or LNs of the mice by MACS (Mouse CD8 + T Cell Isolation Kit, BD Biosciences) and were cultured in DMEM containing interleukin-2 (100 U/ml) with activation by the anti-CD3/CD28 beads for further use.

Techniques: Incubation, Double Staining, Expressing, Western Blot

Journal: Science Advances

Article Title: Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy

doi: 10.1126/sciadv.abq4659

Figure Lengend Snippet: ( A ) Schematic illustration of the animal experiment design. s.c., subcutaneous; i.t., intratumoral. ( B ) Photothermal curve of saline and IR780 after laser irradiation for 5 min (1 W/cm 2 , 808 nm). Data are means ± SD ( n = 3). ( C and D ) Tumor growth curve of 4T1 tumor model after treatment with saline, PTT, RNase A, or PTT + RNase A for three doses. Data are means ± SD ( n = 5). ( E ) Ex-tumor images and ( F ) tumor weight on day 10. Data are means ± SD ( n = 5). NS, not significant. ( G ) survival curve of 4T1-bearing mice ( n = 8). *** P < 0.001 [log-rank (Mantel-Cox) test]. ( H ) Hematoxylin and eosin (H&E) staining and tumor infiltration of CD8 + T cells analysis of the sections on day 10. Scale bars of H&E staining images, 200 μm. Scale bars of CD8 + T cell immunofluorescence (IF) images, 100 μm. ( I ) tumoral CD8 + T cells filtration analysis by FCM on day 10 (gated on CD45 + T cells).

Article Snippet: After 5 days, the 4T1-specific CD8 + T cells were isolated from the spleen or LNs of the mice by MACS (Mouse CD8 + T Cell Isolation Kit, BD Biosciences) and were cultured in DMEM containing interleukin-2 (100 U/ml) with activation by the anti-CD3/CD28 beads for further use.

Techniques: Saline, Irradiation, Staining, Immunofluorescence, Filtration

Journal: Science Advances

Article Title: Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy

doi: 10.1126/sciadv.abq4659

Figure Lengend Snippet: ( A ) Schematic illustration of the animal experiment design. ( B ) Tumor growth curve of 4T1 tumor model after treatment with saline, IRA/αPD-L1 + L, IRA gel + L, or IRA/αPD-L1 gel + L for single-dose and three times of laser irradiation (1 W/cm 2 , 808 nm, 5 min). Data are means ± SD ( n = 5). ( C ) Ex-tumor images on day 18. ( D ) Survival curve of 4T1-bearing mice after therapy ( n = 8). *** P < 0.001 [log-rank (Mantel-Cox) test]. ( E and F ) Tumoral CD8 + T cells filtration analysis by FCM on day 10 (gated on CD45 + T cells). Data are means ± SD ( n = 3). ( G and H ) CD80 + /CD86 + DC maturation ratios in the LNs on day 10 (gated on CD11c + DC cells). Data are means ± SD ( n = 3).

Article Snippet: After 5 days, the 4T1-specific CD8 + T cells were isolated from the spleen or LNs of the mice by MACS (Mouse CD8 + T Cell Isolation Kit, BD Biosciences) and were cultured in DMEM containing interleukin-2 (100 U/ml) with activation by the anti-CD3/CD28 beads for further use.

Techniques: Saline, Irradiation, Filtration