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: Nature Communications

Article Title: CTL-mediated immunotherapy can suppress SHIV rebound in ART-free macaques

doi: 10.1038/s41467-019-09725-6

Figure Lengend Snippet: Phenotypic and functional characteristics pre- and post CTL infusion. a Functional profiles of CD8 + T-cells pre- and post- CTL were evaluated by peptide antigen-specific responses and shown as the frequencies of Env-specific CD8 + T-cells producing CD107a, IFN-γ, MIP-1β and TNF-α ( p < 0.0001 using Wilcoxon signed-rank test, n = 6). b Activation profile of Env-specific, IFN-γ-producing CD8 + T-cells for both pre- and post-infusion responses, based on surface expression of CD69, HLADR, CCR5 and intracellular expression of Ki67 (* p < 0.05 and ** p < 0.01 using unpaired Students t test, Mean ± s.d. are presented in all graphs, n = 6). c Env-peptide functional profiles of CD8 + T-cells in controllers, non-controllers and untreated animals ( p < 0.0001 using Wilcoxon signed-rank test, n = 8). d Env-specific, IFN-γ + CD3 + CD8 + central memory T cells pre-and post-CTL infusion among controllers, non-controllers and untreated animals ( p = 0.038 using paired Students t-test, n = 8). e PD-1 + Ki67 + expression on Env-specific CD8 + T-cells pre- and post-CTL infusion among controllers, non-controllers and untreated animals ( p = 0.01 using paired Students t-test, n = 8). f Env-specific CD8 + T-cells expressing PD-1 + Ki67 + were positively correlated with viral load post infusion (Spearman’s correlation coefficient (R) and P values are indicated. Note that x-axis is log 10 scales, n = 8). g The SHIV-suppressive activity of circulating CD8 + T-cells from controllers and non-controllers were measured on autologous activated CD4 + T-cells infected with SHIV-162P3. SHIV suppression was reported as the Log inhibition in p27 titers in CD4 + T-cells supernatants when autologous ex vivo Env-stimulated CD8 + T-cells were added at a 1:1 ratio to the culture ( p = 0.0001 using unpaired Students t test, Mean ± s.d. is presented, n = 6). h Relationship between SHIV-suppressive activity (% inhibition) of circulating CD8 + T-cells and set point of viral load (Spearman’s correlation coefficient (R) and P values are indicated. Note that x- and y-axis are log 10 scales, n = 6)

Article Snippet: Following two rounds of peptide stimulation, the Env-specific CD8 + T cells were isolated by non-human primate CD8 + T-cell isolation kit (Miltenyi Biotec, Catalogue# 130-092-143) and expanded using the Rapid Expansion Protocol in a sterile 25-cm 2 tissue culture flasks.

Techniques: Functional Assay, Activation Assay, Expressing, Activity Assay, Infection, Inhibition, Ex Vivo

Journal: Nature Communications

Article Title: CTL-mediated immunotherapy can suppress SHIV rebound in ART-free macaques

doi: 10.1038/s41467-019-09725-6

Figure Lengend Snippet: Phenotypic and functional characterization of CTL-lines. a In vitro specific antigen recognition of CTL-lines. Data is shown as Grand median for each group. Each symbol represented one animal ( n = 6). b P27 titers in the culture supernatants of PHA-activated CD4 + T-cells infected in vitro with replicative SHIV-SF162P3, in the absence (CD4 alone) or presence of autologous CTL Lines (1:1, 2:1 and 5:1 of ET ratio). Each symbol represented one animal ( n = 6). c The capacity of CTL-lines to suppress SHIV replication in autologous CD4 + T-cells. The Log of p27 decreased when CD4 + T-cells were cocultured with CTL-lines. Each symbol represented one animal ( n = 6). d Correlation between cytotoxic capacity (% inhibition) and frequencies of Env-specific IFN- γ producing (Spearman’s correlation coefficient (R) and P values are indicated. Note that y-axis is log 10 scales, n = 6). e Memory phenotypic characterization of CTL-lines. Data is shown as Grand median for each group. Each symbol represented one animal ( n = 6). f Relationship between PBMCs Env-specific, IFN-γ + CD3 + CD8 + central memory T cells post-infusion and Env-specific memory response of CTL-lines (Spearman’s correlation coefficient (R) and P values are indicated, n = 6)

Article Snippet: Following two rounds of peptide stimulation, the Env-specific CD8 + T cells were isolated by non-human primate CD8 + T-cell isolation kit (Miltenyi Biotec, Catalogue# 130-092-143) and expanded using the Rapid Expansion Protocol in a sterile 25-cm 2 tissue culture flasks.

Techniques: Functional Assay, In Vitro, Infection, Inhibition

Journal: Nature Communications

Article Title: CTL-mediated immunotherapy can suppress SHIV rebound in ART-free macaques

doi: 10.1038/s41467-019-09725-6

Figure Lengend Snippet: Kinetics of Env-specific immune response and the correlates of virologic control. a The frequency of circulating antigen-specific CD8 + T-cells in PBMCs was measured at the indicated time points from SHIV controllers (Animals 1–3, n = 3), non-controllers (Animals 4–6, n = 3) and untreated animals (Animals 7 and 8, n = 2). y -axis, from Lift to Right, frequency of HIV CN54 Env-specific CD8 + T-cell producing IFN-γ + and frequency of SIV mac239 Gag-specific CD8 + T-cell producing IFN-γ + . wk, weeks. b Correlations between Env-specific CD8 + T-cell responses at 2 weeks after CTL infusion and set point log SHIV RNA after CTL infusion (Spearman’s correlation coefficient (R) and P values are indicated. Note that x-axis is log 10 scales, n = 8). c Depletion of CD8 + T cells in controller macaques results in the rapid induction of plasma viremia. CD8 + T-cell count and plasma viral load were shown in SHIV controller macaques following CD8 depletion (Animal 1 and 3, n = 2). x-axis, days post- ART interruption. The shaded gray indicated time points post-CD8 depletion

Article Snippet: Following two rounds of peptide stimulation, the Env-specific CD8 + T cells were isolated by non-human primate CD8 + T-cell isolation kit (Miltenyi Biotec, Catalogue# 130-092-143) and expanded using the Rapid Expansion Protocol in a sterile 25-cm 2 tissue culture flasks.

Techniques: Control, Clinical Proteomics, Cell Counting

Journal: GeroScience

Article Title: Effective virus-specific T-cell therapy for high-risk SARS-CoV-2 infections in hematopoietic stem cell transplant recipients: initial case studies and literature review

doi: 10.1007/s11357-023-00858-7

Figure Lengend Snippet: Emerging T-cell-based adoptive immunotherapy strategies to treat COVID-19 infection. Main methods: A direct selection with IFNγ CCS CliniMACS® Prodigy device. B Direct selection with CliniMACS® Plus device. C Ex vivo T-cell expansion. D Ex vivo cell expansion and CRISPR gene-modified T-cells. E T-cell receptor-engineered CD8 + T-cell. F Treg/Th2 hybrid T-cells. Abbreviations: PBMC, peripheral blood mononuclear cell; IFNγ, interferon-γ; HLA, human leukocyte antigen; Th, T helper cell; T-reg, T-regulatory cell; NCT, National Clinical Trial; DPC-OHII, Central Hospital of Southern-Pest, National Institute of Hematology and Infectious Diseases; CCS, cytokine capture system; MoAb, monoclonal antibody; ETT-TUKEB, Research Ethics Committee of the Hungarian National Medical Scientific Council; HSCT, hematopoietic stem cell transplantation; IL, interleukin; SOT, solid organ transplantation; CRISPR, RNA-controlled clustered regularly interspaced short palindromic repeats; Cas-9, caspase-9; NR3C1, nuclear receptor subfamily 3 group C member 1; PD1, programmed cell death protein1; ACE2, angiotensin-converting enzyme 2; FKBP12, FK506 binding protein 1A, 12 kDa; KO, knockout; TReAT, Tacrolimus-resistant antiviral T-cell therapy; ARDS, acute respiratory distress syndrome; TCR, T-cell receptor; NA, not available

Article Snippet: For the detection of the IFN-γ, the Rapid Cytokine Inspector (CD4-CD8 T-cell) Kit (ref: 130-097-343, Miltenyi Biotec, Bergisch Gladbach, Germany), supplemented with anti-cytokine antibody RCI Anti-IFN-c-PE (ref: 130-097-600, Miltenyi Biotec Bergisch Gladbach, Germany) was used.

Techniques: Infection, Selection, Ex Vivo, CRISPR, Modification, Transplantation Assay, Binding Assay, Knock-Out