Journal: Microbiology Spectrum

Article Title: Attenuation of Avian Flavivirus by Rewiring the Leucine and Serine Codons of Its E-NS1 Protein toward Stop Mutation To Redirect Virus Evolution

doi: 10.1128/spectrum.02921-22

Figure Lengend Snippet: Immunogenicity characteristics of CQW1-OTS in 25-day-old ducks. (A) Survival rate of duck postinfection. The statistical significance of survival was analyzed using a survival curve and the log-rank (Mantel-Cox) test, with significance defined. Ns, not significant. n = 5. (B) Viral copies (at 3 and 5 dpi) in the heart, liver, spleen, lung, kidney, brain, and thymus of 25-day-old ducks were detected by RT-qPCR. (C) Relative mRNA expression of IFN-α, IFN-β, and IL-1β in duck spleen at 5 dpi. (D) Peripheral T-lymphocyte (from infected ducks) proliferation activated by TMUV infection. (E) Serum (14 dpi) levels of IFN-γ and IL-4 were determined using ELISA. (F) Neutralization antibody levels in the serum were determined with PRNT. Statistical significance was determined using an unpaired t test. Ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Lymphocytes were separated from blood using a peripheral blood lymphocyte separation kit (Solarbio, Beijing, China) following the manufacturer’s recommendations.

Techniques: Immunopeptidomics, Quantitative RT-PCR, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Neutralization

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: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Diagram depicting the culture model used to generate time course samples from healthy human CD4+ memory T cells. Cells were stimulated with CD3/CD28 Dynabeads for 12 days, with fresh beads plus full media change every other day (fresh media alone added on alternate days). Samples were collected on days highlighted in bold. (B) Multiomics workflow (see for details). Experiments were performed in biological triplicate, with proteomics, phosphoproteomics, and metabolomics done on same samples. (C) ELISA-based quantification of secreted cytokines (IL-2, IFN-γ, TNF-α) on days 0, 6, 10, and 12, 3 replicates per time point. (D) Cell counts were taken each time point, and average cell division compared to previous time point calculated. Cell viability remained stable throughout at ~75%–85%. (E) Flow cytometric analysis of inhibitory receptors PD-1, TIM-3, and TIGIT at resting, day 6, and day 12 time points. Data normalized to mode for accurate population comparisons to account for differences in number of cells analyzed. The data underlying can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: Mean fluorescence intensity of PD-1 in CD4+ T cells from both HIV+ and HIV- patients (grouped due to observing same effect in both) for CD4+ cells that are CD276+ or FLT-1+. The data underlying this figure can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Fluorescence, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Causal network analysis leveraging annotated interactions (transcription factor-target, kinase-substrate, phosphatase-substrate) as a framework to evaluate protein expression changes (late exhaustion (day 12) versus resting, taking into account previous time point comparisons) to predict key nodes (regulators) impacting most changes seen across the data set. (B) Pruned version of a large subnetwork with p300 (black outline) at main hub. Orange circles mark proteins of interest mentioned in main text. (C) Depiction of experimental design of p300 inhibition studies. (D) Flow cytometric analysis of PD-1 and TIM-3 showing shifts in fluorescent intensity of cell populations in treated versus untreated (cell number normalized to mode). Cell numbers shown for each plot are 20,312 for p300 treated and 30,438 for DMSO, which corresponds to all CD4+ T cells from the sample (excluding doublets and debris, see for gating), corresponding to >95% of sample. (E) Bar chart of mean fluorescent intensity showing significant changes ( T test) in PD-1 and TIM-3 during p300 inhibition versus controls (antigen removed for 2 days, or treatment with T-cell activation inhibitor Dasatinib); *, p -value <0.05, ***, p -value <0.005. (F) Heatmap showing recruitment of cofactors (p300, class I HDACs, KAT2B) to transcription factor family DNA motifs as measured using CASCADE protein-binding microarrays. The data underlying this figure can be found in and Data (A, B), supplemental flow cytometry files uploaded to flowrepositroy.org (D), and the (E, F). Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Expressing, Inhibition, Activation Assay, Protein Binding, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: UMAPs of CD4+ subtypes and CD8+ T exhausted tumor lymphocytes showing gene expression of (A) exhaustion-associated genes, and (B) gene expression of proteins identified in the model presented by Lawton and colleagues. UMAPs made using Single Cell Portal, Tarhan and colleagues. The data from this figure and tool used to make it can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP1039/a-single-cell-and-spatially-resolved-atlas-of-human-breast-cancers . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Gene Expression