dnam 1 fc  (R&D Systems)

Journal: bioRxiv

Article Title: Using peptide-exchange systems to interrogate peptide-specific KIR binding to HLA Class I

doi: 10.64898/2026.03.03.708729

Figure Lengend Snippet: (A) Display of ULBP1 and CD155 on CombiCells detected by mAbs and NKG2D-Fc, and DNAM-1-Fc, respectively. (B&C) KIR2DL1+ and KIR2DL1-NK-cell degranulation (CD107a upregulation) in response to ULBP1, CD155, and HLA-C*05:01 displayed on CombiCells. (C) Three types of HLA-C*05:01 were tested, all containing P2 (IIDKSGSTV); wild-type, open and dipeptide exchanged. Data from three independent experiments with NK cells from separate donors are shown.

Article Snippet: For binding assays, APC-conjugated Fc fusion protein (NKG2D-Fc (R&D Systems, Cat. No: 1299-NK) or DNAM-1-Fc (R&D Systems, Cat. No: # 666-DN) or KIR-Fc (R&D Systems, KIR2DL1-Fc (Cat. No: 1844-KR-050), KIR2DL3-Fc (Cat. No: 2014-KR-050), KIR2DS4-Fc (Cat. No: 1847-KR-050) (3.6 μg mL -1 ) was added at 25 μL per well and incubated for 30 min at 4 °C in the dark.

Techniques:

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: The proportion of CD226 + B cells is correlated with disease progression in T1D patients. (a and b) Representative flow cytometry plots (a) and columnar scatter plots (b) displaying the expression of CD226 on B cells in HC (n = 40), T2D (n = 20), LADA (n = 20), and T1D (n = 40). One-way analysis of variance (ANOVA) followed by adjustments was used for multiple comparisons. Each point represents an individual. Horizontal bars represent the mean ± SEM. (c–f) Columnar scatter plots displaying the expression of CD226 on naive B cells (c), IgD + -memory B cells (d), switched memory (SM) B cells (e), and plasmablasts (f) in HC (n = 40), T2D (n = 20), LADA (n = 20), and T1D (n = 40), as assessed by flow cytometric analysis. One-way ANOVA followed by adjustments was used for multiple comparisons. Each point represents an individual. Horizontal bars represent the mean ± SEM. (g–j) Relationships between CD226 + B cells and clinical parameters from T1D (n = 40) and LADA (n = 20). Correlations between the percentage of CD226 + B cells and FBG (g), FCP (h) in T1D patients. Correlations between the percentage of CD226 + B cells and FBG (i), FCP (j) in LADA patients. (k and l) Relationships between CD226 + B cells and clinical parameters from T2D (n = 20) and HC (n = 40). Correlations between the percentage of CD226 + B cells and FBG in T2D (k) patients and HC (l). Pearson or Spearman’s rank correlation was used for correlation analyses. Linear regression is shown with 95% CIs (dotted area). (m and n) ROC curves for CD226 + B cells were used to predict T1D (m) and LADA (n). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. Abbreviations: HC, healthy controls; T2D, type 2 diabetes; LADA, latent autoimmune diabetes in adults; T1D, type 1 diabetes; SEM, standard error of the mean; IgD + -m B cells, IgD + -memory B cells; SM B cells, switched memory B cells; FBG, fasting blood glucose; FCP, fasting C-peptide; ROC, receiver operating characteristic curve; AUC, area under the curve.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Biomarker Discovery, Flow Cytometry, Expressing

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: Functional and metabolic analysis of CD226 + B cells and CD226 − B cells in T1D patients. (a) Peripheral blood B cells were categorised into CD226 + and CD226 − B cells on the basis of CD226 expression in T1D. (b–d) Representative flow cytometry plots and scatter plots for paired t-tests of CD69 (n = 11), CD86 (n = 11) (b), HLA-DR (n = 11) (c), TNF-α, IFN-γ, IL-6, and IL-12 (n = 5) (d) expression in CD226 + and CD226 − B cells from T1D patients. (e) Representative flow cytometry plots and scatter plots for paired t-tests of TNF-α and IL-6 expression in B cells between control group and anti-CD226 group (n = 6). (f) Representative flow cytometry plots and scatter plots for paired t-tests of 2-NBDG, BODIPY, and MITO expression in CD226 + and CD226 − B cells from T1D patients (n = 5). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001. Abbreviations: T1D, type 1 diabetes; SSC-H, side scatter height; 2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; BODIPY, boron-dipyrromethene; MITO, MitoTracker® Deep Red FM.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Functional Assay, Expressing, Flow Cytometry, Control

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: Functional analysis of CD226 + B cells and CD226 − B cells in NOD mice. (a) The expression levels of CD226 on B cells in PLN at 4, 8, and 12 weeks of age in NOD mice and 10–12 weeks of age in C57BL/6 mice (n = 5). One-way ANOVA followed by adjustments was used for multiple comparisons. (b) B cells were categorised into CD226 + and CD226 − B cells on the basis of CD226 expression in the spleen and PLN of NOD mice. (c) Representative flow cytometry plots and scatter plots for paired t-tests of CD80, CD86, MHC II, and Ki67 expression in CD226 + and CD226 − B cells from the spleen and PLN of NOD mice (n = 5). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. Abbreviations: NOD, non-obese diabetic; SSC-H, side scatter height; PLN, pancreatic lymph node.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Functional Assay, Expressing, Flow Cytometry

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: NF-κB signalling drives the heightened inflammatory response in CD226 + B cells. (a) Volcano plot illustrating the differential expression of genes between CD226 + B cells and CD226 − B cells in NOD mice (n = 5). The adjusted P value of each gene was determined by DESeq2 with Benjamini-Hochberg false discovery rate (FDR) correction. Genes meeting the criteria of adjusted P < 0.05 and fold change > 2.0 were considered significant. (b and c) Functional annotation analysis of DEGs using GO (b) and KEGG (c) analyses. Statistical disparities between enriched terms and pathways were adjusted utilising Bonferroni’s test, applying FDR adjusted P-value ≤ 0.05 to ensure accuracy. (d) GSEA for inflammatory response, positive regulation of cytokine production, positive regulation of cell cycle and NF-kappa B signalling pathway associated genes in CD226 + B cells versus CD226 − B cells. (e) Quantitative real-time PCR (qPCR) analysis for NF-κB target genes expression in B cells from T1D patients and HC (n = 11). Student’s t-test was used for comparing two groups. (f) Representative flow cytometry plots and scatter plots for paired t-tests demonstrating the inhibition of CD226 expression in B cells upon QNZ treatment (n = 6). (g and h) Representative flow cytometry plots and scatter plots for paired t-tests demonstrating the inhibition of activation (g) and inflammatory cytokine production (h) in CD226 + B cells upon QNZ treatment (n = 6). (i) Representative flow cytometry plots and scatter plots for paired t-tests demonstrating the inhibition of CD226 expression in B cells upon BAY 11-7082 treatment (n = 5). (j and k) Representative flow cytometry plots and scatter plots for paired t-tests demonstrating the inhibition of activation (j) and inflammatory cytokine production (k) in CD226 + B cells upon BAY 11-7082 treatment (n = 5). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. Abbreviations: DEGs, differential express genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; NES, normalised enrichment score; HC, healthy controls; T1D, type 1 diabetes; ns, not significant; SSC-H, side scatter height.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Quantitative Proteomics, Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Inhibition, Activation Assay

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: IL-15 promotes the generation and activation of CD226 + B cells. (a) Forecasting the regulator of the CD226 gene. (b) Representative flow cytometry plots and line charts of CD226 expression on B cells isolated from T1D patients following 48-h culture with either medium control, 20 ng/ml IL-21, 20 ng/ml IL-4, or 20 ng/ml IL-15R alpha & IL-15 (n = 6). One-way repeated measures ANOVA followed by adjustments was used for multiple comparisons. Lines connect the same sample. (c–f) Representative flow cytometry plots and line charts of CD69 (c), CD86 (d), TNF-α (e), and IL-6 (f) expression in CD226 + B cells from T1D patients after 48-h indicated stimulation (n = 6). One-way repeated measures ANOVA followed by adjustments was used for multiple comparisons. Lines connect the same sample. (g) The levels of IL-15 in the serum of HC and T1D patients (n = 8). Student’s t-test was used for comparing two groups. (h) The expression of IL-15R (CD122 and CD132) was compared between CD226 + B cells and CD226 − B cells in T1D patients (n = 10). Paired t-test was used for comparing two groups. (i) The expression of IL-15R (CD122 and CD132) was compared between HC and T1D patient B cells (n = 12). Student’s t-test was used for comparing two groups. ∗P < 0.05. ∗∗P < 0.01. Abbreviations: SSC-H, side scatter height; HC, healthy controls; T1D, type 1 diabetes.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Activation Assay, Flow Cytometry, Expressing, Isolation, Control

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: Monocyte/macrophage-derived IL-15 promotes the generation and activation of CD226 + B cells. (a) IL-15 expression in peripheral blood cell types. The relative expression of IL-15 across immune cell types was analysed using the publicly available dataset GEO: GSE107019 as referenced ( https://doi.org/10.1016/j.celrep.2019.01.041 ), and visualised in a bar chart. (b) IL-15 secretion of monocytes and CD8 + T cells in T1D patients and HC, as assessed by ELISA (n = 5). Student’s t-test was used for comparing two groups. (c) Representative flow cytometry plots and bar graphs of IL-15 expression in monocytes from T1D patients and HC (n = 7). Student’s t-test was used for comparing two groups. (d) Representative flow cytometry plots and bar graphs of IL-15 expression by macrophages in PLN from NOD mice and C57BL/6 mice (n = 6). Student’s t-test was used for comparing two groups. (e) Representative flow cytometry plots and line charts of CD226 expression on B cells isolated from T1D patients following 48-h culture with either medium control, 20 ng/ml IL-15R alpha & IL-15, 20 nM QNZ, or 20 ng/ml IL-15R alpha & IL-15 and 20 nM QNZ (n = 6). One-way repeated measures ANOVA followed by adjustments was used for multiple comparisons. Lines connect the same sample. (f and g) Representative flow cytometry plots and line charts of CD69 (f) and TNF-α (g) expression in CD226 + B cells from T1D patients after 48-h indicated stimulation (n = 6). One-way repeated measures ANOVA followed by adjustments was used for multiple comparisons. Lines connect the same sample. ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001. Abbreviations: HC, healthy controls; T1D, type 1 diabetes; SSC-H, side scatter height; NOD, non-obese diabetic.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Derivative Assay, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Control

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: Combination therapy with anti-CD3 and anti-CD132 synergistically reverts the type 1 diabetes in NOD mice. (a) Experimental design for the four indicated groups. (b) Diabetes remission rates among the four indicated groups (n = 15). Diabetes remission rates were compared by cox proportional hazards regression test. (c) Blood glucose levels among the four indicated groups (n = 15). Blood glucose levels were compared by two-way ANOVA followed by Tukey’s multiple comparison test. (d) Histopathological H&E staining images of pancreatic islets and insulitis scores among the four indicated groups. Scale bar: 50 μm. Insulitis scores were compared by the chi-square test. (e) Representative flow cytometry plots and bar graphs of the proportions of CD226 + B cells in the PLN among the four indicated groups (n = 5). One-way ANOVA followed by adjustments was used for multiple comparisons. (f) Representative flow cytometry plots and bar graphs of the expression of CD80 in B cells in the PLN among the four indicated groups (n = 5). One-way ANOVA followed by adjustments was used for multiple comparisons. (g) Representative flow cytometry plots and bar graphs of the expression of CD69 in CD4 + T cells in the PLN among the four indicated groups (n = 5). One-way ANOVA followed by adjustments was used for multiple comparisons. (h) Representative flow cytometry plots and bar graphs of the expression of CD69 in CD8 + T cells in the PLN among the four indicated groups (n = 5). One-way ANOVA followed by adjustments was used for multiple comparisons. ∗P < 0.05. ∗∗P < 0.01. ∗∗∗∗P < 0.0001. Abbreviations: NOD, non-obese diabetic; CYC, cyclophosphamide; i.p., intraperitoneally; Anti-CD3, anti-CD3 monoclonal antibody; Anti-CD132, anti-CD132 monoclonal antibody; H&E, haematoxylin and eosin; SSC-H, side scatter height.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Comparison, Staining, Flow Cytometry, Expressing

Journal: eBioMedicine

Article Title: Monocyte/macrophage-derived interleukin-15 mediates the pro-inflammatory phenotype of CD226 + B cells in type 1 diabetes

doi: 10.1016/j.ebiom.2025.105946

Figure Lengend Snippet: Working model of the monocyte/macrophage-IL-15-CD226 + B cell axis in the immunopathogenesis of T1D. There is increased secretion of IL-15 by monocytes or macrophages in T1D. IL-15 secreted by monocytes or macrophages binds to IL-15Rβ/γc on B cells. This further enhances the expression of CD226 on B cell surfaces and increases their pro-inflammatory cytokine production, activation, and proliferative capacity. The NF-κB signalling pathway promotes the generation and pro-inflammatory responses of CD226 + B cells. Targeting the IL-15-IL-15R signalling pathway reverses inflammatory phenotypes, mitigating disease severity, thereby representing a promising therapeutic strategy for preventing T1D. Abbreviations: T1D, type 1 diabetes; GLUT1, glucose transporter type 1.

Article Snippet: We cultured PBMCs from T1D donors for 2 days using monoclonal anti-human CD226 antibody (10 μg/mL; R&D Systems, Cat#MAB666, RRID:AB_2072624) or mouse IgG1 isotype control (10 μg/mL; R&D Systems, Cat#MAB002, RRID:AB_357344) to inhibit CD226 on B cells in vitro.

Techniques: Expressing, Activation Assay