Journal: Nature Communications

Article Title: Type I interferon drives T cell responses to amyloid beta in the central nervous system

doi: 10.1038/s41467-026-72262-6

Figure Lengend Snippet: a Representative confocal images of fresh frozen paraffin-embedded (FFPE) cortical sections from APP23-tg mice aged 8, 12, 16, 20, and 24 months. Regions of interest are shown at 20× magnification (LSM700 confocal microscope). T cells (CD3⁺) are indicated by arrows and identified based on marker expression and characteristic morphology. The strong signal observed in the DAPI channel at plaque sites reflects the known intrinsic blue fluorescence of plaque cores . b Quantification of CD3⁺ T cells normalized to total cell count across five timepoints (8, 12, 16, 20, and 24 months). Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. c Quantification of parenchymal and vascular amyloid deposits per tile scan across the same five timepoints. Two-way ANOVA with Tukey’s post hoc test was used for statistical analysis. For 2 (b) + (c) data are presented as mean ± SD. d T-cell count per amyloid plaque (parenchymal vs vascular) in APP23-tg mice aged 16, 20, and 24 months. Statistical significance was assessed using two-way ANOVA with Tukey’s post hoc test. For analyses in ( b – d ), three tile scans per section from three sections per mouse ( n = 3 mice per timepoint) were used. Each tile scan covered an area of 960.25 µm². e Fixed frozen immunofluorescence (IF) images of cortical sections from one representative 24-month-old APP23-tg mouse (top) and age-matched wild-type control (bottom). Left: tile scan at 20× magnification; right: zoomed-in area of interest. f Fixed frozen IF images of a 24-month-old APP23-tg mouse showing a plaque-rich region (top) and a plaque-devoid region (bottom). Left: tile scan at 20× magnification; right: corresponding zoom-in. For 2 (e) + (f) three tile scans per section from three sections per mouse ( n = 3 mice per timepoint) were used. g Spearman correlation analysis between T-cell frequency and amyloid plaque burden, including parenchymal (middle), vascular (right), and total plaque counts (left). The solid line shows the linear regression fit (least-squares), with curved lines indicating the 95% confidence interval of the fit. Spearman’s ρ , p -value, and R ² are indicated in each plot.

Article Snippet: CD3+ T cells were isolated from spleens of Rosa26-Cas9 mice using mouse Pan T-cell isolation kit l (Miltenyi Biotec) according the manufacturer protocol.

Techniques: Microscopy, Marker, Expressing, Fluorescence, Cell Characterization, Immunofluorescence, Control

Journal: Nature Communications

Article Title: Type I interferon drives T cell responses to amyloid beta in the central nervous system

doi: 10.1038/s41467-026-72262-6

Figure Lengend Snippet: a Volcano plot of differential gene expression (DGE) between ISG-expressing microglia and other immune subsets using the Wilcoxon rank-sum test. P -values: Benjamini-Hochberg (BH) adjusted. b Bar plot showing ISG⁺ microglia frequency within total CD45⁺ cells across cohorts ( n = 4 mice/cohort). Statistics: two-way ANOVA. Data are presented as mean + SD. c Volcano plot of DGE between ISG⁺ CD8⁺ T cells and other immune subsets using the Wilcoxon rank-sum test (BH-adjusted P -values). d Bar plot showing ISG⁺ CD8⁺ T cell frequency within CD45⁺ cells ( n = 4 mice/group). Statistics: two-way ANOVA. Data are presented as mean + SD. e Dot plot of average RNA expression and frequency for canonical ISGs across immune subsets. f Left: UMAP showing ISG CD8⁺ T-cell-specific ISG density. Right: Average RNA expression and frequency of the ISG CD8⁺ T-cell signature per mouse (M1-M4). g Spatial transcriptomic visualization of selected ISGs in the cortex of a 24-month-old APP23-tg mouse. h Spatial quantification of ISG expression relative to plaque proximity in 12–14- and 22–24-month-old APP23-tg mice ( n = 5). Regions: inside, adjacent (≤69 μm), and distant (>69 μm). Statistics: Kruskal–Wallis test with Dunn’s correction (* p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001). (i) Spatial maps of ISG⁺ CD8⁺ T cells and ISG⁺ microglia in a 24-month-old APP23-tg mouse (left: slide overview; right: zoom-in with segmented cells in white). j Spatial distance quantification between ISG expression and plaque-associated microglia (left) or T cells (right). k , l Heatmaps of inferred cell–cell communication (ligand–receptor interactions) showing interaction number (left) and strength (right) with signaling sources on the y -axis. Comparisons include ( k ) APP23-tg vs. Wt (early stage) and l early vs. late stage (APP23-tg).

Article Snippet: CD3+ T cells were isolated from spleens of Rosa26-Cas9 mice using mouse Pan T-cell isolation kit l (Miltenyi Biotec) according the manufacturer protocol.

Techniques: Gene Expression, Expressing, RNA Expression

Journal: Nature Communications

Article Title: Type I interferon drives T cell responses to amyloid beta in the central nervous system

doi: 10.1038/s41467-026-72262-6

Figure Lengend Snippet: a Differential analysis of enriched ligand–receptor interactions using ISG⁺ CD8⁺ T cells as the signaling source. Dot color: communication probability; dot size: p-value (one-sided permutation test, p < 0.05). b UMAP showing Cxcl10 and Cxcr3 RNA expression density across CD45⁺ immune cells. c Violin plots of average Cxcl10 and Cxcr3 expression across immune subsets. d Spatial RNA expression of Cxcl10 in representative regions of a 24-month-old APP23-tg mouse. e Spatial quantification of Cxcl10 relative to amyloid plaques: intraplaque, adjacent (≤69 μm), and distant (>69 μm) in 12–14 and 22–24-month-old mice ( n = 5). Statistics: Kruskal–Wallis with Dunn’s correction (* p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001). f Chord diagram of Cxcl10-Cxcr3 signaling in late-stage APP23-tg mice. Bar size indicates total signal strength. g Spatial distribution of ISG marker expression near Cxcl10⁺ plaque-associated cells in early-stage (top) and late-stage (bottom) mice. h Transwell migration assays of CXCR3 knockout (KO) vs. non-targeting (NT) control CD8⁺ T cells. Left: schematic (Created in BioRender. D170, P. (2026) https://BioRender.com/ccvdxlm ); right: bar graph of migrated cells/µl ± stimulation (CXCL10, medium, ± blocking antibody). Data: mean ± SEM; dots: biological replicates ( n = 3 per cohort). i Quantification of migrated CXCR3 KO and NT CD8⁺ T cells toward untreated (UT) or ISG-induced CD8⁺ T cell supernatants. Data: mean ± SEM; dots: biological replicates ( n = 3 per cohort). Statistics for ( h , i ): two-way ANOVA with Tukey’s correction (* p < 0.0322; ** p < 0.0021; *** p < 0.0002; **** p < 0.0001; ns = not significant).

Article Snippet: CD3+ T cells were isolated from spleens of Rosa26-Cas9 mice using mouse Pan T-cell isolation kit l (Miltenyi Biotec) according the manufacturer protocol.

Techniques: RNA Expression, Expressing, Marker, Migration, Knock-Out, Control, Blocking Assay

Journal: Nature Communications

Article Title: Type I interferon drives T cell responses to amyloid beta in the central nervous system

doi: 10.1038/s41467-026-72262-6

Figure Lengend Snippet: a Ingenuity Pathway Analysis (IPA) identifying the top upregulated immune signaling pathways in CD45⁺ immune cells from APP23-tg mice versus Wt controls. Significance was determined using a right-tailed Fisher’s exact test and is shown as –log₁₀( p -value). Pathways are ranked by significance ( p -value) and color-coded by activation z-score. b Heatmap showing normalized average RNA expression of selected T-cell activation markers across all cohorts. Color intensity indicates relative expression levels. c Heatmap showing normalized RNA expression of the same activation markers specifically within CD8⁺ T-cell subsets. d Spatial transcriptomic maps of T-cell activation markers in 24-month-old APP23-tg mice. Whole-slide views are shown on the left, with zoomed-in regions of interest on the right. e Number of T-cell clones grouped by clone size (visualized by color gradient) in identified T cell subsets between all cohorts. f Z-score depicting upregulated genes from differential expression analysis in non-expanding clones (absolute clone numbers ≥ 5) and highly expanding clones (absolute clone numbers >200). g Left: UMAP plot of CD45⁺ immune cells overlaid with an expression density gradient of a defined T-cell exhaustion gene signature (Pdcd1, Lag3, Tox). Right: Average RNA expression and expression frequency of the exhaustion signature across all cohorts. h Violin plots comparing average expression levels of exhaustion-associated genes between late-stage APP23-tg and wild-type mice. Statistical significance was assessed using the Wilcoxon rank-sum test. i Spatial transcriptomic visualization of Pdcd1 (PD-1) expression in two selected regions of interest from 24-month-old APP23-tg mice. j Quantification of Pdcd1 RNA expression in relation to plaque proximity: intraplaque, plaque-adjacent (≤69 μm from plaque boundary), and plaque-distant (>69 μm). Analysis was performed in 22–24-month-old APP23-tg mice. Statistical testing was performed using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons; significance levels are shown as *= p < 0.0332, **= p < 0.0021,***= p < 0.0002,****= p < 0.0001) ( k ) Spatial distribution of Pdcd1 expression relative to T-cell markers in a 24-month-old APP23-tg mouse, visualized by density plots highlighting proximity to T-cell-enriched regions.

Article Snippet: CD3+ T cells were isolated from spleens of Rosa26-Cas9 mice using mouse Pan T-cell isolation kit l (Miltenyi Biotec) according the manufacturer protocol.

Techniques: Protein-Protein interactions, Activation Assay, RNA Expression, Expressing, Clone Assay, Quantitative Proteomics

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function of B cells is closely associated with their FA metabolism in metastatic OvCa. A GSVA pathway enrichment analysis of APC function low and APC function high B cells in patients with metastatic OvCa ( GSE235951 , GSE147082 and GSE154600 dataset in the GEO database, n = 13). B Representative images of the IHC staining of TLS structure (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC), CD80, and CD86, respectively, in the area adjacent to or away from tumor or adipose tissues in clinical HGSOC specimens (n = 20 for TLS and n = 5 for CD80 or CD86). Red dashed line area: TLS structure. Magnification × 200. C Representative image of lymphoid aggregates (white dashed line area) by immunofluorescent staining in ascites of OvCa mice with 3 w and 6 w in tumor-bearing mice. D Mean fluorescence intensity of CD80, CD86, CD83, and MHC class II molecules in ascitic CD19 + B cells of OvCa mice were detected by flow cytometry. E Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. F Protein expression of β-actin and FA metabolic proteins in ascitic B cells in OvCa mice was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of FA metabolic proteins. G Mean fluorescence intensity of Bodipy C16 in ascitic B cells of OvCa mice detected by flow cytometry. H Expression of A-CoA in ascitic B cells of OvCa mice detected by ELISA. I Expression of ATP in ascitic B cells of OvCa mice detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in ascitic B cells of OvCa mice detected by flow cytometry. I Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. Data are presented as the mean ± SD of three independent experiments. FA, fatty acid; TLS, tertiary lymphoid structure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Immunohistochemistry, Staining, Fluorescence, Flow Cytometry, Comparison, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function and resulting anticancer immunity of B cells can be enhanced by oleic acid (OA) via reprogramming FA metabolism in vitro. A Mean fluorescence intensity of CD80 and MHC Class II molecules in CD19 + B cells from peripheral blood of healthy volunteers (n = 3) treated with OA and PA (both 150 μM), respectively. B Mean fluorescence intensity of CD80, CD86, CD83, MHC Class II molecules, and Ki67 in splenic CD19 + B cells of WT mice treated with 150 μM OA. C Mean fluorescence intensity of CD80, CD86, CD83, and MHC Class II molecules in ascitic CD19 + B cells from 3 w OvCa-bearing mice when treated with 150 μM OA. D Analysis of FA metabolism-related signaling pathways based on RNA-seq results. GSEA was used to analysised the FA metabolic pathways. E Comparison of mRNA levels of main FA metabolic genes in ascitic B cells from 3 w tumor-bearing mice when treated with 150 μM OA. The relative expression of each gene was calculated using β -actin as the internal reference. F Experimental scheme to detect the influence of inhibiting OA uptake on ascitic B cells. G Comparison of protein expressions of main FA metabolic molecules in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA. β -Actin was used as the internal control to calculate the relative expression level of the main FA metabolic molecules. H Mean fluorescence intensity of Bodipy C16 in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. I The expression of A-CoA, ATP and the FAO activity in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS treated with OA was detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. K Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was detected by flow cytometry. L Mean fluorescence intensity of Bodipy C16, CD80, CD86 and CD83 in FABP4-knockdown CD19 + B cells, which are from the ascites of 3 w OvCa-bearing mice, treated with OA, was detected by flow cytometry. PBMC, peripheral blood mononuclear cell; SP, spleen; AS, Ascites; OA, oleic acid; PA, palmitic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Protein-Protein interactions, RNA Sequencing, Comparison, Expressing, Control, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: Reprogramming of the FA metabolism of B cells by OA can improve anticancer immunity in vitro. A Experimental scheme to detect the in vitro effects of ascitic B cells treated with OA on anticancer immunity. B Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells after pulsed with ID8-Luc-cell-prepared antigenic peptides and pretreated with BMS and treated with OA, T cells and ID8-Luc. C Levels of IFN- γ , GZMB, and TNF-α in the supernatant of the coculture system constructed by ID8-Luc cells and 3 w OvCa-bearing mouse ascitic B and splenic T cells were detected by ELISA. D Cytotoxicity of T cells in the coculture system mentioned above was detected by luciferase assay. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Construct, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The enhanced APC function of B cells by OA in vitro is achieved through H3K27ac-mediated upregulation of PPAR γ expression. A Protein expression of β -actin and H3K27ac in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of H3K27ac. B The enrichment percentage of H3K27ac at the PPAR γ , CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. C Correlation analysis of PPAR γ and APC function-related genes (CD80, CD86, CD83, MHC II) in B cells in OvCa patients in the TCGA database (n = 426). D Analysis of protein interaction among FABP4, PPAR γ , CD80, CD86, and CD83. E The enrichment percentage of PPAR γ at the CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. F Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS, GW9662/Trog, and treated with OA was detected by flow cytometry. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Expressing, Control, ChIP-sequencing, Fluorescence, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The combination of adoptive immunotherapy with APC function-enhanced B cells and LDC improved anticancer immunity in metastatic OvCa mice. A Experimental scheme to evaluate the effects of combining adoptive immunotherapy with APC function-enhanced B cells and LDC in metastatic OvCa mice. B Representative BLI images and comparison of OvCa progression in each group (n = 3). C Representative images ( a ) and the number of tumor nodules ( b ) in the abdominal wall of mice in each group (n = 3). Areas marked by yellow dashed lines: Representative tumor nodules in the abdominal wall. D (a) Representative images of lymphoid aggregates (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC) in the ascitic cells of mice in each group. (b) Comparison of the amount of CD3 + T, CD8 + T, Ki67 + CD3 + T, Ki67 + CD8 + T, CD19 + B, CD80 + B, CD86 + B, and lymphoid aggregates respectively in the ascitic cells of mice in each group (n = 3) in frozen sections detected by immunofluorescence. E Comparison of the proportion of CD3 + T, CD8 + T, CD19 + B, CD19 − CD138 + B cells, mean fluorescence intensity of Ki67 in CD3 + T and CD8 + T, and mean fluorescence intensity of CD80, CD86, CD83, MHC II in CD19 + B, respectively, in ascitic cells of each group of mice (n = 3) detected by flow cytometry. F Mean fluorescence intensity values of IL-2, IFN- γ , GZMB, and CTLA-4 in CD8 + T cells, respectively. G Kaplan Meier analysis of survival time of each group of mice (n = 7). LDC, low-dose chemotherapy; B OA , B cells that have been treated with OA for 24h; L-DDP, low-dose DDP (1 mg/kg); H-DDP, high-dose DDP (2 mg/kg); BLI, bioluminescence imaging. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Comparison, Immunofluorescence, Fluorescence, Flow Cytometry, Imaging

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Peritoneal CD5 + CD19 + CD43 + B1a cells differentiate into TCR + CD19 + DP cells (A) Total PECs from muMT mice were cultured in vitro , and the frequencies of TCR + CD19 + DP cells were assessed on days 0 and 5. (B) TCR + CD19 + DP cells were evaluated in B1 (CD19 + CD43 + ) and B2 (CD19 + CD43 − ) subsets in total PECs. (C) Time-course analysis of TCR + CD19 + DP cells from B1 (CD43 + ) and B2 (CD43 − ) subsets during in vitro culture on days 0, 3, 5, and 7. (D) FACS-sorted B1 and B2 cells were cultured separately, and the frequencies of TCR + CD19 + DP cells were analyzed on day 5. (E) Gating strategy for B cell subset analysis: total PECs initially gated on CD5 + CD19 + (red rectangle) and CD5 − CD19 + populations (blue), which were further subdivided into B1a (CD5 + CD19 + CD43 + ; green), B1b (CD5 − CD19 + CD43 + ; purple), and B2 (CD5 − CD19 + CD43 − ; orange) subsets. The frequencies of DP cells were quantified within each subset. (F) MACS-purified B1a cells were cultured in vitro , and the frequency of TCR + CD19 + DP cells was analyzed on days 0, 3, and 5.

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Purification

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Adoptively transferred B1a cells differentiate into DP cells and extrathymic T cell lineages (A) Two weeks after the adoptive transfer of B1a cells into muMT mice, the frequencies of TCR + CD19 + DP cells and B1a cells were assessed in the PEC, spleen, liver, small intestine, and thymus by flow cytometry. (B) Four weeks after the adoptive transfer of CD45.2 + B1a cells into CD45.1 + recipients, flow cytometric analysis was performed to evaluate the frequencies of CD45.2 + extrathymic T cells, DP cells, and B1a cells in the indicated tissues.

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Flow Cytometry

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Cooperative regulation of B1a cell-derived T cell differentiation by IL-2 family cytokines via STAT activation, Notch signaling, and Thy1.2 expression (A) FACS-sorted Thy1.2 + or Thy1.2 − DP cells isolated from GFP + mice were co-cultured with WT total PECs for 7 days. CD19 and TCR expression in GFP + cells was analyzed, and Thy1.2 expression of GFP + DP cell-derived T cells is represented as histograms. (B) GFP + B1a cells were co-cultured with either WT PEC-derived stromal cells or OP9-DLL1 cells for 14 days, and their differentiation into T cells (red polygon) within the GFP + population was evaluated based on CD19 and TCR expression. (C) WT B1a cells were co-cultured with OP9-DLL1 cells in the presence of 5 ng/mL concentration of IL-2, IL-4, IL-7, or IL-21 for 14 days. Their differentiation into DP cells and extrathymic T cells was analyzed. (D) B1a cells were co-cultured with or without OP9-DLL1 and treated with a cytokine cocktail (5 ng/mL each of IL-2, IL-4, IL-7, and IL-21) for 14 days. (E) B1a cells were stimulated with IL-2 family cytokines, and the phosphorylated and total forms of STAT1, STAT3, STAT5, ERK, NF-κB, AKT, and p38 were analyzed by western blot, with β-actin used as a loading control (top). Phosphorylated STAT1 and STAT3 were quantified using MultiGauge software and are expressed as fold changes after normalization to total STAT1 and total STAT3 levels, respectively (bottom). (F) Nuclear extracts from IL-2 family cytokine-stimulated B1a cells were analyzed by EMSA, using a biotin-labeled STAT-binding probe (left). Band intensities were quantified using MultiGauge software and are shown as bar graphs (right).

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Differentiation, Activation Assay, Expressing, Isolation, Cell Culture, Concentration Assay, Western Blot, Control, Software, Labeling, Binding Assay

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Surface marker profiling and t-SNE analysis reveal transitional DP subsets during B1a-derived extrathymic T cell differentiation (A) Total PECs were analyzed by flow cytometry, and CD43 + cells were initially gated. Within the gated population, CD5 + CD19 + B1a cells were further subdivided into two subsets: CD5 dim B1a (blue polygon) and CD5 br B1a (red polygon). TCR, Thy1.2, and CD3 expression levels were compared between the subsets by using histogram overlays. (B) CD43 + cells gated from total PECs were separated into CD3 − and CD3 + populations, which were subsequently analyzed for TCR and CD19 expression to identify DP cells. (C) t-SNE analysis of total PECs revealed distinct clusters based on CD19 and TCR expression. Populations were visualized as CD19 + (purple), TCR + (orange), and TCR + CD19 + DP (cyan) cells. Within αβ TCR + DP (αβ-TCR + CD19 + ) cells, three clusters (G1–G3) were identified: G1 (green), G2 (red), and G3 (blue), whereas γδ TCR + DP (γδ-TCR + CD19 + ) cells were classified as G4. G1–G4 clusters are displayed on a CD19 versus TCR dot plot. (D) TCR + CD19 + DP cells (gated, Red rectangle) were further analyzed by t-SNE, and the relative expression of CD43, CD5, CD3, and Thy1.2 was visualized using color-coded intensity maps. Among αβ TCR + DP cells, two phenotypically distinct subsets were identified: CD43 br CD5 br CD3 + Thy1.2 + cells (black arrows) and CD43 dim CD5 dim CD3 − Thy1.2 − cells. γδ TCR + DP cells constituted a minor CD43 br CD5 br CD3 + Thy1.2 + population (white). (E) t-SNE-defined G1, G2, and G3 DP subsets were isolated and analyzed for proliferative capacity and functional T cell properties. Ki-67 expression was assessed after 48 h of in vitro culture. T cell-associated cytokine (IFN-γ, IL-4, and IL-17) production and Foxp3 expression were analyzed by intracellular staining following PMA and ionomycin stimulation.

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Marker, Derivative Assay, Cell Differentiation, Flow Cytometry, Expressing, Isolation, Functional Assay, In Vitro, Staining

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Sequential induction of Thy1.2 expression during B1a-derived extrathymic T cell and thymocyte differentiation (A) Flow cytometric analysis was performed to evaluate Thy1.2 expression in distinct cell populations: T cells (TCR + CD19 − ; blue), DP cells (TCR + CD19 + ; orange), TCR high DP cells (TCR high CD19 + ; red), and B1a cells (TCR − CD19 + ; green). Representative histograms show Thy1.2 expression in each population, and overlay histograms are used to compare MFI values. (B) Thymic T cell subsets were classified as DN (CD4 − CD8 − ; cyan), DP (CD4 + CD8 + ; blue), CD4 + single-positive (SP) (CD4 + CD8 − ; purple), and CD8 + SP (CD4 − CD8 + ; pink) cells. DN subsets were further subdivided into DN1 (CD25 − CD44 + ; red), DN2 (CD25 + CD44 + ; orange), DN3 (CD25 + CD44 − ; green), and DN4 (CD25 − CD44 − ; sky blue) subsets. Thy1.2 expression within thymocyte subsets was assessed by flow cytometry and compared using histogram overlays.

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Flow Cytometry

Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: B1a-derived extrathymic T cells undergo lineage reprogramming associated with TCR rearrangement and exhibit CD8 lineage bias and functional effector capabilities (A) Expression of recombination-activating genes Rag1 and Rag2 was analyzed by quantitative PCR in MACS-sorted B1a cells on day 0 and on days 3, 5, and 9 of culture. Relative mRNA expression levels are shown normalized to day 0. (B) Relative levels of T cell receptor excision circles (TRECs) in peritoneal B1a cells after 5 days of in vitro culture with or without a cytokine cocktail (IL-2, IL-4, IL-7, and IL-21; 5 ng/mL each) and 5% (v/v) supernatant from OP9-DLL1 cultures. TREC levels were normalized to MyoD1 . (C) B1a cells were differentiated under the same conditions as in A, and B1a-derived DP cells (blue) and B1a-derived T cells (red) were identified by flow cytometry (left). Surface IgM expression of the gated populations was then analyzed and is displayed as overlaid histograms (right), with isotype control staining shown in orange. (D) Phenotypic comparison of B1a-derived T cells and splenic T cells. Surface expressions of CD5, IgM, CD1d, Thy1.2, CD3, CD4, and CD8 were analyzed by flow cytometry in B1a-derived extrathymic T cells and splenic T cells. Representative histogram overlays compare the expression profiles between B1a-derived T cells and splenocytes. (E) Cytokine production by B1a-derived extrathymic T cells and splenic T cells following stimulation with PMA and ionomycin was quantified by ELISA. Levels of IFN-γ, IL-4, IL-17, and IL-10 were compared between the two groups and are presented as bar graphs. (F) Cytotoxic activity of B1a-derived extrathymic T cells and splenic T cells was evaluated using a lactate dehydrogenase (LDH) release assay. Effector cells were co-cultured with MC38 target cells at various effector-to-target (E:T) ratios, and specific lysis was calculated. Data are presented as the mean ± standard error of the mean (SEM) and represent three independent experiments. Statistical significance was determined by unpaired Student’s t tests or one-way ANOVA. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: B1a cells were purified from total PECs using the B1a Cell Isolation Kit (130-097-413; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Functional Assay, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Flow Cytometry, Control, Staining, Comparison, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Cell Culture, Lysis