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

Journal: Acta biomaterialia

Article Title: Early Changes in Cartilage Pericellular Matrix Micromechanobiology Portend the Onset of Post-Traumatic Osteoarthritis

doi: 10.1016/j.actbio.2020.05.005

Figure Lengend Snippet: a) Schematic illustration of cartilage matrix molecular constituents highlighting the distinctive structure and composition of the PCM in comparison to the T/IT-ECM, as well as the pivotal role of the PCM in mediating cell-matrix interactions. The schematic is inspired by Ref. [7, 10]. b) Representative immunofluorescence images of matrix molecules that are localized and/or preferentially distributed in the PCM at 8 weeks post-surgery, including collagen VI, perlecan, biglycan (green) and aggrecan (red; blue: DAPI). Internal negative controls are shown in the right panel.

Article Snippet: Sections were then incubated with secondary antibodies directed against the primary antibodies for collagen type VI (28903, Rockland, Pottstown, PA, 1:1,000), perlecan (A11006, Invitrogen, Carlsbad, CA, 1:1,000), biglycan and aggrecan (RE234142, Invitrogen, 1:1,000), respectively, for 1hr at room temperature.

Techniques: Comparison, Immunofluorescence