hbegf  (Bioss)

Journal: iScience

Article Title: Serial intravital microscopy reveals temporal dynamics of autoreactive germinal centers in the spleen

doi: 10.1016/j.isci.2026.115340

Figure Lengend Snippet: An AIW to track FDC network dynamics in the spleen over 2 weeks using serial intravital microscopy (A) Graphical overview of surgery steps of AIW (abdominal imaging window) implantation: 1. surgical landmarks, 2. purse string suture, 3. spleen mobilization, 4. placement of AIW, and 5. recovered mouse after surgery. (B) Recovered mouse 8 days after AIW implantation. (C) Global treatment timeline: Foxp3 DTR−GFP mice were treated with R848 three times per week for four weeks to induce an autoimmune response. An AIW was implanted either shortly after R848 treatment initiation or after 4 weeks of treatment. GCs were then followed on six days over 15 days total during or after treatment, respectively. To visualize FDCs in the light zone, mice received αCD35-iFluor647 i.v. 24 h before every imaging session. (D) Maximum intensity projection of a 3D-stitched overview z-stack (100–250 μm below capsule, 150 μm thick) of the entire AIW FOV (8×10 tiles). Asterisks indicate the most superficial FDC networks. White arrowheads point to a large blood vessel, used as a landmark for reorientation over time. Green arrows indicate the borders of AIW FOV. Scale bar, 500 μm. Image was processed with background subtraction, median filtering (Despeckle), linear brightness, and contrast adjustment. See also and .

Article Snippet: Foxp3 DTR−GFP (B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J) breeding pairs were purchased from The Jackson Laboratory (strain no. 016958) and then bred in-house.

Techniques: Intravital Microscopy, Imaging

Journal: iScience

Article Title: Serial intravital microscopy reveals temporal dynamics of autoreactive germinal centers in the spleen

doi: 10.1016/j.isci.2026.115340

Figure Lengend Snippet: Processing of deep 3D data to enhance single-cell resolution, and spatiotemporal tracking using PA-GFP (A–C) Imaging frames of the same FOV 75, 145, and 185 μm below the capsule (dual track overlay from λ Ex 840 and λ Ex 940). FoxP3-GFP + cells are mostly visible, but with a low signal-to-noise ratio (SNR). Scale bars, 100 μm. (D–F) Same images as A-C, after background subtraction and median filtering (Despeckle). (G–I) Same images as D-F, after 3D Maximum filter. White arrow identifies a FoxP3 + cell. Scale bars, 100 μm, calculated by pixels as precise scaling is not possible after 3D maxima filtering due to the spatial enlargement of single spots. (J) Longitudinal tracking of PA-GFP-stimulated area (collagen trabecula) for 2 weeks. Scale bars, 100 μm. Experimental n = 3. All micrographs were adjusted in brightness and contrast (linear); any further processing is indicated in panel legends above.

Article Snippet: Foxp3 DTR−GFP (B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J) breeding pairs were purchased from The Jackson Laboratory (strain no. 016958) and then bred in-house.

Techniques: Single Cell, Imaging

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: Regulatory T cell dynamics in lungs during fibrosis. (A) The previously described SP-C I73T mouse pulmonary fibrosis model was utilized with an induction protocol containing 2 sequential doses of tamoxifen delivered by oral gavage that produces 2 distinct phases of overt disease development – alveolitis or peak inflammation (1–2 weeks) and fibrogenesis or fibrotic remodeling (2–6 weeks); (B) Lungs from control and SP-C I73T mice were analyzed for Foxp3+ Tregs by flow cytometry at indicated time points post Tam induction. Data pooled from three independent experiments with a total of 3–12 mice in each group as presented in the scatter plots. Kruskal-Wallis multiple comparison one-way ANOVA was performed. *** p = 0.0006 and **** p < 0.0001; (C) Representative immunohistochemical staining of paraffin-fixed SP-C I73T lungs obtained 4-weeks post induction and stained for murine Foxp3 (mFoxp3) expression. Arrows indicate mFoxp3+ Tregs localized in perivascular and fibrotic areas, high power magnification of insets 1,2. Bar: a = 100 μm, b-d = 10 μm,1–2 = 10 μm; Images are representative of 3–5 animals per group; (D) Uniform manifold approximation and projection (UMAP) analysis of 1,341 CD3+ T cells from lung scRNAseq data ( GSE234604 ) reveals a Foxp3+ Treg cluster following mutant SP-C expression. Gradient dot plots show expression of Treg-specific genes Foxp3 and Il2ra in SP-C I73T mice at 2- and 4-weeks post tamoxifen induction (n = 2 per genotype per time point); (E) Representative immunohistochemical staining for human Foxp3 (hFoxp3) in a paraffin embedded section from a human IPF lung explant. Insets 1–4 are magnified views of boxed regions. Bar 1–4 = 100 μm, a = 1000 μm (F) UMAP visualization of 8,404 CD3+ CD4+ T cell subtypes from lung cells analyzed by scRNA-seq in accession number GSE227136 , comparison of relative frequency of Treg (Foxp3+) clusters between control (n = 12) and IPF (n = 17) patients, a threshold cut-off > 50 cells was used for analysis; ** p = 0.004 by two-tailed Mann-Whitney test.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: Control, Flow Cytometry, Comparison, Immunohistochemical staining, Staining, Expressing, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: Regulatory T cells in fibrotic lungs have increased expression of effector and activation markers (A) Foxp3 eGFP reporter mice were crossed with previously published SP-C I73T mice to generate SP-C I73T - Foxp3 eGFP mice; (B) CD4+ Foxp3-GFP+ T regs were analyzed by flow cytometry in lungs harvested at 2, 3 and 4 weeks post Tamoxifen induction of mutant SP-C. Ordinary one-way ANOVA was performed. ** p < 0.005 (n = 3–5 mice in each group); (C-G) Control and SP-C I73T -Foxp3 eGFP lungs were digested, and samples were enriched for T cells using magnetic bead depletion as described in . Flow cytometric analysis and quantification of geometric mean fluorescence intensity of effector and activation markers expressed by CD4+ Foxp3-GFP+ T regs in lung tissue of SP-C I73T -Foxp3 eGFP mice 2 and 4-weeks post mutant Sftpc induction: (C) CD44; (D) CD279 (PD-1); (E) CD103; (F) GITR; (G) TIGIT. For each marker, ordinary one-way ANOVA was performed and *p < 0.05 and ** p < 0.005, n = 3–5 mice per group.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: Expressing, Activation Assay, Flow Cytometry, Mutagenesis, Control, Fluorescence, Marker

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: Depleting T regs during fibrogenesis worsens the disease phenotype in I ER -SP-C I73T -Foxp3 DTR mice. (A) I ER -SP-C I73T -Foxp3 DTR knock-in mice generated as described in . SP-C I73T -Foxp3 DTR mice were first induced with Tamoxifen administered via oral gavage on Days 0 and 4. 5 μg of DT or PBS was then administered i.p. every 48 h starting at day 17 post Tam induction and weight loss was recorded every day in SP-C I73T and SP-C I73T -Foxp3 DTR mice. Data are pooled from three independent experiments. ***p < 0.0001 by two-way ANOVA; (SP-C I73T uninduced – no tam+ DT n = 10, SP-C I73T + DT n = 16, SP-C I73T - Foxp3 DTR + PBS n = 13, SP-C I73T -Foxp3 DTR + DT n = 27); (B) Kaplan Meier survival curves of SP-C I73T -Foxp3 DTR mice that either received PBS (n = 13) or DT (n = 25). Log-rank (Mantel-Cox) test was performed with p value 0.0391; (C) Lungs from SP-C I73T -Foxp3 DTR mice receiving DT (n = 9) or PBS (n = 9) were harvested and cell suspensions analyzed by flow cytometry for GFP+ Tregs. *** p value < 0.0001 by unpaired t -test with Welch’s correction; (D) Quantification of total BALF cell counts between SP-C I73T -Foxp3 DTR mice treated with PBS (n = 7) or DT (n = 12) 26 days after Tam induction. * p = 0.01 by unpaired t -test with Welch’s correction; (E)(Left) Representative Picrosirius red stained fields from lung sections from DT or PBS treated SP-C I73T -Foxp3 DTR mice 26 days after Tam induction. Bar = 200 μm. (Right) Quantification was performed using ImageJ and data expressed as a percentage area staining for PSR. ** p = 0.004 by unpaired t -test with Welch’s correction; (F) BALF TGFβ quantified using ELISA (PBS n = 11, DT n = 11); (G) qRT-PCR analysis of whole lung mRNA for Col3a (PBS n = 12, DT n = 8); For E- G, scatter plots with individual values data was analyzed by unpaired t -test *p < 0.05 ** p < 0.01; (H) Box and Whiskers plot of static lung compliance (Cst) measured using Flex-Vent as described in (PBS n = 11, DT n = 12). Individual values for each animal are depicted and analyzed by unpaired t -test *p < 0.05 ** p < 0.01.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: Knock-In, Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: Tregs in SP-C I73T mice display a type 2 signature post injury. (A) Reanalysis of a previously published and deposited scRNAseq data set ( GSE234604 ). (Top) UMAP clustering of 1,341T cells in SP-C I73T and uninduced control mouse lungs and an accompanying gradient plot depicting genes used for their classification (Lower) identifies identified 2 populations of Foxp3+ T regs as proliferating Gata 3 low and activated Gata 3 high plus CD4+ and CD8+ T cells at 2- and 4-weeks post injury; (B)) UMAP projection T cell populations identifies the non-proliferating Foxp3 Treg population as expressing Gata3 but low in Tbx21 and Rorc; (C) Gradient plot depicting Gata3-regulated genes – in lung Tregs at 2 and 4 weeks post-injury and uninduced control (“WT”) mice; (D) Representative FACS plot of lung cell suspension obtained 3 weeks post-Tam induction showing CD25hi Foxp3+ T regs co-stained for transcription factors – Tbet (Th1), GATA3 (Th2) and Rorgt (Th17) as labeled. Spleens (n = 3) from of SP-C I73T animals served as controls. Unpaired t -test was performed and *p = 0.01; (E) Lungs from uninduced control (“WT”) and SP-C I73T mice were analyzed and quantified by flow cytometry for transcription factors – Foxp3 and GATA3 flow cytometry at indicated time points post Tam induction. Ordinary one-way ANOVA was performed. * p < 0.05 ** p < 0.005. n = 3–5 mice per group; (F) Comparative analysis of deposited scRNAseq data set ( GSE234604 ) for genes associated with Th2 immune response 4-weeks post Tam induction. Shown are data from uninduced control (“WT”) and SP-C I73T mice represented in a gradient plot. Genes from the gradient plot were combined to generate a Th2 gene score calculated for T regs from SP-C I73T and uninduced control mouse lungs.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: Control, Expressing, Suspension, Staining, Labeling, Flow Cytometry

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: T regs in SP-C I73T mice are proliferative and make pro-reparative factors. (A) Schematic for two dose Tamoxifen induction (days 0 and 4) of I ER -SP-C I73T -Foxp3 eGFP mice. Foxp3-GFP+ Tregs were FACS purified at 2-, 3- and 4-weeks post Tam induction and total RNA isolated. (B-D) Scatter plots of qRT-PCR analysis of Treg mRNA for expression of: (B) Ki67; (C) Top2a (Topoisomerase2a); (D) Areg. For each gene ordinary one-way ANOVA was performed. * p < 0.05 *** p < 0.0001. n = 3–7 mice per group; (E) Foxp3-GFP+ Tregs were FACS sorted from lungs and spleen of SP-C I73T mice 4 weeks post Tam induction and plated overnight. Cells were then treated with a cell stimulation cocktail that includes Brefeldin A, a protein transport inhibitor for 4 h, fixed and stained for GATA3 and intracellular amphiregulin (Areg). Identified Gata3+/Areg+ cells were expressed as a percentage of total Tregs. Comparisons were made using an unpaired t -test. *p = 0.01 n = 3–8 mice per group; (F) Areg levels of BALF collected 4wks after Tam induction and quantitated by ELISA. ***p = 0.0002 by unpaired t -test; n = 7–8 per group; (G) Gradient plots from the previously published scRNA-seq dataset GSE234604 were re-analyzed to compare the expression of Areg, GATA3 and Ki67 among Tregs, proliferating Tregs, CD4 + , and CD8 + T cells identified in single cell suspensions prepared from lung tissue 2- and 4-weeks post-Tam induction.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: Purification, Isolation, Quantitative RT-PCR, Expressing, Cell Stimulation, Staining, Enzyme-linked Immunosorbent Assay, Single Cell

Journal: Mucosal immunology

Article Title: Regulatory T cells protect against aberrant remodeling in a mouse model of pulmonary fibrosis

doi: 10.1016/j.mucimm.2025.12.004

Figure Lengend Snippet: Lung SP-C I73T T regs augment alveolar organoid growth supported by adventitial fibroblasts in vitro . (A) Schematic depicting tri-cellular organoid cultures generated using lung Tregs derived from SP-C I73T -Foxp3 eGFP mice 3–4 weeks after Tam induction, AT2 cells FACS sorted from SP-C CreER2 -Rosa26 Tdt mice, and adventitial fibroblasts isolated from Pdgrfa GFP mice; (B) Tricellular organoids were cultured for 2 weeks with activated CD3+ CD28+ Tregs replenished every week. Organoid cultures were imaged using EVOS FL auto and images were quantified using ImageJ an “analyze particle” macro with minimum area threshold set to1500 μm 2 . The experiment was independently repeated at least three times, each with three biological and three technical replicates. ****p < 0.00005 done by Ordinary one-way ANOVA; (C) Gradient plot from a previously published scRNA-seq dataset ( GSE234604 ) re-analyzed for expression of potential ligands made by immune cells as labeled (CD8+ T cell or Foxp3+ T regs) and cognate receptor expression on two fibroblast sub-populations (adventitial or alveolar) as labeled that are each increased in SP-C I73T mouse lungs at 2- and 4-week post Tam induction; (D) Bi-cellular organoids containing FACS purified TdTom+ AT2 and ScaI+ adventitial fibroblast were cultured with either 100 nM Areg, 200 nM Opn, or Areg+ Opn for 2 weeks. For comparison, tricellular organoids containing lung or spleen Tregs were prepared and co-cultured for 2 wks. (Left) Representative immunofluorescence staining of fixed wholemount organoid cultures co-stained for proSP-C and RAGE as labeled. Bars 50 μm; (Right) Live organoid cultures were images as in (B); (E) Representative immunofluorescence staining of fixed, frozen lung sections prepared form SP-C I73T -Foxp3 eGFP mice at 26 days post Tam induction stained for: (a) adventitial fibroblasts as CD39L1+ (white); (b) nuclei as DAPI+ (blue); (c) inherent Foxp3+ GFP+ fluorescence identifying Tregs (green); (d) composite overlay. Bars 100 μm. Insets 1 and 2 are magnified views of the boxed perivascular regions of lung with GFP+ Tregs and CD39L1+ adventitial fibroblasts.

Article Snippet: To generate an I ER -SP-C I73T -Foxp3 DTR knock-in mouse line, we used B6.129(Cg)- Foxp3 tm3(Hbegf/GFP)Ayr /J purchased from The Jackson Laboratory (strain # 016958).

Techniques: In Vitro, Generated, Derivative Assay, Isolation, Cell Culture, Expressing, Labeling, Purification, Comparison, Immunofluorescence, Staining, Fluorescence