Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 2 In vivo administration of TL1A + IL-18 results in acute thymic atrophy and increased thymic neutrophil numbers in neonatal and adult mice. A Schematic of the experimental model of TL1A + IL-18 injection in neonatal mice. Wild-type (WT) neonatal mice (P3) were IP injected with either PBS (vehicle) or TL1A [250 ng/day] + IL-18 [100 ng/day] for 4 consecutive days in a final volume of 20 µl. B Schematic of the experimental model of TL1A + IL-18 injection in adult mice (8 weeks old). WT mice were IP injected with either vehicle (PBS) or TL1A [1 µg/ day] + IL-18 [750 ng/day] for 4 consecutive days in a final volume of 200 µl. C Body weights of neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. D Pictures of thymuses from neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18. E Thymic cellularity of neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18. (n = 10). Data representative of one of at least five independent experiments are shown. F Quantification of neutrophil numbers in neonatal mice (P7) injected with PBS (gray) or TL1A + IL-18 (blue). Neutrophils were defined as Lin-CD11b+Ly-6G+ cells. The number of neutrophils in the complete thymus was quantified (n = 10). Data are representative of one of at least five independent experiments. G Body weights of adult model mice on Day 5 (D5) that were injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. H Pictures of thymuses from adults (D5) injected with either PBS (vehicle) or TL1A + IL-18. I Thymic cellularity of adult thymuses (D5) injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. J Quantification of the neutrophil numbers in adult mice (D5) injected with PBS (gray) or TL1A + IL-18 (blue). Neutrophils were defined as Lin-CD11b+Ly-6G+ cells. Neutrophil numbers were quantified from complete thymuses (n = 8). Data representative of one of at least five independent experiments are shown. Statistics: (C, E, G, I) Unpaired t test with Welch’s correction. E–J The error bars represent the SDs. F–J The Mann–Whitney U test was performed, as the standard deviations were different between treatment groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, MANN-WHITNEY

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 3 Thymic neutrophils develop and mature in situ in the neonatal thymic organ culture, and NOTCH restricts their development. A UMAP representation of scRNAseq cell cycle analysis of the three neutrophil clusters. Phase G2/M cells are shown in green. Phase G1 cells are shown in red. Phase S cells are shown in blue. The black arrow highlights neutrophils in phase G2/M (undergoing mitosis), defined as “pre-Neutrophils” (preNeu) in Fig. 1F. B Slingshot trajectory analysis of thymic neutrophils in organ culture. A trajectory from preNeu →imm. Neu →mat. Neu was identified. C Pseudotime analysis (“Slingshot” package) of genes and transcription factors involved in thymic neutrophil development. Kinetics of neutrophil numbers in the NTOC lobes (D) and supernatant (E) during the 6 days of culture with different cytokine treatments. (n = 3). Data representative of one of at least three independent experiments are shown. Error bars represent the SEM. F Heatmap of genes associated with neutrophil maturation. G Flow cytometry characterization of the expression of CD62L, CXCR4, and CD101 in newly generated neutrophils in the NTOC lobes from Day 3 to Day 6 after treatment with TL1A + IL-18. Histograms were set in modal mode. (n = 3). Data representative of one of at least three independent experiments are shown. H EM images from magnetically isolated neonatal bone marrow neutrophils and sorted Ly-6G+ cell NTOC supernatant treated with TL1A + IL-18 on Day 6. The different nuclear morphologies reveal distinct maturation stages of neutrophil development. I NOTCH negatively regulates neutrophil development in the thymus. NTOCs were treated with (1) control (PBS), (2) 1 µM, (3) 5 µM, or (4) 10 µM of the γ-secretase inhibitor LY411575, which prevents NOTCH signaling activation. (n = 3). Data representative of one out of three independent experiments are shown. The error bars represent the SDs. Statistics: (I) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. TEM transmission electron microscopy

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: In Situ, Organ Culture, Cell Cycle Assay, Flow Cytometry, Expressing, Generated, Isolation, Control, Activation Assay, Transmission Assay, Electron Microscopy

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 5 Thymic neutrophils can phagocytose, produce ROS, migrate, and form NETs similar to benchmark peritoneal neutrophils. A Schematic of the experimental design used to assess the ROS production capacity, phagocytosis, migration, and NET formation of thymic neutrophils compared to those of peritoneal neutrophils. Thymic neutrophils were isolated from NTOC supernatants treated for 6 days with TL1A + IL-18. Adult peritoneal neutrophils were isolated 4 h after IP injection of 1 ml of 3% Brewer thioglycolate. Neutrophils were purified using an EasySep™Mouse Neutrophil Enrichment Kit. Isolated peritoneal and thymic neutrophils were treated with PBS, eBioscience™Cell Stimulation Cocktail (1:500), DHR 123 [5 µM], pHrodo particles from S. aureus [1 mg/mL], cytochalastin D [2 µM], DMSO, LPS [4 µg/ml] from Klebsiella pneumoniae, and ionomycin [2.5 µg/ml]. B ROS production assay. Both TL1A- and IL-18-induced NTOC-derived and peritoneal neutrophils were treated with DHR 123 [5 µM] at 37 °C and 5% CO2 for 30 min. The fluorescence intensities were measured in channel B530 and are displayed as histograms. The Y-axes are normalized to the mode. Data representative of one of two experiments are shown. C Heatmap of manually curated neutrophil ROS-related genes. D Phagocytosis assay. TL1A + IL-18-induced, NTOC-derived, and peritoneal neutrophils were incubated with PE-conjugated pHrodo particles from S. aureus for 60 min and treated with cytochalastin D [2 µM] to inhibit phagocytosis. The number of engulfment events was normalized to the cell count. Data representative of one of two experiments are shown. E Heatmap of manually curated neutrophil phagocytosis genes. F Migration assay. TL1A + IL-18-induced, NTOC-derived, and peritoneal neutrophils were seeded at 150,000 cells/well on top of a 12-well Transwell plate with 5.0 µm pores and incubated with the neutrophil chemoattractants CXCL1 [50 ng/ mL] and fMLP [10 µM] at 37 °C and 5% CO2 for 90 min. The migrated cells were counted in a BD FACSVerse™Cell Analyzer. Data representative of one of two experiments are shown. G Heatmap of manually curated neutrophil migration-related genes. H Confocal images of thymic neutrophils isolated from the supernatants of TL1A + IL-18-stimulated NCTs (Day 6) and treated with DMSO (vehicle) or ionomycin for 4 h. The cells were stained with DAPI, SYTOX Green, and antibodies for detecting neutrophil elastase and citrullinated histone 3 (citH3). The scale bar represents 10 µM. (n = 3). Data representative of one of three independent experiments are shown. I Heatmap representation of the genes associated with primary/azurophilic, secondary/specific, tertiary/gelatinase or secretory granule release by NTOC-derived neutrophils. Statistics: (F) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. C, E, G The Y-axes were subjected to hierarchical clustering. NET Neutrophil extracellular trap

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: Migration, Isolation, Injection, Cell Stimulation, Derivative Assay, Phagocytosis Assay, Incubation, Cell Counting, Staining

Journal: Frontiers in Immunology

Article Title: TL1A/DR3 Axis, A Key Target of TNF-a, Augments the Epithelial–Mesenchymal Transformation of Epithelial Cells in OVA-Induced Asthma

doi: 10.3389/fimmu.2022.854995

Figure Lengend Snippet: Effects of the TL1A/DR3 axis on the EMT in Beas-2B cells were detected. TNF-a (50 ng/mL) stimulation for 48 h as the best cell model based on the above experimental results. The effect of sTL1A on EMT was detected by Western blotting (A, C) . The effects of TL1A siRNA on TL1A induced by TNF-a were detected by immunofluorescence (E) . Effects of TL1A siRNA on the EMT formation induced by TNF-a were measured by Western blotting analysis (G) . (E) : magnification 200 ×, scale bar 50 µm. (B, D, F, H) intensity analysis of (A, C, E, G), respectively. Data are expressed as the means ± SD for three independent experiments. # p < 0.05 versus the control group. # *p < 0.05 versus the TNF-a group.

Article Snippet: Recombinant human sTL1A (1319-TL) was obtained from RD Systems.

Techniques: Western Blot, Immunofluorescence, Control

Journal: Frontiers in Immunology

Article Title: Roles of tumor necrosis factor-like ligand 1A in γδT-cell activation and psoriasis pathogenesis

doi: 10.3389/fimmu.2024.1340467

Figure Lengend Snippet: Imiquimod (IMQ) treatment activates dermal γδT cells through TL1A. (A–D) Mouse ears were smeared with IMQ cream or control Vaseline for 3 consecutive days. One day after the last treatment, single-cell suspensions from ears were analyzed for cytokine production (n = 5). (A, B) Frequencies of cytokine-producing cells in the CD45 + gate. (C, D) TCR usage of IL-17 + and IL-22 + lymphocytes in IMQ-treated mice. DN, double negative. (E–I) Ears were smeared with IMQ for 3 (E, F) or 4 (G–I) consecutive days. Anti-TL1A antibodies were injected from day −1 until the day before the analysis. (E, F) CD3 int γδTCR int cells from ears on day 3 were analyzed for cytokine production (n = 5). (G) Ear swelling was calculated as changes in thickness from day 0 (IMQ-treated n = 6; Vaseline-treated n = 3). (H) H&E staining sections were prepared from ears on day 4. Scale bar, 100 μm. (I) Epidermal keratinocyte layers and epidermal and dermal thicknesses were measured in the sections. Representative of three (A, C, E) and two (H) independent experiments. Accumulated from three (B, D, F) and two (G, I) independent experiments. Error bars, mean ± standard error (SE). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Where indicated, mice were intraperitoneally injected once daily with 20 mg/kg anti-TL1A monoclonal antibodies (clone 5G4.6; Bio X Cell) from day −1 until a day before analysis ( ).

Techniques: Cream, Control, Injection, Staining

Journal: Frontiers in Immunology

Article Title: Roles of tumor necrosis factor-like ligand 1A in γδT-cell activation and psoriasis pathogenesis

doi: 10.3389/fimmu.2024.1340467

Figure Lengend Snippet: TL1A activates splenic γδT17 cells synergistically with IL-23. (A) CD3 + γδTCR + cells were subdivided into four fractions based on CD27 and Vγ4 expression. (B) Expression of DR3, type I IL-1 receptor (IL1-R), and IL-23R in indicated γδT-cell subsets. (C) Expression levels of cytokine receptors in each γδT-cell subset are shown as relative fluorescence intensities normalized with the mean fluorescence intensity in total γδT cells defined as 100 (n = 6). (D) CD27 + and CD27 − γδT cells isolated by sorting were analyzed by qPCR for the expression of the indicated genes (n = 3). mRNA expression in CD27 − γδT cells is shown as relative levels in CD27 + γδT cells defined as 1. The statistical significance of the expression levels between CD27 + and CD27 − cells is shown by asterisks. (E, F) Spleen cells were cultured with the indicated cytokines for 24 (h) A protein transport inhibitor was added for the last 4 (h) IL-17 and IL-22 production was analyzed by intracellular staining. (E) Representative flow cytometry profiles of γδT cells at the end of culture. (F) Frequencies of IL-17 + , IL-22 + , and IL-17 + IL-22 + γδT cells (n = 5). Representative of three (A, B) and four (E) independent experiments. Cumulative results from three (C, D) and four (F) independent experiments. Error bars, mean ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Where indicated, mice were intraperitoneally injected once daily with 20 mg/kg anti-TL1A monoclonal antibodies (clone 5G4.6; Bio X Cell) from day −1 until a day before analysis ( ).

Techniques: Expressing, Fluorescence, Isolation, Cell Culture, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Roles of tumor necrosis factor-like ligand 1A in γδT-cell activation and psoriasis pathogenesis

doi: 10.3389/fimmu.2024.1340467

Figure Lengend Snippet: Dermal γδT cells are ready to respond to IL-23 and TL1A. (A) CD45 + gate in single-cell suspensions from ears contained CD3 hi γδTCR hi dendritic epidermal T cells (DETCs) and CD3 int γδTCR int conventional dermal γδT cells. CD3 int γδTCR int dermal γδT cells were divided into Vγ4 + and Vγ4 − cells. (B) Expression of DR3, type I IL-1R, and IL-23R in DETCs and dermal γδT cells. (C) Expression levels of cytokine receptors in Vγ4 + and Vγ4 − γδT cells are shown as relative fluorescence intensities normalized with the mean fluorescence intensity in DETCs defined as 100 (n = 5). (D, E) Mouse ears were intradermally injected with IL-23 alone or combined with TL1A. Twenty-two hours later, DETCs and γδT cells were analyzed for IL-17 (D) and IL-22 (E) by intracellular staining (n = 7 per group). Representative data of three independent experiments (A, B) . Cumulative results from four (C–E) independent experiments. Error bars, mean ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Where indicated, mice were intraperitoneally injected once daily with 20 mg/kg anti-TL1A monoclonal antibodies (clone 5G4.6; Bio X Cell) from day −1 until a day before analysis ( ).

Techniques: Expressing, Fluorescence, Injection, Staining

Journal: Frontiers in Immunology

Article Title: Roles of tumor necrosis factor-like ligand 1A in γδT-cell activation and psoriasis pathogenesis

doi: 10.3389/fimmu.2024.1340467

Figure Lengend Snippet: TL1A exacerbates symptoms in the IL-23-induced murine psoriasis model. Mouse ears were intradermally injected with PBS, IL-23 alone, or the combination of IL-23 and TL1A for 4 consecutive days from days 0 to 3. (A) Thickness of ears was monitored (n = 4). Swelling was calculated as changes in thickness before the treatments. (B–E) H&E staining sections were prepared on day 4. (B) Representative view with low magnification. Scale bar, 100 μm. (C) Epidermal layers of keratinocytes and epidermal and dermal thicknesses were measured (n = 6). (D) Magnified view of the epidermis. Scale bar, 50 μm. (E) Number of microabscesses per section (n = 6). nd, not detected. (F) Immunohistochemical analysis. Scale bar, 25 μm (top), 50 μm (middle), and 100 μm (bottom). (G) PCNA + and Gr1 + cells were counted in epidermal and dermal areas, respectively (n = 6). Cumulative results from two (A) and three (C, E, G) independent experiments. Representative data of three independent experiments (B, D, F) . Error bars, mean ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Where indicated, mice were intraperitoneally injected once daily with 20 mg/kg anti-TL1A monoclonal antibodies (clone 5G4.6; Bio X Cell) from day −1 until a day before analysis ( ).

Techniques: Injection, Staining, Immunohistochemical staining

Journal: Frontiers in Immunology

Article Title: Roles of tumor necrosis factor-like ligand 1A in γδT-cell activation and psoriasis pathogenesis

doi: 10.3389/fimmu.2024.1340467

Figure Lengend Snippet: Involvement of TL1A-mediated γδT-cell activation in early psoriasis. Mouse ears were intradermally injected with indicated cytokines. One day later, ears were harvested. (A–D) Single-cell suspensions were prepared for flow cytometry analysis (n = 7). (A, B) Frequencies of cytokine-producing cells in the CD45 + gate. (C, D) IL-17 + and IL-22 + lymphocytes were analyzed for CD3 and γδTCR expression. (E–G) Comparison of wild-type and Tcrd -knockout mice. (E) Ear thickness was measured before and a day after the treatments (n = 4–5). (F, G) Histological analysis of H&E sections. (F) Representative views from mice injected with IL-23 plus TL1A. Scale bar, 50 μm. (G) Epidermal layers and epidermal and dermal thicknesses (n = 4–5). (H) Ear samples were subjected to qPCR analysis (n = 4–6). mRNA expression is shown relative to the average values in wild-type mice injected with IL-23 alone defined as 1. Representative of two (F) and four (A, C) independent experiments. Accumulated from two (E, G, H) and four (B, D) independent experiments. Error bars, mean ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Where indicated, mice were intraperitoneally injected once daily with 20 mg/kg anti-TL1A monoclonal antibodies (clone 5G4.6; Bio X Cell) from day −1 until a day before analysis ( ).

Techniques: Activation Assay, Injection, Flow Cytometry, Expressing, Comparison, Knock-Out

Journal: International journal of molecular sciences

Article Title: Increased Serum Levels of Tumor Necrosis Factor-like Ligand 1A in Atopic Dermatitis.

doi: 10.3390/ijms24031813

Figure Lengend Snippet: Figure 1. Serum TL1A protein levels in patients with AD and normal controls. The measured values from individual patients were plotted by dots. Each bar means the average. ** p < 0.01. (a) Serum TL1A protein levels in AD (n = 36) or normal controls (n = 10). (b) Serum TL1A levels in mild (n = 14) or severe (n = 22) AD patients and normal controls (n = 16).

Article Snippet: Serum TL1A levels were quantified using Human TL1A ELISA kit (DY1319–05 and DY008, R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: International journal of molecular sciences

Article Title: Increased Serum Levels of Tumor Necrosis Factor-like Ligand 1A in Atopic Dermatitis.

doi: 10.3390/ijms24031813

Figure Lengend Snippet: Figure 2. Correlation between TL1A protein levels and clinical markers in TL1A positive patients of AD. (a) Correlation between serum TL1A protein levels and Eczema Area and Severity Index (EASI). (b) Correlation between serum TL1A levels and the age of each patient. (c) Correlation between serum TL1A levels and serum thymus and activation-regulated chemokine (TARC) levels. (d) Correlation between serum TL1A levels and serum immunoglobulin E (IgE) levels. (e) Correlation between serum TL1A levels and serum lactate dehydrogenase (LDH) levels. (f) Correlation between serum TL1A levels and the number of eosinophils in peripheral blood.

Article Snippet: Serum TL1A levels were quantified using Human TL1A ELISA kit (DY1319–05 and DY008, R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay

Journal: International journal of molecular sciences

Article Title: Increased Serum Levels of Tumor Necrosis Factor-like Ligand 1A in Atopic Dermatitis.

doi: 10.3390/ijms24031813

Figure Lengend Snippet: Figure 4. Immunohistochemistry of TL1A in atopic dermatitis (AD) or normal skin samples.

Article Snippet: Serum TL1A levels were quantified using Human TL1A ELISA kit (DY1319–05 and DY008, R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemistry

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Two days post-confluent MEFs or 3T3-L1 cells were cultured in adipogenic induction medium supplemented with TL1A recombinant protein at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL). (A) Oil Red O staining was conducted after adipogenic induction for 7 days. (B) The average optical density (OD) was measured using a Universal Microplate Spectrophotometer at 490 nm. (C) Measurement of triglyceride (TG) content in MEFs 7 days after adipogenic differentiation. (D-E) Both Oil Red O staining and triglyceride assays in 3T3-L1 cells were performed on day 7 of differentiation. (F-G) qRT-PCR analysis was conducted to evaluate the expression of adipogenic markers aP2, Plin1, Plin2 and PPARγ in 3T3-L1 cells 7 days after adipogenic differentiation. (H) qRT-PCR analysis was conducted to evaluate the expression of VEGFA and several adipogenic markers C/EBPα/β/δ, PPARγ2, aP2, CD36, Plin1, Plin2, adispin, Glut4 and LPL in MEFs 7 days after adipogenic differentiation. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Cell Culture, Recombinant, Staining, Spectrophotometry, Quantitative RT-PCR, Expressing

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: (A-D) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 3 days in the presence of the specified concentrations of TL1A. Total protein was extracted to determine C/EBPα, C/EBPβ, PPARγ2, PPARγ1 and aP2 protein expression by Western blotting with quantitation of band density. (E-H) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 7 days with the indicated concentrations of TL1A. Total protein was extracted to determine PPARγ2, PPARγ1, CD36 and aP2 protein expression by Western blottingwith quantitation of band density. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: After two days post-confluence, MEFs were treated with standard differentiation medium and TL1A recombinant protein (0, 10, 20, 50, 100, 200 ng/mL). At specified time points, cells were harvested for RNA isolation and subsequent qRT-PCR analysis. * P < 0.05 vs . the group of adipocytes without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Recombinant, Isolation, Quantitative RT-PCR

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: MEFs were treated with standard differentiation medium and TL1A at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL) for 7 days. (A) Total cellular proteins were extracted, and the expression levels of β-catenin, phosphorylated YAP1 S127 (p-YAP1 S127 ) and total YAP1 proteins were determined by Western blotting. (B) The relative amounts of β-catenin protein expression were calculated according to the grayscale values and were showed in the histogram. (C) The quantitation of ratio (p-YAP1 S127 /total YAP1) to reflect the inactivation of YAP1 signaling pathway. (D-E) At the end of adipogenesis induction, protein expression of phosphorylated β-catenin (p- β-catenin) and YAP1 in the cytoplasm extract from MEFs was determined by Western blotting. (F-G) Nuclear extracts were isolated from the MEFs and analyzed for protein expression by Western blotting. Nuclear proteins were isolated to evaluated the expression of β-catenin, YAP1 and PPARγ proteins through Western blotting, accompanied by a quantitative analysis of band density. GAPDH and Lamin A/C served as the internal controls for cytoplasmic and nuclear extract, respectively. All the histograms represent the relative expression levels of proteins normalized by GAPDH or Lamin A/C. * P <0.05, ** P <0.01, *** P <0.001 vs . the group of MEFs without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay, Isolation

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Exogenous TL1A triggers the phosphorylation of YAP1 at Ser127, resulting in its cytoplasmic retention and functional inactivation. This event subsequently reduces β-catenin stability and prevents its nuclear translocation, thereby triggering the expression of essential adipogenic regulators and lipid metabolism-related proteins. Additionally, TL1A-mediated regulation of ABCA1 and ABCG1 expression facilitates cholesterol homeostasis, providing essential substrates for lipid droplet biogenesis. Collectively, these molecular mechanisms orchestrate the differentiation of MEFs and 3T3-L1 preadipocytes into terminally differentiated adipocytes.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Phospho-proteomics, Functional Assay, Translocation Assay, Expressing