Journal: The Journal of Experimental Medicine

Article Title: Cell circuits between B cell progenitors and IL-7 + mesenchymal progenitor cells control B cell development

doi: 10.1084/jem.20180778

Figure Lengend Snippet: List of reagents, DNA constructs and mouse strains, sources, and catalog numbers

Article Snippet: Rat monoclonal anti-mouse IgG1 PE , BD Biosciences , Clone A85-1; Cat#550083.

Techniques: Construct, Subcloning, Recombinant, Plasmid Preparation, Software

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: Immunocytochemical detection of MMCs in epithelial whole mounts and frozen sections of jejunum from T. spiralis-infected BALB/c mice. A to D: Whole mounts of jejunal epithelium were probed with anti-mMCP-1 (pseudo-colored red) or isotype-matched control antibodies together with anti-IgE (pseudo-colored green) or isotype-matched control antibodies and counterstained with DRAQ5 (pseudo-colored blue). IgE surface labeling was almost exclusively restricted to mast cells exhibiting strong intracellular mMCP-1 staining in samples probed with mMCP-1- and IgE-specific antibodies (A and D). Single-negative controls for IgE (B) and mMCP-1 (C) were used to confirm labeling specificity. Frozen sections of jejunum from uninfected (E) and T. spiralis-infected (F) BALB/c mice were probed with rabbit anti-tryptase (pseudo-colored green), anti-IgE (pseudo-colored red), and anti-integrin-α6 antibodies (pseudo-colored blue). No IgE or tryptase-positive cells were detected in uninfected animals (E). Abundant IgE+ve cells were detected in the samples from infected mice, the majority of which were located intraepithelially (F). Although significant numbers of intraepithelial IgE+ve cells were tryptase-negative (F, arrowheads), most IgE+ve cells detected in the lamina propria expressed tryptase (F, arrow). Staining specificity was confirmed using single-positive controls for tryptase (G), integrin-α6 (H), and IgE (I), in which the two other antigen-specific antibodies were replaced with control IgG. Identical excitation and exposure settings were used to acquire images of multilabeled and control samples. Scale bar length (μm) is provided in the bottom left corner of each image.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Infection, Labeling, Staining

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: IgE+ve cells in the jejunal mucosa of T. spiralis-infected mice express integrin-αEβ7. Frozen sections of jejunum from infected BALB/c mice were triple-labeled with anti-integrin-αE or anti-integrin-β7 together with anti-IgE and anti-integrin-α6 (see Figure 4). Using integrin-α6 staining to differentiate the epithelium (filled bars) and lamina propria (open bars), IgE+ve cells mm−2 (A) and the relative frequencies of IgE+ve cells expressing integrin-αE or integrin-β7 (B) were determined for each location using five randomly selected 0.31-mm2 fields of view per sample. The mean percentage of intraepithelial IgE+ve cells ± 1 SEM is provided in parentheses (A). Data are expressed as mean values ± 1 SEM (n = 4). Differences observed between the epithelial and lamina propria compartments were significant at P < 0.01 (**).

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Infection, Labeling, Staining, Expressing

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: Immunofluorescent detection of integrin-αE and -β7 expression by IgE+ve cells and PCNA-specific staining of toluidine blue-positive mast cells in the jejunal mucosa of S129 β6−/− and β6+/+ mice. A to J: Frozen sections of jejunum were probed with monoclonal rat antibodies specific for integrin-α6 (pseudo-colored blue), IgE (pseudo-colored green), and integrin-β7 or -αE (pseudo-colored red). Representative images are shown from uninfected β6+/+ (A) and β6−/− (B) mice stained for IgE, integrin-α6, and integrin-β7; uninfected β6+/+ (C) and β6−/− (D) mice stained for IgE, integrin-α6, and integrin-αE; T. spiralis-infected β6+/+ (E) and β6−/− (F) mice stained for IgE, integrin-α6, and integrin-β7; and T. spiralis-infected β6+/+ (G) and β6−/− (H) mice stained for IgE, integrin-α6, and integrin-αE. Arrowheads in D and H highlight occasional cells that exhibit low-level integrin-αE immunoreactivity in sections from β6−/− mice (inset in D has been contrast enhanced). Higher magnification images from T. spiralis-infected β6+/+ (I) and β6−/− (J) mice stained for IgE, integrin-α6, and integrin-αE are annotated with arrows (intraepithelial IgE+ve cells) and arrowheads (lamina propria IgE+ve cells). Note that although large numbers of IgE+ve cells are present in the mucosa of T. spiralis-infected β6−/− mice, they are predominantly located in the lamina propria. Single-positive controls from β6+/+ mice for integrin-β7 (K), integrin-αE (L), IgE (M), and integrin-α6 (N), in which two of the three antigen-specific antibodies were substituted with isotype-matched controls, were used to confirm staining specificity. Identical excitation and exposure settings were used to acquire images A to D. Constant excitation and exposure settings were also maintained during acquisition of images E to N. Bright-field images of toluidine blue-labeled mast cells probed with anti-PCNA antibodies from T. spiralis-infected β6+/+ (O) and β6−/− (P) mice, arrow indicates a PCNA-positive mast cell in O. Scale bars, 25 μm.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Expressing, Staining, Infection, Labeling

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: IgE+ve cell recruitment and integrin-αEβ7 expression in the jejunal mucosa of T. spiralis-infected β6−/− and β6+/+ S129 mice. Frozen sections of jejunum from infected β6−/− (open bars) and β6+/+ (filled bars) S129 mice were triple-labeled with anti-integrin-αE or anti-integrin-β7 together with anti-IgE and anti-integrin-α6 (see Figure 4). Data are presented as mean values ± 1 SEM (n = 3) for IgE+ve cells mm−2 (A); the percentage of IgE+ve cells located intraepithelially (B); and the relative frequencies of IgE+ve cells expressing integrin-αE (C) or integrin-β7 (D). Differences observed between β6−/− and β6+/+ S129 mice were significant at P < 0.05 (*) and P < 0.01 (**).

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Expressing, Infection, Labeling

Journal:

Article Title: Expression of Integrin-? E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-? 1 -Activating Integrin ? v ? 6

doi:

Figure Lengend Snippet: RT-PCR analysis of integrin-αE and -β7 transcript abundance and toluidine blue analysis of MMC distribution in the jejunal mucosa of T. spiralis-infected β6+/+ and β6−/− S129 mice. A: RT-PCR products for integrin-αE, integrin-β7, and the housekeeping gene GAPDH from reverse-transcribed total jejunal RNA from infected β6−/− (lanes 1 to 4) and β6+/+ (lanes 5 to 8) mice. B: RT-PCR product signal intensity expressed as a percentage of GAPDH signal intensity. Differences in PCR product abundance between β6+/+ and β6−/− S129 mice were significant at P < 0.05 (*). Mast cells were identified using toluidine blue staining (pH 0.5) in Carnoy’s-fixed/paraffin-embedded sections from β6+/+ (C) and β6−/− (D) S129 mice. Note that mast cells are present in greater numbers in β6−/− mice but are predominantly restricted to the lamina propria. Scale bars, 50 μm.

Article Snippet: Purified monoclonal rat IgG 2a anti-human integrin-α 6 (clone GoH3), control rat IgG 2a (clone R35-95), control rat IgG 1 (clone R3-34), and FITC-conjugated control rat IgG 1 (clone R3-34) were purchased from BD Biosciences, Cowley, UK.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Staining

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3A and Nrp-1 in human lung tumour cell lines and CTL clone. a Sema-3A expression in human lung tumour cell lines. Total protein extracts were analysed by western blot using anti-Sema-3A mAb. The 16HBE cell line was included as a control. Full length and proteolytically processed proteins are indicated. b Co-expression of Nrp-1 and CD25, and Nrp-1 and PD-1 on P62 T cells stimulated with immobilised anti-CD3. c The P62 clone was unstimulated or stimulated with anti-CD3, pre-incubated with Sema-3A-Fc, and then labelled with anti-human IgG-Fc secondary mAb. Results are gMFI mean ± SEM of triplicate samples. d Sema-3A-Fc inhibits CTL migration toward a CXCL12 gradient. The T-cell clone was stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwell plates and exposed to a gradient of CXCL12 loaded in the lower chambers. The number of T cells that had migrated into the lower chambers was determined. Results are mean chemotaxis index ± SEM of triplicate samples. e Sema-3A-Fc inhibits CD8 + TIL migration toward CXCL12. CD8 + TIL isolated from three human NSCLC tumours were stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwells and exposed to a CXCL12 gradient. Results are mean chemotaxis index ± SEM of triplicates. f Cytotoxicity of the CTL clone toward autologous tumour cells. The P62 clone was stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward the cognate IGR-Pub cell line was determined. g Cytotoxic activity of freshly isolated TIL toward autologous tumour cells. TIL, freshly isolated from a NSCLC tumour, were stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward freshly isolated autologous tumour cells was determined. Data shown for cytotoxic assay correspond to one of three independent experiments. Means ± SEM two-tailed Student’s paired t test c , one-way ANOVA test with Bonferroni correction d , e or two-way ANOVA test with Bonferroni correction f , g . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Western Blot, Control, Incubation, Migration, Chemotaxis Assay, Isolation, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3B and Nrp-1 in B16F10 mouse melanoma model. a Expression of Sema-3B in B16F10 tumour cells. Total protein extracts from B16F10 cells cultured in vitro or isolated ex vivo from tumour grafts were analysed by western blot using anti-Sema-3B mAb. b Surface expression of Nrp-1 on CD4 + and CD8 + T cells infiltrating B16F10 melanoma engrafted in C57BL/6 mice. TIL from individual tumours were isolated at day 15 after tumour cell inoculation. T lymphocytes from spleens and TdLN of tumour-bearing mice were analysed in parallel. Percentages of positive cells are included. Right: percentages of Nrp-1 + cells among CD8 + and CD4 + T cells in TIL ( n = 29 and 31), splenocytes ( n = 29 and 31) and TdLN ( n = 11 and 16). c Expression of Nrp-1 and FoxP3 in CD4 + T cells. T lymphocytes from tumours ( n = 20), spleens ( n = 22) and TdLN ( n = 11) of B16F10 melanoma-bearing mice were analysed at day 15 by flow cytometry. Right: percentages of Nrp-1 among FoxP3 + and FoxP3 − CD4 + T lymphocytes from B16F10. d Expression of CD44 and CD62L on Nrp-1 + and Nrp-1 − CD8 + T cells from B16F10 TIL. Right: Distribution of naive, effector and memory T cells populations among Nrp-1 − and Nrp-1 + CD8 + TIL ( n = 10). Results are representative of 3–5 independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction b or two-way ANOVA test with Bonferroni correction c . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a , d

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Cell Culture, In Vitro, Isolation, Ex Vivo, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: The Nrp-1 + PD-1 hi TIL subset is enriched with activated antigen-specific CD8 + T cells. a Staining of CD8 + TIL with dextramers. C57BL/6 mice were engrafted with B16F10 and then vaccinated with Trp2 and gp100 peptides. On day 15, TIL were isolated from tumours. Right: Percentages of Trp2 ( n = 8) and gp100 ( n = 5) dextramer-positive T cells among Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + TIL. b Expression of IFNγ ( n =20) and TNF ( n =18) in Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + T cells. TIL were stimulated for 4 h with autologous tumour cells. c Degranulation of CD8 + TIL. TIL were stimulated with autologous tumour cells; then, T-cell subsets were analysed for expression of CD107a ( n = 18). d Cytotoxicity of freshly isolated CD8 + TIL. CD8 + TIL were pre-incubated in medium or with anti-Nrp-1, anti-PD-1 or a combination of both mAb; then, cytotoxicity toward autologous tumour cells was determined. e Increase in MHC-I and PD-L1 expression on tumour cells co-cultured with autologous CD8 + TIL. Kinetic studies of H-2-K b /-D b and PD-L1 expression on B16F10 co-cultured with CD8 + TIL. Expression profiles (left), percentages of positive cells (middle) and gMFI (right) of MHC-I (upper panels) and PD-L1 (lower panels) are shown. f Expression of perforin in CD8 + T cells. TIL were stimulated with autologous tumour cells in the absence or presence of neutralising anti-Nrp-1, anti-PD-1 or anti-Nrp-1 plus anti-PD-1 then, T-cell subsets were analysed for expression of perforin ( n = 5). g Anti-Nrp-1 re-establishes migration of Nrp-1 + PD-1 hi T cells toward B16F10. TIL were pre-incubated in medium or with neutralising anti-Nrp-1, -PD-1, a combination of both mAb or an isotype control. Cells were seeded in the upper chambers of transwells and then exposed to a gradient of B16F10 supernatant, enriched in Sema-3B, loaded in the lower chambers. The numbers of Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − T cells that had migrated were determined by flow cytometry. Results are representative of three independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction a – g . * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Staining, Isolation, Expressing, Incubation, Cell Culture, Migration, Control, Flow Cytometry

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: Stem Cell Research & Therapy

Article Title: Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes

doi: 10.1186/s13287-020-01980-5

Figure Lengend Snippet: Establishment and molecular characterization of NPC lines and mature neuronal cultures. Investigation of target genes KHSRP and LRRC7. a Changes in gene expression patterns in NPCs and neurons derived from the case-parent trio. b , c NPCs and neurons derived from hiPSCs by the hippocampal neuronal differentiation protocol were investigated by immunofluorescence staining and visualized by confocal fluorescent microscopy. Immunocytochemical staining shows Nestin/Sox2 ( b ) and Map 2/Prox1 ( c ) positivity in these established neural cell types. Scale bars = 100 μm. d , e Immunofluorescence staining for KHSRP and LRRC7 in neurons. KHSRP ( d ) shows nuclear and cytoplasmic localization, while LRRC7 ( e ) localized postsynaptically in neurons. Scale bars = 50 μm

Article Snippet: The samples were then incubated for 1 h at room temperature with antibodies anti-SOX2 (monoclonal/mouse, 1:20 dilution; MAB2018, R&D Systems, Minneapolis, USA) and anti-Nestin (polyclonal/rabbit, 1:250 dilution; ab92391, Abcam, Cambridge, UK) or for overnight at 4 °C with antibodies anti-PROX1 (polyclonal/rabbit, 1:500 dilution; ab101851, Abcam, Cambridge, UK) and anti-MAP 2 (monoclonal/mouse, 1:500 dilution; M1406, Sigma/Merck, Darmstadt, Germany or polyclonal/rabbit, 1:1000 dilution; ab5622, Millipore, MA, USA).

Techniques: Gene Expression, Derivative Assay, Immunofluorescence, Staining, Microscopy

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: A small population of ACC cells H295R overexpresses Ptch1 at the plasma membrane . ( A ) H295R were labeled with an anti-Ptch1 antibody directed against the extracellular loop and cells presenting Ptch1 at their plasma membrane (H295R-PM-Ptc+ AF594+ cells) were sorted. AF594+ in blue represents the percentage of cells with Ptch1 at the cell surface (H295R-PM-Ptc+ cells). ( B ) Surface labeling of Ptch1 using anti-Ptch1 antibody directed against the extracellular loop of Ptch1 (Alexa 594 in red) on nonpermeabilized parental H295R and H295R-PM-Ptc+ cells. Nuclei were stained with DAPI (in blue). The histogram represents the mean ± SEM of Alexa 594 fluorescence intensity per cell (****: p -value < 0.00005 ( p -value = 2 × 10 −36 )).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Clinical Proteomics, Membrane, Labeling, Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: H295R-PM-Ptc+ cells are more resistant to chemotherapy than parental cells . ( A ) Doxorubicin (dxr) cytotoxicity. H295R and H295R-PM-Ptc+ cells were treated for 48 h with increasing concentrations of dxr before cell viability measure. ( B ) Doxorubicin IC50 of H295R-PM-Ptc+ and H295R parental cells in the absence or the presence of 10 μM of the Ptch1 efflux inhibitor methiothepin. ( C ) H295R-PM-Ptc+ cells accumulate less doxorubicin than parental H295R cells. Cells on coverslips were incubated with 2 μM dxr for 15, 30, 60, 180 and 240 min and immediately fixed with PFA. Dxr fluorescence was acquired using a filter for Alexa 594 and quantified using ImageJ software. About 100 cells (from three wells) were scored per condition per experiment. All data presented are the mean ± SEM of at least 3 independent experiments. Significance is attained at p -value < 0.05 (*), (**** p < 0.00005).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Incubation, Fluorescence, Software

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Differentially expressed genes (DEG) between H295R-PM-Ptc+ and parental H295R cells selected for their role in cancer. Genes overexpressed are indicated in red and genes underexpressed are in blue.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Activation Assay, Expressing, Inhibition, Gene Expression, Migration, Marker, Biomarker Discovery

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Composition of active modules containing one or more of the identified genes of interest listed in Table 1 (in bold) with genes upregulated in red and genes downregulated in blue, representative enrichment and role of differentially expressed genes (DEGs) in cancers.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Migration, Cell Differentiation, Activation Assay, Membrane, Activity Assay