Journal: bioRxiv

Article Title: Pathogenic IgE-fated B cell memory retains functional plasticity

doi: 10.1101/2023.11.28.567094

Figure Lengend Snippet: (A) Overview of analyzed datasets. (B) UMAP of B cell clusters from scRNA-seq data sets. Colored points indicate class-switched cluster used for downstream analyses. (C) Expression level of MBC2-related genes in MBC clusters. (D) Representative contour plots of OVA + MBC2s from inguinal LNs of epicutaneously sensitized mice four weeks after re-exposure and antibody treatment. Pre-gated as OVA + B cells > CD38 + GL7 - > IgM - IgD - . (E) Summary plots of MBC2 and CD23 + IL-4R low MBC frequency from IC and A4RA-treated epicutaneously sensitized mice. (F) MFI of CD23 within OVA + (left) and OVA - (right) MBCs from IC and A4RA-treated epicutaneously sensitized mice. Pre-gated as OVA +/- B cells > CD38 + GL7 - > IgM - IgD - . (G) Representative contour plots of MBC2s from human PBMCs 8 days following in vitro allergen stimulation and treatment with IC or A4RA. Pre-gated as live > singlet > CD3 - CD14 - CD16 - CD19 + > IgD - IgM - > CD38 low-med . (H) Summary plots of human MBC2 frequency in G , separated by CD27 expression. (I) MFI of CD23 within CD27 +/- class-switched B cells from human PBMCs 8 days following in vitro allergen stimulation and treatment with IC or A4RA. Data represents 2-3 experiments with 5 mice per group and are presented as median ± min/max (E, F) or 2 experiments ( n = 3 blood donors) with each line representing a single donor (H, I). * p < 0.05, ** p < 0.01, *** p < 0.001. Ab, antibody; Ag, antigen; LSC, lymphoid stromal cell.

Article Snippet: The following anti-mouse fluorochrome-conjugated antibodies were used: B220-AF700 (RA3-6B2), CD138-PE/Dazzle 594 (281– ), CD3-BV711 (17A2), CD3-BV510 (17A2), F4/80-BV711 (BM8), F4/80-BV510 (BM8), CD38-PE/Cy7 (90), GL7-PerCP.Cy5.5 (GL7), GL7-BV421 (GL7; BD, Franklin Lakes, NJ), IgD-Pacific Blue (11-26c.2a), IgD-BV605 (11-26c.2a), IgM-BV786 (II/41; BD), IgM-FITC (II/41), IgG1-BV650 (RMG1-1), IgG2b-PECy7 (RMG2b-1), IgG2c-FITC (polyclonal; SouthernBiotech, Birmingham, AL), IL-4Rα-PE (REA235; Miltenyi Biotec), and CD23-BV421 (B3B4).

Techniques: Expressing, In Vitro

Journal:

Article Title: Hodgkin lymphoma therapy with interleukin-4 receptor-directed cytotoxin in an infiltrating animal model

doi: 10.1182/blood-2004-08-3216

Figure Lengend Snippet: Cytotoxicity of IL-4 cytotoxin to Hodgkin/Reed-Sternberg cells

Article Snippet: The slides were subjected to indirect immunocytochemical analysis using Vector ABC kit (Vector Laboratories, Burlingame, CA) with polyclonal anti–IL-4Rα antibody ( c -20; Santa Cruz Biotechnology) and were developed with 3,3′-diaminobenzidine substrate (Vector Laboratories).

Techniques: Expressing

Journal:

Article Title: Hodgkin lymphoma therapy with interleukin-4 receptor-directed cytotoxin in an infiltrating animal model

doi: 10.1182/blood-2004-08-3216

Figure Lengend Snippet: Expression of IL-4R subunits and STAT6 activation induced by IL-4 in human H-RS cell lines. (A) Expression of IL-4Rα chain on 5 H-RS cell lines was assessed by flow cytometry using phycoerythrin-conjugated anti–IL-4Rα monoclonal antibody (open curve). Staining with isotype-matched IgG served as control (shaded curve). (B) Cell lysates prepared from H-RS cell lines were subjected to immunoblotting analysis to assess the expression of IL-4R subunits. (C) Antibody to Tyr-641–phosphorylated STAT6 or whole STAT6 proteins was reacted with membranes with cell lysates from H-RS cells stimulated with or without IL-4.

Article Snippet: The slides were subjected to indirect immunocytochemical analysis using Vector ABC kit (Vector Laboratories, Burlingame, CA) with polyclonal anti–IL-4Rα antibody ( c -20; Santa Cruz Biotechnology) and were developed with 3,3′-diaminobenzidine substrate (Vector Laboratories).

Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: mTOR inhibition rescues osteopenia in mice with systemic sclerosis

doi: 10.1084/jem.20140643

Figure Lengend Snippet: T H 2/IL4 infiltration in Fbn1 -deficient mice. (A) IHC staining of distal femoral in WT and Fbn1 +/− mice showed the number of IL4 + cells (arrowheads) in BM. Bar, 25 µm. (B) ELISA showed the serum level of IL4 in WT and Fbn1 +/− mice. (C–F) Flow cytometry showed T H 2 subsets in peripheral blood (PB) and BM of WT and Fbn1 +/− mice. (G and H) qPCR and ELISA analysis showed the levels of IL4 in WT and Fbn1 +/− BMMSCs compared with T cells. (I) IF staining showed MSC marker CD73 coexpressed with IL4Rα in WT and Fbn1 +/− BMMSCs. Bar, 25 µm. (J) Western blotting showed expression mTOR signaling genes, including IL4Rα, PI3K-p110, p-Akt, and p-mTOR in WT and Fbn1 +/− BMMSCs. (K) qPCR analysis of IL4Rα in WT and Fbn1 +/− BMMSCs. (L) Western blot showed efficacy of Il4rα and mTOR shRNAs in Fbn1 +/− BMMSCs. (M) Western blot showed that rapamycin treatment induced a dose-dependent inhibition of p-P70S6K in Fbn1 +/− BMMSCs. (N) Western blot showed the expression levels of p-mTOR and p-P70S6K in shRNAs and rapamycin-treated Fbn1 +/− BMMSCs. Shown are representative of all experimental data verified in at least three independent experiments. Error bars represent the SD from the mean values. ***, P < 0.005.

Article Snippet: BMMSCs (0.5 × 10 6 ) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz Biotechnology, Inc.), P70s6k siRNA (Santa Cruz Biotechnology, Inc.), Il4rα shRNA (Santa Cruz Biotechnology, Inc.), or Mtor shRNA (Addgene), according to the manufacturers’ instructions.

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Marker, Western Blot, Expressing, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: mTOR inhibition rescues osteopenia in mice with systemic sclerosis

doi: 10.1084/jem.20140643

Figure Lengend Snippet: Fbn1 deficiency-induced activation of IL4Rα/mTOR signaling regulates osteogenic/adipogenic lineage differentiation of BMMSCs. (A) Alizarin red staining showed the capacity to form mineralized nodules in scrambled shRNA (sh-Scr)–treated, Il4rα , and mTOR shRNA- and rapamycin-treated Fbn1 +/− BMMSCs. (B) Western blot showed the expression of the osteogenic genes RUNX2, ALP, and OCN in Fbn1 +/− BMMSCs with shRNAs and rapamycin treatment. (C) H&E staining showed BMMSC-mediated bone (B) and BM regeneration when subcutaneously implanted into immunocompromised mice with HA carrier after shRNAs and rapamycin treatment in Fbn1 +/− BMMSCs. Bars, 50 µm. A semiquantitative analysis showed the amount of bone formation in different implants. (D) Oil red O staining showed the capacity to differentiate into adipocytes in shRNAs and rapamycin-treated Fbn1 +/− BMMSCs, as well as the expression levels of adipogenic genes PPARγ2 and LPL. Bar, 50 µm. (E) IL4Rα neutralizing antibody (NAb) treatment showed the expression levels of p-mTOR and RUNX2 in Fbn1 +/− BMMSCs. (F) Western blot showed the level of p-P70S6K in Fbn1 +/− BMMSCs. (G) Western blot showed the expression level of RUNX2 in Fbn1 +/− BMMSCs after knockdown of P70s6k by siRNA. (H) Western blot analysis showed the expression of p-mTOR after knockdown of P70s6k . (I) To avoid off-target effects, two P70s6k siRNAs were used to show they both elevated expression of RUNX2 in Fbn1 +/− BMMSCs. (J–M) Knockdown of P70s6k by siRNA, Fbn1 +/− BMMSCs showed increased mineralized nodule formation by alizarin red staining (J), elevated expression of RUNX2, ALP, and OCN (K), decreased Oil red O + cells (bar, 50 µm; L), and decreased expression of PPARγ2 and LPL (M). All experimental data verified in at least three independent experiments. Error bars represent the SD from the mean values. ***, P < 0.005; **, P < 0.01.

Article Snippet: BMMSCs (0.5 × 10 6 ) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz Biotechnology, Inc.), P70s6k siRNA (Santa Cruz Biotechnology, Inc.), Il4rα shRNA (Santa Cruz Biotechnology, Inc.), or Mtor shRNA (Addgene), according to the manufacturers’ instructions.

Techniques: Activation Assay, Staining, shRNA, Western Blot, Expressing, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: mTOR inhibition rescues osteopenia in mice with systemic sclerosis

doi: 10.1084/jem.20140643

Figure Lengend Snippet: TGF-β and STAT6 synergistically enhance the expression of IL4Rα in Fbn +/− BMMSCs. (A) ELISA assay showed the level of TGF-β in Fbn1 +/− mouse serum and Fbn1 +/− BMMSCs cultured medium compared with WT group. (B) Western blotting showed the expression levels of p-SMAD3 and SP-1 in WT and Fbn1 +/− BMMSCs. (C and D) Western blotting showed that TGF-β treatment elevated IL4Rα expression in BMMSCs, which could be blocked by TGF-β neutralizing antibody (NAb). (E and F) Western blot showed that Fbn1 knockdown by siRNA in BMMSCs elevated IL4Rα expression, which could be blocked by TGF-β NAb. (G) Western blot showed the expression of IL4Rα after Smad2/3 siRNA treatment compared with vehicle-treated group. (H) Il4rα promoter luciferase fusions were examined in WT, TGF-β–treated, and Fbn1 +/− BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of cotransfected Renilla luciferase. (I) ChIP-qPCR assay showed enrichment of direct association of SP1 and STAT6 on Il4rα promoter in TGF-β–treated and Fbn1 +/− BMMSCs. (J) Western blot showed the expression of p-JAK1 and p-STAT6 in Fbn1 +/− BMMSCs. (K and L) Western blotting showed the expression of IL4Rα and p-mTOR after TGF-β, IL4, or combinatorial treatment with TGF-β and IL4. All experimental data verified in at least three independent experiments. Error bars represent the SD from the mean values. ***, P < 0.005.

Article Snippet: BMMSCs (0.5 × 10 6 ) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz Biotechnology, Inc.), P70s6k siRNA (Santa Cruz Biotechnology, Inc.), Il4rα shRNA (Santa Cruz Biotechnology, Inc.), or Mtor shRNA (Addgene), according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Knockdown, Luciferase, Activity Assay, ChIP-qPCR

Journal: The Journal of Experimental Medicine

Article Title: mTOR inhibition rescues osteopenia in mice with systemic sclerosis

doi: 10.1084/jem.20140643

Figure Lengend Snippet: Conditional KO of IL4Rα in BMMSC ameliorates osteopenia phenotype by rescuing impaired osteogenic/adipogenic differentiation. (A) Experimental outline describing the use of Il4rα f/f crossed with Fbn1 +/− mice to generate Il4rα f/+ ; Fbn1 +/− mice. Prrx1 - Cre mice were crossed with Il4rα −/− mice to generate Prrx1-Cre;Il4rα +/− mice. Il4rα f/+ ; Fbn1 +/− mice were then crossed with Prrx1-Cre;Il4rα +/− mice to generate Il4rα f/- ; Fbn1 +/− (DKO, n = 9) and Prrx1-Cre;Il4rα f/- ; Fbn1 +/− (TKO, n = 7) conditional KO mice. Il4rα f/− littermates ( n = 15) were used as control. (B) qPCR analysis showed the efficacy of Cre -medicated deletion of the floxed allele. (C) MicroCT analysis showed the BMD of DKO and TKO mice. Bar, 1 mm. (D) H&E (bar, 1 mm) and Oil red O (bar, 50 µm) staining showed the TB volume and percentage of adipocytes in distal femur. (E) Alizarin red staining showed the mineralized nodule forming capacity in Adenovirus-Cre–treated BMMSCs derived from DKO mice (Ad-Cre) compared with Adenovirus-GFP–treated DKO group (Ad-GFP). (F) Western blot analysis showed the expression of p-mTOR, RUNX2, ALP, and OCN in Ad-Cre compared with Ad-GFP. (G) Western blot showed the expression of RUNX2 in IL4Rα NAb-treated DKO BMMSCs. (H) When implanted into immunocompromised mice subcutaneously with HA carrier, Ad-Cre showed significantly increased bone formation. Bar, 50 µm. (I) Oil red O staining showed the number of adipocytes in Ad-Cre. Bar, 50 µm. (J) Western blotting showed the expression of adipogenic genes PPARγ2 and LPL in Ad-Cre. All experimental data verified in at least three independent experiments. Error bars represent the SD from the mean values. ***, P < 0.005.

Article Snippet: BMMSCs (0.5 × 10 6 ) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz Biotechnology, Inc.), P70s6k siRNA (Santa Cruz Biotechnology, Inc.), Il4rα shRNA (Santa Cruz Biotechnology, Inc.), or Mtor shRNA (Addgene), according to the manufacturers’ instructions.

Techniques: Control, Staining, Derivative Assay, Western Blot, Expressing

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Surface staining of IL-13-displaying yeast with biotinylated IL-13Rα1 and IL-4Rα ECDs. IL-13Rα1 binds IL-13 displayed on yeast independent of IL-4Rα (second panel from the left), whereas IL-4Rα on the other hand requires the presence of IL-13Rα1 to bind IL-13 recapitulating the cooperativity that has been observed previously 15 (right two panels). (B) Crystal structure of the IL-13 ternary complex (IL-13Rα1 in green, IL-4Rα in blue, and IL-13 in gold). Magnifications of the site II (A–D helixes) and site III (C–D loop) binding interfaces highlighting the amino acids on IL-13 involved in IL-13Rα1 binding are shown. (C) Sequential enrichment of IL-13 agonists. The site-specifically mutagenized library was selected against decreasing concentrations of IL-13Rα1 for five rounds. Clones were selected from each round to isolate variants with a range of IL-13Rα1 binding affinities.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Staining, Binding Assay, Clone Assay

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Crystal structure of the IL-13 agonist A11 (brown) in complex with IL-13Rα1 (green) and IL-4Rα (blue). (B) Overlay of the IL-13 wt and IL-13 A11 ternary complex crystal structures. Note that IL-13 A11 engages the two receptor chains with identical geometry to that of IL-13 wt (RMSD=0.738). (C–D) Schematic representation of ligand-receptor contact interaction of IL-13Rα1 with IL-13 (top half) versus IL-13 A11 (bottom half). Black lines and arrows represent van der Waals interactions, blue lines represent hydrogen bonds and red lines designate aromatic amino acid interactions.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques:

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A–C) IL-13 receptor dimerization detected by dual color single molecule imaging and co-locomotion-analysis. Images identifying an individual IL-13Rα1/IL-4Rα dimer in the presence of 200 nM IL-13 at different time points of a 500 frame (16 s) movie (A), the distance between the two molecules in each frame (B) and an overlay of the individual trajectories of IL-13Rα1 (magenta) and IL-4Rα (green) (C) are presented. Scale bars: 1μm in panels A and C. (D) Diffusion properties represented as step length distribution obtained for IL-13Rα1 in the absence (red) and presence (orange) of IL-13. For comparison, the step length distribution obtained for receptor dimers identified by co-locomotion analysis is shown (blue). (E) Cumulative probability of detecting an IL-13Rα1 molecule in the vicinity of an IL-4Rα molecule as a function of the squared distance, corrected for the statistical probability, in the presence and absence of IL-13 wt. For comparison, the cumulative correlation probability obtained in the presence of IL-13 DN is shown. (F) Correlation fraction for the IL-13Rα1 and IL-4Rα receptor subunit as a function of IL-13 agonist affinity. (G) Assembly and dissociation of an individual IL-13Rα1/IL-4Rα dimer in presence of agonist D7. Overlaying trajectories are shown in white. Scale bar 0.5 μm. (H) Average lifetime of the IL-13Rα1/IL-4Rα dimer as a function of agonist affinity.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Imaging, Diffusion-based Assay