recombinant human madcam 1 ig  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: A CD25–chemokine receptor complex initiates noncanonical IL-2 signaling

doi: 10.1016/j.jbc.2025.110981

Figure Lengend Snippet: hCD25–chemokine receptor complex triggers integrin activation. A and B, IL-2(E52K) does not trigger MACS-dependent integrin activation in a CD25-expressing T-cell line or Tregs. A, hCD25-Jurkat T cells or ( B ) primary human Tregs were incubated with 5 μg/ml VCAM-1 Fc (these cells express primarily α4β1 rather than α4β7) and 5 μg/ml 7G7B6 or isotype control in the presence or the absence of 1 μg/ml IL-2(WT) or IL-2(E52K) for 30 min at 37 °C before measurement of bound VCAM-1 Fc by flow cytometry. C, IL2RA + -YT1 cells express ∼8-fold more CCR5 than hCD25 Jurkat T cells. Depicted are flow cytometry histograms and geometric mean fluorescence intensity of staining with anti-CCR5 or isotype control antibody. D, a CCR5 antagonist blocks assembly of the 7G7B6-induced CD25–CCR5 complex. HEK293T-HA-hCD25 cells were transfected with cDNA encoding the indicated FLAG-tagged chemokine receptor and then incubated with 5 μg/ml 7G7B6 and 1 μg/ml IL-2 (WT), IL-2(E52K), or IL-2(E52K)+ 50 nM maraviroc for 1 h at 37 °C. Cells were lysed, and immunoprecipitates were fractionated by SDS-PAGE followed by immunoblotting for HA-hCD25 ( E ) 7G7B6-induced the CCR5–CD25 complex triggers integrin activation. IL2RA + -YT1 cells (express α4β7) were incubated with 5 μg/ml MAdCAM-1 Fc in the presence or the absence of 5 μg/ml 7G7B6 and/or isotype control (0) in the presence or the absence of 1 μg/ml IL-2, IL-2(E52K), or 50 nM maraviroc (Mara) + IL-2(E52K) for 30 min at 37 °C before measurement of bound VCAM-1 Fc by flow cytometry: ∗ p < 0.05, ∗∗ p < 0.01, by one-way ANOVA with Tukey’s correction, Scatter plots depict mean (horizontal line) from at least three independent experiments. cDNA, complementary DNA; HA, hemagglutinin; hCD25, human CD25; HEK293T, human embryonic kidney 293T cell line; IL-2, interleukin 2; Treg, regulatory T cell; VCAM-1, vascular cell adhesion molecule 1.

Article Snippet: Cells were plated in a 96-well plate and incubated with the following ligands for 30 min at 37 °C in Hanks' balanced salt solution (HBSS) with Ca/Mg (Gibco; catalog no.: 14-025-092): 5 μg/ml human MAdCAM-1 Fc (R&D Systems; catalog no.: 6056-MC) for YT-1 hCD25 cells or human VCAM-1 Fc (R&D Systems; catalog no.: 862-VC) with human primary CD4 + T cells, 0.5 μg/ml IL-2, 10 μg/ml IgG control or 7G7B6, 5 μg/ml HS, 200 ng/ml pertussis toxin, 100 nM phorbol 12-myristate 13-acetate (Sigma; catalog no.: P8139), and 10 mM EDTA (Invitrogen; catalog no.: AM9260G).

Techniques: Activation Assay, Expressing, Incubation, Control, Flow Cytometry, Fluorescence, Staining, Transfection, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A CD25–chemokine receptor complex initiates noncanonical IL-2 signaling

doi: 10.1016/j.jbc.2025.110981

Figure Lengend Snippet: HS induces an IL-2-dependent CD25–CCR7 complex to trigger MACS. A, IL-2(E52K) does not support HS-induced CD25–CCR7 association. Membrane lysates from HA-hCD25–expressing HEK293T cells were incubated with 5 μg/ml HS in the presence (+) or absence (−) of 2.5 μg/ml IL-2 (WT) or IL-2 (E52K) for 30 min at 37 °C. Anti-HA immunoprecipitates were fractionated by SDS-PAGE, and blots were probed with either anti-HA or anti-CCR7. Quantification of data by LI-COR infrared spectroscopy from three independent experiments is shown to the righ t of the blot. B, IL-2(E52K) does not support HS-induced integrin activation. hCD25-Jurkat cells were incubated with 5 μg/ml HS in the presence or the absence (−) of 2.5 μg/ml IL2 (WT) or IL-2(E52K) and 5 μg/ml VCAM-1 for 30 min at 37 °C, and bound VCAM-1 was measured by flow cytometry. C, HS-induced CCR5–CD25 complex triggers integrin activation. IL2RA + -YT1 cells were incubated with 5 μg/ml MAdCAM-1 Fc in the presence or the absence of 40 μg/ml HS and/or buffer control (0) in the presence or the absence of 1 μg/ml IL-2, IL-2(E52K), 50 nM maraviroc (Mara) + IL-2(E52K) for 30 min at 37 °C before measurement of bound MAdCAM-1 Fc by flow cytometry. D, HS did not affect canonical IL-2 signaling. IL-2Rα + YT-1 cells were incubated with varying concentrations of IL-2 WT in the presence ( blue ) or the absence (0, black ) of 40 μg/ml HS for 30 min at 37 o C followed by analysis of phospho-STAT5 staining by flow cytometry. Curves were fitted to a simple agonist response model by nonlinear regression on GraphPad Prism, version 10. ∗ p < 0.05, ∗∗ p < 0.01 by unpaired t tests ( A ) or one-way ANOVA with Tukey’s correction ( B , C ), mean ± SEM from at least three independent experiments. HA, hemagglutinin; hCD25, human C25; HEK293T, human embryonic kidney 293T cell line; HS, heparan sulfate; IL-2, interleukin 2; MACS, monoclonal antibody (or matrix)–directed alternative cytokine signalling; STAT5, signal transducer and activator of transcription 5; VCAM-1, vascular cell adhesion molecule 1.

Article Snippet: Cells were plated in a 96-well plate and incubated with the following ligands for 30 min at 37 °C in Hanks' balanced salt solution (HBSS) with Ca/Mg (Gibco; catalog no.: 14-025-092): 5 μg/ml human MAdCAM-1 Fc (R&D Systems; catalog no.: 6056-MC) for YT-1 hCD25 cells or human VCAM-1 Fc (R&D Systems; catalog no.: 862-VC) with human primary CD4 + T cells, 0.5 μg/ml IL-2, 10 μg/ml IgG control or 7G7B6, 5 μg/ml HS, 200 ng/ml pertussis toxin, 100 nM phorbol 12-myristate 13-acetate (Sigma; catalog no.: P8139), and 10 mM EDTA (Invitrogen; catalog no.: AM9260G).

Techniques: Membrane, Expressing, Incubation, SDS Page, Spectroscopy, Activation Assay, Flow Cytometry, Control, Staining