Journal: bioRxiv

Article Title: A CD25-CCR7 complex initiates non-canonical IL-2 signaling

doi: 10.1101/2025.02.03.636356

Figure Lengend Snippet: (A) 7G7B6 triggers IL-2 dependent hCD25-CCR7 complex . 5 ug/mL anti human CD25 antibodies 7G7B6 or BC96 were incubated with HA-hCD25 expressing HEK293T cells in the presence or absence of 1 ug/mL IL-2 and following lysis and immunoprecipitation with anti-HA were assayed for captured CCR7 or HA-hCD25 by immunoblotting. (B) IL-2(E52K) does not support 7G7B6-induced hCD25-CCR7 complex . IL-2(E52K) or wild type IL-2 were assayed for capacity to support 7G7B6-induced CD25-CCR7 complex as described in panel A. (C) IL2(E52K) mutation does not disrupt CD25-IL-2 interaction . Microplates coated with recombinant hCD25 extracellular domain were incubated with the indicated concentrations of IL-2 or IL-2(E52K) and bound IL-2 was quantified by ELISA. (D) IL-2(E52K) does not trigger MACS-dependent integrin activation . IL-2Rα YT-1 cells. were incubated with indicated reagents in the presence of 5 μg/mL hMAdCAM-1 Fc for 30 min at 37 o C, followed by APC-anti-human IgG Fc and FACS to assess MAdCAM-1 binding. (E) IL-2(E52K) does not trigger MACS-dependent integrin activation Freshly isolated human Tregs were incubated with 7G7B6 and 5 μg/ml VCAM-1 Fc (these cells express little α4β7) and 7G7B6 in the presence or absence of IL-2(WT) or IL-2(E52K) (E52K) for 30 min at 37 o C before measurement of bound VCAM-1 Fc by FACS. In some experiments 200 ng/ml Pertussis toxin was added (PTX) or an irrelevant antibody (iso) was added in place of 7G7B6. (F) IL-2(E52K) triggers canonical IL-2 signaling . IL2Rα YT-1 cells were stimulated with varying concentrations of IL-2 or IL-2(E52K) for 30 min at 37 o C, followed by staining with APC-anti phospho-STAT5 and analysis by FACS. *: p<0.05, **: p<0.01, NS: not significant by one-way ANOVA.

Article Snippet: Microplates (Greiner Bio-One) were coated with hCD25 (2μg/ml) in carbonate/bicarbonate coating buffer (pH 9.2) and blocked with 2% BSA.

Techniques: Incubation, Expressing, Lysis, Immunoprecipitation, Western Blot, Mutagenesis, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Binding Assay, Isolation, Staining