Fig 2: IL-2(E52K). does not support CD25-CCR7 complex.(A) Design Strategy. Depicted are space filling models of the trimeric IL-2 receptor (gray) with the helical elements of IL-2 depicted as a gold ribbon. Charged residues in unburied IL-2 loops are labelled and are targeted for mutagenesis. (B) Schematic of ELISA. HEK293T-HA-mCD25 cells were plated on anti-HA-coated wells and then incubated with 1 μg/mL IL-2 (red) and 5 μg/mL PC61 (green) for 1 h at 37 °C. Cells were lysed and washed plates were probed by antiCCR7 or anti-HA followed by chemiluminescence reagents. (C) ELISA screen identifies mutants that fail to support CD25-CCR7 complex. Note that CCR7 association is IL-2 dependent and reduced in mutants 1,5, and 6. (Mean ± SEM. N=3). D) IL-2(E52K) fails to support CD25-CCR7 complex. Experiment was performed as in panels B and C with detection by immunoprecipitation of HA-CD25 followed by immunoblotting.

Fig 3: Assembly of human CD25-CCR7 complex triggers integrin activation(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 °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 °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 °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.

Fig 4: HS induces an IL-2 dependent CD25-CCR7 complex to trigger MACS.(A) IL-2(E52K) does not support HS induced CD25-CCR7 association. Membranes from HA-CD25 expressing HEK293T cells were incubated with 5 μg/mL HS in the presence or absence (−) of 2.5 μg/mL IL2 (WT) or IL-2 (E52K (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. (B) Quantification of data from experiments performed as in panel B. Blots from 3 such experiments were quantified by Licor infrared spectroscopy. (C) IL-2(E52K) does not support HS induced integrin activation. IL2Rα YT-1 cells were incubated with 5 μg/mL HS in the presence or absence (−) of 0.5 μg/mL IL2 (WT) or IL-2(E52K) (E52K) and 5 μg/ml MAdCAM-1 for 30 min at 37°C and bound MAdCAM-1 was measured by FACS. None signifies no additions. (D) 5 μg/mL HS did not affect STAT5 phosphorylation. IL2Rα YT-1 cells were incubated with varying concentrations of IL-2 or IL-2(E52K) in the presence or absence of 5 μg/ml HS for 30 min at 37° followed analysis of phsopho-STAT5 staining by FACS. *: p<0.05, **: p<0.01 by one-way ANOVA.

Fig 5: 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 right 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 oC 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.