Fig 2: 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 3: 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 4: Human CXCR4 or CCR5 can form a complex with hCD25.A, schematic of the assay. HEK293T-HA-mCD25 cells were transfected with cDNA encoding the indicated FLAG-tagged murine chemokine receptor and then incubated with 1 μg/ml IL-2 (red) and 5 μg/ml PC61 (purple) for 1 h at 37 °C. Cells were lysed, and immunoprecipitates were fractionated by SDS-PAGE followed by immunoblotting for HA-mCD25 (B) or FLAG-murine chemokine receptor (C). Bar graphs depict quantified intensities of HA-mCD25 or FLAG-chemokine receptors from three independent experiments (mean ± SEM; N = 3). D, IL-2(E52K) supports the hCD25–CCR5 complex assembly. The experiment was performed as in B and C with HEK293T cells expressing HA-hCD25, human chemokine receptors, and the use of 7G7B6 in place of PC61. FLAG-human chemokine receptor was immunoprecipitated, followed by fractionation by SDS-PAGE and immunoblotting for HA-hCD25 or FLAG-chemokine receptor. The black vertical line indicates the site at which two irrelevant lanes were removed, and the remaining lanes were joined. cDNA, complementary DNA; HA, hemagglutinin; hCD25, human CD25; HEK293T, human embryonic kidney 293T cell line; IL-2, interleukin 2; mCD25, murine CD25.

Fig 5: 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.