1 Supplementary Figure 1 Protease allergens induce IgE and IgG1 production. (a-c) Serum IgG1 (a), IgM (b) and IgG2 (c) concentrations in response to papain immediately before primary immunization (day 0), before secondary immunization (day 14) and at day 21 in Balb/c mice. (d) Activity of E64-inactivated papain as measured by IgG cleavage assay. (e,f) Total (e) and antigen-specific (f) IgE responses in Balb/c mice immunized with E64-inactivated papain. (g) Total IgE response in Balb/c mice immunized with bromelain. For each timepoint, n=5-10. Data are representative of at least three separate experiments. Error bars represent s.e.m.; P- values are calculated via the Student s t-test, and represent comparisons to HSA immunizations at the indicated timepoints; ***, P<0.0001; **, P<0.001, *, P<0.01. If not indicated P>0.05.
2 Supplementary Figure 2 IL-4-eGFP expression marks T H 2 cells. T cell populations were sorted from 4get mice immunized as indicated, and were restimulated with anti- CD3 and anti-cd28 for 3 days in vitro. Cytokine concentrations were determined via ELISA. ND: not detected; error bars represent s.e.m. Data are representative of three separate experiments.
3 Supplementary Figure 3 Dermal DCs migrate to the popliteal LN 22 hours postsubcutaneous footpad immunization with papain. (a) Gating strategy for identification of dermal DCs. The CD8α cell population is gated as indicated. (b) Dermal DCs as a portion of total DCs in the popliteal LN after papain immunization. White bar, dermal DCs; gray bar, remaining CD11c + cells. (c) Upregulation of CD86 on bone marrowderived DCs from Balb/c Tlr4 / mice stimulated with papain in vitro. Filled histograms represent unstimulated DCs, black histograms represent indicated stimulus. P-values determined using Student s t-test; *, P<0.05; **, P<0.01. Unless indicated, P>0.05. Plots and percentages are representative of at least three experiments.
4 Supplementary Figure 4 The role of mast cells and basophils in papain-induced T H 2 response initiation. (a) Incubation of bone marrow-derived basophils with FcεR1αspecific MAR-1 in vitro blocked subsequent staining with MAR-1. (b) Bone marrowderived basophils were treated with the indicated stimuli and IL-4 production was measured by intracellular cytokine staining. (c) MAR-1 mediated basophil depletion from spleen and liver, even after papain immunization. (d) Basophil depletion from peripheral blood, spleen, bone marrow and liver after intravenous treatment with MAR- 1. (e) Left, reduced basophil migration into the draining LN 3 days after subcutaneous papain immunization in MAR-1-treated mice. Right, preservation of mast cell populations in the peritoneal lavage and skin after MAR-1 treatment. (f) Impaired T H 2 differentiation in MAR-1-treated mice 8 days after papain immunization; plots are gated on CD4 + DX5 cells. (g) Basophil migration to draining LNs in response to immunization with papain in mast cell (MC) deficient mice (Kit W/W-v ). Plots and percentages are representative of at least three experiments. P-values determined using Student s t-test; *, P<0.05.
5 Supplementary Figure 5 Immunofluorescence of basophils in the popliteal LN following subcutaneous footpad immunization with indicated stimuli. (a) B220 (purple), FcεRI (red), IL-4-eGFP (green); basophils can be identified as FcεRI + IL-4- egfp +. Original magnification 40; insets original magnification 63. (b) Left panel, B220 (purple), right panel, CD4 (purple). FcεRI (red), IL-4-eGFP (green). Original magnification 63. Data represent at least three experiments.
6 Supplementary Figure 6 IL-4 and TSLP dependence of papain-induced T H 2 differentiation. (a) T H 2 differentiation in vivo in response to papain immunization in the presence or absence of antibodies blocking IL-4Rα or neutralizing TSLP. (b) T H 2 development of OVA-specific DO11 4get T cells transferred into Balb/c or IL-4- deficient recipients, which were immunized as indicated. (c) Naïve T cells were stimulated for 48 hours in vitro in the presence of the indicated cytokines and/or neutralizing antibodies, and indicated cytokines were measured by ELISA. ND, none detected; N/A, not tested. (d) CFSE dilution in CD4 + cells stimulated for 2 days in vitro with plate-bound anti-cd3 and anti-cd28 and in the presence of the indicated cytokines and/or neutralizing antibodies. Filled histogram represents the no cytokine control, open histogram corresponds to indicated stimulation. Percentages are of total CD4 + DX5 cells (a) or of OVA-specific CD4 + cells (b); percentages and graphs are representative of two experiments, with 2-4 animals per immunization, per experiment. P-values determined using Student s t-test with comparison to isotype control; *, P<0.05; **, P<0.01.
7 Supplementary Figure 7 Model for protease activation of T H 2 responses. Proteases derived from allergens or parasites cleave a proteolytically sensitive sensor expressed by basophils. Cleavage activates basophils to produce IL-4 and TSLP, which leads to T H 2 differentiation.