Nature Immunology: doi: /ni Supplementary Figure 1. Overexpression of Ym1 reduces eosinophil numbers in the lungs.

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1 Supplementary Figure 1 Overexpression of Ym1 reduces eosinophil numbers in the lungs. (a-d) Absolute numbers per mg of tissue of CD8 + T cells (a), CD4 + T cells (b), CD19 + CD11b - B cells (c) and SigF + CD11c - CD11b + eosinophils (d) in the lungs of BALB/c wild-type mice transfected intranasally with 20 µg pcdna3.1 control or plasmids encoding BRP- 39, Ym1 or Ym2. Lungs were collected 48 h after transfection. NS not significant; *P < 0.05 compared to pcdna3.1 transfected mice (analysis of variance with Tukey-Kramer HSD multiple comparison test). Data are pooled from two independent experiments (a-d; mean ± s.e.m., five to nine mice per group).

2 Supplementary Figure 2 Model of acute allergic inflammation. BALB/c wild-type mice were sensitized to OVA-Alum (20 µg, i.p.) on day 0 followed by aerosol challenge with PBS or OVA on days Mice were treated daily with IgG2a isotype control or α-ym1 (200 µg, i.p.) prior to and during aerosol challenge. Samples were collected 24 h after the last challenge.

3 Supplementary Figure 3 Ym1 overexpression increases IL-17A production in γδ T cells in an IL-1-dependent manner. (a) Percentage of IFNγ + γδ T cells in PMA and ionomycin stimulated PECs collected at 48 h from BALB/c wild-type mice administered with pcdna3.1 and plasmids encoding BRP-39, Ym1 or Ym2 (20 µg) or glucose (5 %). (b) Absolute numbers of IL-17 - or IL-17 + TCRγδ + cells per gram of tissue from the lungs of C57BL6 wild-type mice administered with glucose (5 %), pcdna3.1 or a plasmid encoding Ym1 (20 µg) intranasally. Lung was collected 48 h post-transfection and single cells stimulated ex vivo with PMA and ionomycin. (c-d) Percentage of IFNγ + (c) and IL-17A + (d) γδ T cells in PMA and ionomycin stimulated PECs collected at 48 h from BALB/c mice administered with pcdna3.1 and plasmids encoding BRP-39, Ym1 or Ym2 (20 µg) or glucose (5 %) and treated with PBS or anakinra (Ank, 100 mg/kg, i.p.). NS not significant; *P < 0.05 and ** P < 0.01 compared to pcdna3.1 mice (a), pcdna3.1 PBS or Ym1 PBS (cd) (analysis of variance with Tukey-Kramer HSD multiple comparison test). Data are representative from two independent experiments (a; mean ± s.e.m., six mice per group), one experiment (b; mean ± s.e.m., 3 mice per group) or pooled from two independent experiments (c-d; mean ± s.e.m with ten to thirteen mice per group).

4 Supplementary Figure 4 Ym1 does not alter IL-17A production in ILCs. (a) IL-17A expression in PMA and ionomycin stimulated PECs collected 48 h after transfection of BALB/c wild-type (WT) or Rag2 -/- mice with pcdna3.1 or a plasmid encoding Ym1 (20 µg, i.p.). (b) Absolute numbers of IL-17 - or IL-17 + ILCs (Lineage - CD CD25 + ) from PECs of mice as in a. (c) Absolute numbers of Ly6G + CD11b + F4/80 + neutrophils from PEC of mice as in a. Bars show mean values ± SEM. *P < 0.05; compared to pcdna3.1 transfected mice, (analysis of variance with Tukey-Kramer HSD multiple comparison test). Data are representative of two independent experiments (mean ± s.e.m. five mice per group).

5 Supplementary Figure 5 Ym1 reduces secretion of IL-17A and IL-13 from splenocytes following N. brasiliensis infection. (a) IL-17A secretion in splenocytes cultured with medium, NES antigen (Ag, 1 µg/ml) or α-cd3 (1 µg/ml) from uninfected or N. brasiliensis (500 L3s) infected BALB/c wild-type treated intraperitoneally with anti-ym1 or isotype IgG2a. (b) IL-13 secretion measured in mice as per a. Data show Ag and α-cd3 responses normalized to the amount of IL-13 secreted in medium for individual mice. *P < 0.05 and **P < 0.01 and ***P < compared to IgG2a groups (analysis of variance with Tukey s test). Data are representative of two independent experiments (mean ± s.e.m. six mice per group).

6 Supplementary Figure 6 Expression of eyfp in T cell populations from Il17a Cre Rosa26R eyfp mice. eyfp expression in PMA and ionomycin stimulated PECs from uninfected or N. brasiliensis (500 L3s) infected Il17a Cre Rosa26R eyfp mice. Data are representative from two independent experiments.

7 Supplementary Figure 7 A model of chitinase-like protein function during innate immune responses. Expression of Ym1 and Ym2 regulates IL-1β and IL-18 expression leading to the expansion of IL-17A-producing γδ T cells triggering epithelial cells to secrete neutrophil-chemotactic factors CXCL1, CXCL5 and CCL3 resulting in neutrophil infiltration. BRP-39 stimulates IL-18 also leading to IL-17A-production by γδ T cells but in a way that does not trigger downstream neutrophil chemotaxis. During these innate settings, Ym1 and Ym2 cause neutrophils to accumulate, a function that can be host-protective by limiting helminth parasite integrity but at the same time is costly to the host by promoting acute lung.

8 Gene Primer Sequence Rpl13a For CATGAGGTCGGGTGGAAGTA GCCTGTTTCCGTAACCTCAA Chil1 For AGCAGTATTTCTCCACCCTGAT ACCTTTCCTGCTGACAAAGC Chil3 For ACCTGCCCCGTTCAGTGCCAT CCTTGGAATGTCTTTCTCCACAG Chil4 For CAGAACCGTCAGACATTCATTA ATGGTCCTTCCAGTAGGTAATA Il17a For GCTCCAGAAGGCCCTCAGACT CCAGCTTTCCCTCCGCATTGA Cxcl5 For GGGAAACCATTGTCCCTGA TCCGATAGTGTGACAGATAGGAAA Nbactin For ACGACGTGGCAGCTCTCGTTGTGG GGTGCTTCGGTCAGCAGCACGGGA Il1b For TGAGCACCTTCTTTTCCTTCA GCAGCTGTCTAATGGGAACG Il23p19 For CACCAGCGGGACATATGAA CCTTGTGGGTCACAACCAT Il18 For ACAGGCCTGACATCTTCTGC CCTTGAAGTTGACGCAAGAGT Supplementary Table 1 Primer sequences used for quantitative RT-PCR 8