Supplementry Informtion Cutneous immuno-surveillnce nd regultion of inflmmtion y group 2 innte lymphoid cells Ben Roediger, Ryn Kyle, Kwok Ho Yip, Nitl Sumri, Thoms V. Guy, Brin S. Kim, Andrew J. Mitchell, Szun S. Ty, Rohit Jin, Elizeth Fores-Bm, Xi Chen, Philip L. Tong, Holly A. Bolton, Dvid Artis, Willim E. Pul, Brr Fzeks de St. Groth, Michele A. Grimldeston, Grhm Le Gros & Wolfgng Weninger
Supplementry Figure 1 Spleen B22 81 5.93 82.3 4.5 79.7 7.38 3.1 83.2 Spleen B22 CD11 NK1.1 11.6 1.54 NK1.1 13.2.123 35 (NKp46) 1.4 2.53 CD49 (DX5) 12 1.77 CD9 NK1.1 1.7 Supplementry Figure 1. expression y nturl killer (NK) nd NKT cells. () Representtive dotpts depicting expression y NK1.1, NKp46, DX5 nd CD9 NK cells in wild-type C57BL/6 mice. () expression y splenic NK1.1 NKT cells. Results re representtive of 2 independent experiments (n = 4).
Supplementry Figure 2 Whole skin CD9 hi CD9 hi CD9 hi CD9 hi 4.88 2.99.621.69 CD4 2 3 4 5 1 1 1 1 CD5 CD8 NK1.1 CD9 hi CD9 hi CD9 hi CD9 hi.275 CD9 3.9 CD9 2.21.57 NKp46 2 3 4 5 1 1 1 1 CD11c Fc R1 Ly6G/C (Gr-1) CD9 hi CD9 hi CD9 hi CD9 hi 98.1 25.8 CD9 T1-ST2 44.8 CD9 CD44 CD13 Ly6A/E (Sc-1) 34.2 CD9 hi CD9 hi CD9 hi CD9 hi 7.34 CD9 19.5 CD9 2.36 CD9 8.2 5 CD132 CD117 (c-kit) 78 (ICOS) Supplementry Figure 2. Phenotype of skin ILC2. Representtive histogrms depicting surfce expression of indicted mrkers y CD9 hi dilc2 isolted from the skin of wild-type mice. Contour pts indicte the presence of other leukocytes ering the mrker of interest within the sme smple, requisite internl positive control to exclude possile misinterprettion due to enzyme clevge rtifcts. Results re compiled from 6 independent experiments (n 2 per mrker).
Supplementry Figure 3 CD9 Whole skin Bl/c C57BL/6 Rg1 / 9.2 6.2 12.1 Tcr / Tcrd / Tcr / Tcrd / 5.7 7.1 17.9 Foxn1 nu/nu 17.7 T cells norml norml reduced reduced ttenuted ttenuted sent sent dilc2 cells present present present present expnded expnded expnded expnded C57BL/6 Bl/c Tcr / Tcrd / NOD SCID Foxn1 nu/nu (nude) Rg1 / Tcr / Tcrd / Supplementry Figure 3. Reltionship etween T cells nd ILC in the skin. () Representtive dotpts of cells in the skin of indicted mouse strins. Gtes indicte CD9 hi ILCs. Numers indicte percent positive cells within ech gte. () Summry of T cell nd ILC composition in indicted mouse strins. Dt re representtive of t lest two independent experiments for ech strin.
Supplementry Figure 4 CD9 Whole skin C57BL/6.6 dsred ethnol 4C13R CD9 hi NK1.1 Il13-dsRed ethnol 32.7 45.4 MC93 Il13-dsRed dilc2 (x ) NS 6 4 2 EtOH MC93 IL-13 (pg/g) NS.5.4.3.2.1 EtOH MC93 Supplementry Figure 4. IL-13 production y dilc2 in response to MC93 in vivo. () Left, representtive dotpts of dilc2 in 4C13R trnsgenic mice topiclly treted with MC93 or vehicle (ethnol, EtOH). Right, solute numer of Il13-dsRed dilc2 in 4C13R mice topiclly treted with MC93 or EtOH. Dt re men ± s.d. nd were pooled from 2 independent experiments (n = 6 for MC93, n = 4 for EtOH) () IL-13 protein detection within skin homogentes of MC93-treted mice nd vehicle (ethnol, EtOH) treted controls, s detected y cytokine ed rry. Dt re men ± s.d. (n = 5 for MC93, n = 4 for EtOH). NS, not significnt.
Supplementry Figure 5 1 5 IL-6 relese (ng/ml) 8 6 4 2 ** TNF relese (pg/ml) 4 3 2 1 * UT 1 1 1 UT 1 1 1 IL-9 (pg/ml) DNP (2ng/ml) IL-9 (pg/ml) DNP (2ng/ml) Supplementry Figure 5. Role of ILC2-derived cytokines on mst cell function. (,) Effect of exogenous IL-9 on cytokine production y mst cells in vitro. Mst cells were pre-incuted with rmil-9 ( 1, pg/ml) nd sensitized with nti-dnp IgE (2 µg/ml) for 16 h. Cells were wshed nd then stimulted with DNP-HSA (2ng/ml) in the presence of rmil-9 ( 1, pg/ml) for 6 h. () IL-6 nd () TNF in superntnt were mesured y ELISA. Dt in () nd () re expressed s men ± s.e.m. from 3 independent experiments. Significnt differences in cytokine relese y DNP ne nd in the presence of IL-9 were determined y one wy ANOVA with Dunnett s post-test. *P <.5, ** P <.1.
Supplementry Figure 6 CD9 UT nti-5 nti-cd9 CD11 CD11 CD11 2.54 2.38.13.2 Spleen (Rg1 / mice) 2.5 2.33.12 CD9 hi ILC (%).8.6.4.2 UT nti- 5 nti- CD9 Whole skin (Rg1 / mice) CD9 UT nti-5 nti-cd9 CD11 CD11 CD11 16 28.4 22.9 CD9 hi ILC (%) 15 1 5 UT nti- 5 nti- CD9 Supplementry Figure 6. ILC in the spleen nd skin of mice treted with nti-5 nd nti-cd9. () Left: Representtive contour pts of CD11 splenocytes from untreted Rg1 / mice nd mice treted with either nti-5 (cne PC61) or nti-cd9.2 (cne 3H12). Mice were treted with 2 µg ntiody per dy for two dys prior to ssessment. CD9-expressing cells were detected using cne 53-2.1. Red oxes indicte CD9 hi ILC. Right: percentge of CD9 hi ILC (of totl cells) in the spleens of ntiody-treted mice. () Left: Representtive contour pts of CD11 skin cells from untreted Rg1 / mice nd mice treted with either nti-5 or nti-cd9 s descried in (). Red oxes indicte CD9 hi dilc2. Right: Percentge of CD9 hi dilc2 (of totl cells) in the skin of ntiody-treted mice. Dt in () nd () re men ± s.e.m. from 2 independent experiments. Symols represent individul mice.
Supplementry Figure 7 NKp46 Lmin propri (Rg1 / Cxcr6 gfp/gfp mice) CD11 CD9 NK1.1 NK1.1 21.5 77.4 NK1.1 17.8 KLRG1 3.6 Cxcr6-eGFP 32.1 NKp46 91.3 2.1 41.2.3 KLRG1 2.6 5 1.8 3.2 87.2 Spleen CD9 Lung Mes. Skin Lmin prop. NK cell ILC2 ILC2 ILC2 ILC2 ILC2 ILC22 hi hi hi hi hi hi hi -/ -/ -/ /- /- /- hi hi /- hi /- /- /- /- /- /- hi hi hi /- hi hi hi /- /- hi NKG2D 7 T1-ST2 8 9 CD49 CD49d CD49e CD69 CD84 IL13R 1 IL15R 44.2 Fc R1 Integrin 7 /- /- 5 ICOS CD13 CD9 Sc-1 Cxcr6-eGFP CD117 NKp46 KLRG1 Supplementry Figure 7. Comprtive phenotype of NK cells, NKp46 ILC nd ILC2 in Rg1 / mice. () Representtive dotpts depicting Cxcr6-eGFP nd 5 expression y NK1.1 NK cells, NKp46 ILC22 nd KLRG1 ILC2 (ref. 38) isolted from the lmin propri of Rg1 / Cxcr6 /gfp mice. Dt re representtive of two independent experiments (n = 2). () Summry of cell surfce expression (fw cytometry) of indicted proteins y NK cells, NKp46 ILC nd ILC2 from spleen, lung, mesentery, skin nd lmin propri of Rg1 / mice., not detected;, positive; hi, high expression;, w expression; /, expression y some ut not ll cells. Dt re summrized from 3 independent experiments (n 2 per mrker).
Supplementry Figure 8 Er skin (Rg1 / Cxcr6 /gfp mice) UT Til skin (Rg1 / mice) UT IL-2 JES6-1 SHG Cxcr6- egfp evns lue IL-2 JES6-1 c Er skin (Rg1 / mice) d Til skin (Rg1 / mice) Supplementry Figure 8. IL-2-induced skin inflmmtion. () Representtive imges of IL- 2 JES6-1 treted nd untreted Rg1 / Cxcr6 /gfp mouse skin y intrvitl multiphoton microscopy. Ech imge is z-projection through volume of 88 m within the dermis, otined 3 min fter injection of Evns lue (red). Extrcellulr mtrix in the dermis ws detected y SHG signls (lue). dilc2 shown in green. () Til sections otined from untreted nd IL-2- treted Rg1 / mice, stined with hemtoxylin nd eosin. Yelw rrows indicte eosinophils. (c) Er section otined from n IL-2-treted Rg1 / mouse, cut through spontneous lesion. Enlrged regions indicted the thickened epidermis with ttched crusts nd leukocyte infiltrte. (d) Til sections otined from n IL-2-treted Rg1 / mouse, stined with hemtoxylin nd eosin (left) or toluidine lue (middle nd right). Enlrged region indictes rounded, hypodense mst cells. Dt in (), (), (c) nd (d) re representtive of two independent experiments (n = 4).