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1 Supplementary Information Memory-type ST2 + CD + T cells participate in the steroid-resistant pathology of eosinophilic pneumonia Naoko Mato 1, 2, Kiyoshi Hirahara 2, Tomomi Ichikawa 2, Jin Kumagai 2, Masayuki Nakayama 1, Hideaki Yamasawa 1, Masashi Bando 1, Koichi Hagiwara 1, Yukihiko Sugiyama 1, 3, and Toshinori Nakayama 2, 1 Division of Pulmonary Medicine, Department of Internal Medicine, Jichi Medical University, Yakushiji, Shimotsuke-city, Tochigi 329-3, Japan 2 Department of Immunology, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba , Japan 3 Department of Respiratory Medicine, Nerima-Hikarigaoka Hospital AMED-CREST, AMED, Inohana Chuo-ku, Chiba , Japan Corresponding author: Toshinori Nakayama Telephone: Fax: Mailing Address: Inohana, Chuo-ku, Chiba-shi, Chiba, Japan

2 Supplemental Figure 1 (A) Representative flow cytometry plots of ST2 + CD + T cells in the lung and spleen. (B) The percentage of ST2 + CD + T cells among total CD + T cells in the lung was depicted. (C) Representative flow cytometry plots of ST2 + and ST2 - CD + T cells in the lung. (D) The expression of CD (left) and CD62L (right) on ST2 + (red) and ST2 - (blue) CD + T cells in the lung are shown. The gray-shaded histogram represents the corresponding isotype-matched staining. Two independent experiments were performed with similar results. (E) Representative flow cytometry plots of CD + T cells in the spleen and peripheral blood after the intravenous injection of anti-cd antibody. The isotype represents the corresponding isotype-matched antibody injection. Control: saline-treated mice, : -treated mice. (F) The protocol of -treatment and the following analyses are depicted. (5 μg) was administered intratracheally. Analyses by flow cytometry were performed at the indicated time points. (G) Representative flow cytometry plots of intracellular staining profiles of IL- and IL-5 in CD + ST2 + CD + T cells after saline() or -administration (left panel). Absolute numbers of IL--producing CD + ST2 + CD + T cells in the lung from the two groups are depicted (right panel). The mean values ± SD are shown for four mice in each group. (H) Lung ILC2s were identified as follows: live cells were separated by viability dye, and lineage marker-negative and CD5 + and

3 Thy1.2 + cells were selected and ultimately confirmed by positive staining by CD25 and CD127. (I) Representative flow cytometry plots of ST2 + ILC2s in the lung at the indicated time points are depicted after -administration. (J) Absolute cell numbers of ST2 + ILC2s in the lung from the two groups at the indicated time points are depicted. (K) Representative intracellular staining profiles of IL-5 and IL-13 of ST2 + ILC2s. (L) Absolute cell numbers of indicated cytokine-producing ST2 + ILC2s are depicted. The mean values ± SD are shown for three to five mice in each group. Two independent experiments were performed with similar results (C-E, G-L). Three independent experiments were performed with similar results (A, B). p <.5; Mann-Whitney U test. Supplemental Figure 2 (A) Experimental protocol of intratracheal -treatment and subsequent analyses. (5 μg) was administered intratracheally, and the BAL and peripheral blood samples were analyzed at the indicated time points. (B) The numbers of total leukocytes (left), eosinophils (middle), and lymphocytes (right) at the indicated time points in peripheral blood are shown. The mean values ± SD are shown for four to seven mice in each group. Pooled data from two independent experiments were provided in (B). One-way ANOVA was performed.

4 Supplemental Figure 3 was administered to Gata1 tm6sho mice (deficient in eosinophils), Kit W /Kit W-v mice (deficient in mast cells and basophils). The BAL fluid was analyzed, and a histological examination was performed. (A) Numbers of total leukocytes and eosinophils in the BAL fluid in Gata1 tm6sho mice and Kit W /Kit W-v mice are depicted. (B) Representative microscopic pictures of the lungs stained with hematoxylin-eosin from Gata1 tm6sho mice are shown. (C) Histological scores from HE stained samples are depicted. (D) Representative microscopic pictures of the lungs stained with hematoxylin-eosin from Kit W /Kit W-v mice are shown. (E) Histological scores from HE stained samples are depicted. (F) Representative flow cytometry plots describing the process of preparation of ST2 + ILC2 cells in the lung from mice and Foxn1 nu mice after saline () or -administration are shown. (G) Absolute cell numbers of ST2 + ILC2 cells in the lung from mice and Foxn1 nu mice are depicted. (H) Representative intracellular staining profiles of IL-5 and IL-13 in ST2 + ILC2 cells from mice and Foxn1 nu mice (upper panel). Absolute cell numbers of the indicated cytokine-producing ST2 + ILC2 cells are depicted from mice and Foxn1 nu mice (lower panel). The mean values ± SD are shown for each group (A, G, and H). Representative data were obtained from two individual experiments (A), and pooled data (n=5-7) from two independent experiments were provided in (G, and H). : wild type BALB/c mice, Kit + /Kit + :

5 littermate of Kit W /Kit W-v mice. p<.5: One-way ANOVA. Supplemental Figure (A) Experimental protocol of adoptive transfer prior to the -administration and subsequent analyses is depicted. Naïve CD + T cells obtained from spleen of Thy1.1 congenic mice or BALB/c mice were transferred to NSG mice, and saline () or (5 μg) was administered intratracheally on the next day, and the FACS was performed at day 1, and BAL, AHR and histological analyses were performed at day1. (B) To distinguish lung tissue-localized CD + T cells and blood-borne CD + T cells, anti-cd antibody was injected intravenously and analyzed three minutes later. In the CD + T cells in the lung, injected antibody-unstained cells (CD (i.v.) - cells) indicate lung tissue-localized CD + T cells. Representative flow cytometry plots of ST2 + CD (i.v.) - CD + T cells in the lung of wild type mice (BALB/c) and NSG mice with adoptive transfer at day1. (C) Percentage of ST2 + CD + T cells in the lung from three groups in Figure A are depicted. (D) Representative flow cytometry plots of lung tissue-localized CD + T cells in the lung at day1 are depicted. The histogram represents the expression of CD on lung tissue-localized CD + T cells. p<.5: one-way ANOVA. Supplemental Figure 5

6 (A) The protocol of administration of with or without dexamethasone is depicted. (B) Absolute cell numbers of the ST2 + ILC2 cells are depicted from three groups. The mean values ± SD are shown for eight to ten mice in each group. (C) Representative intracellular staining profiles of IL-5 and IL-13 in ST2 + ILC2 cells. (D) Absolute numbers of indicated cytokine-producing ST2 + ILC2 cells in the lung from three groups are depicted. The mean values ± SD are shown for eight to ten mice in each group. Representative data were obtained from two individual experiments (C), and pooled data (n=8-1) from three independent experiments were provided in (B, D). p<.5: one-way ANOVA.

7 Supplemental Figure 1 A CD + T cells Spleen 1 3 Lung B ST2 + CD + T cells (%) spleen lung C ST2 CD Lung ST B 2.5 CD62L CD ST TCRβ CD ST2 CD 97.5 D CD + T cells in the lung 1 CD62L CD Max of % Isotype ST2 + ST2 - E CD CD + T cells CD62L day 5 Spleen Isotype Blood ST Isotype..2 CD (i.v.)

8 Supplemental Figure 1 (Continued) F intratracheal administration of (5 μg/mouse) (days) FACS analysis G IL- CD + ST2 + CD + T cells in the lung IL IL- + CD + ST2 + CD + T cells (x1 2 ) 12 6 H Lung gating strategy for defining ILC2s I Lineage Viability dye Thy1.2 CD5 CD127 CD25 J 9.7 ST2 + ILC2s in the lung day day 3 day 1 day ST2 + ILC2 cells (x1 ) 7.5 ST2 + ILC2 cells (x1 ) day 1 ST2 CD5

9 Supplemental Figure 1 (Continued) K ST2 + ILC2s in the lung day day day L IL-13 IL-5 IL-5 + cells 2.5 IL-13 + cells 3 IL-5 + IL-13 + cells IL-5 + ILC2 cells (x1 ) day 3 day IL-13 + ILC2 cells (x1 3 ) day day 1 IL-5 + IL-13 + ILC2 cells (x1 ) 6 3 day 3 day 1 6 3

10 Supplemental Figure 2 A intratracheal administration of (5 μg/mouse) (days) B Peripheral blood Assay Total leukocyte Eosinophil Lymphocyte 6 15 cell numbers (x1 3 ) 3 cell numbers (x1) 7.5 cell numbers (x1 3 ) (days) (days) (days)

11 Supplemental Figure 3 A B D F BALF cell numbers (x1 5 ) Gata1 tm6sho Gata1 tm6sho Kit + /Kit + Kit W /Kit W-V Kit + /Kit + Kit W /Kit W-V Gata1 tm6sho Total leukocyte Eosinophil Total leukocyte Eosinophil Lung Lung Lung Gata1 tm6sho 1μm cell numbers (x1 5 ) KitW/KitW-v 1μm HE stain HE stain C E Histological Score Histological Score cell numbers (x1 5 ) Kit W /Kit W-V cell numbers (x1 5 ) G 1 5 ST2 + ILC2 cells(x1 ) 1 5 Foxn1 nu Lineage Thy1.2 ST2 Viability dye CD5 CD Foxn1nu Viability dye CD ST2 CD Lineage Thy1.2

12 Supplemental Figure 3 (Continued) H ST2 + ILC2s in the lung Foxn1 nu IL-13 IL IL-5 + cells IL-5 + ILC2 cells (x1 ) 2 Foxn1 nu IL-13 + cells IL-13 + ILC2 cells (x1 2 ) 12 6 Foxn1 nu IL-5 + IL-13 + cells IL-5 + IL-13 + ILC2 cells (x1 ) Foxn1 nu

13 Supplementary Figure A adoptive transfer of CD T cells from BALB/c intratracheal administration of (5 μg/mouse) NSG (days) B FACS analysis BAL, AHR, Histology CD + T cells in the lung NSG CD T ST CD (i.v.) C CD + T cells in the lung 25 ST2 + CD + T cells (%) 12.5 NSG CDT+ D Lung tissue-localized CD + Tcells NSG CD T CD CD % of Max CD NSG CD T +

14 Supplemental Figure 5 A intratracheal administration of (5 μg/mouse) (days) Dexamethasone 1 mg/kg Assay B C ILC2s in the lung ST2 + ILC2s in the lung ST2 + ILC2 cells (x1 ) 2 1 DEX +DEX IL IL DEX D IL-5 + cells IL-5 + ILC2 cells (x1 ) 8 DEX +DEX IL-13 + ILC2 cells (x1 2 ) IL-13 + cells 8 DEX +DEX IL-5 + IL-13 + cells IL-5 + IL-13 + ILC2 cells (x1 ) 8 DEX +DEX

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