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Supporting information Supplementary Methods Histological analysis

Supporting information Supplementary Methods Histological analysis

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Supporting information Supplementary Methods Histological analysis

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  1. Supporting information Supplementary Methods Histological analysis Lung and NALT tissue samples from immunized mice were fixed in 4% paraformaldehyde (Nakalai Tesque) and then subjected to a sucrose gradient (10%–30%). Nasal passages were decalcified by incubating in 0.75 M EDTA solution at 60 °C for 2 days. The tissue was embedded in Tissue-Tek compound (Sakura Finetek, Torrance, CA, USA) and cut into 7-μm–thick (lung tissue) or 11-μm–thick (NALT tissue) sections. The sections were stained with 4,6-diamidino-2-phenylindole (Invitrogen), anti-mouse TSLP Ab (Millipore), and biotinylated anti-CD11c Ab (BD Biosciences), and the signal was detected and quantitated by using the TSA (Tyramide Signal Amplification system) (PerkinElmer, Waltham, MA) according to the manufacturer’s instructions. All samples were mounted and then analyzed by DMIRE2 TCS SP2 confocal microscopy (Leica, Wetzlar, Germany). In vitro DC cultures We used the mouse bone marrow–derived DC 2.4 cell line for in vitro stimulation studies. Cells (1 × 105 cells/well) were cultured with medium alone or medium plus 20 ng/ml of rTSLP (R&D Systems) for 48 h in flat-bottomed 48-well plates. Cells were harvested and then subjected to FACS analysis or RT-qPCR.

  2. Primers for qPCR Joo et al. Supplementary table 1.

  3. a PspA only PspA+CT Figure S1. TSLP expression in nasally immunized mice. Each mouse was nasally immunized weekly for three consecutive weeks with 5 µg PspA only or 5 µg PspA plus 1 µg CT. TSLP expression in frozen sections of (a) lung tissue or (b) NALT was detected by using immunofluorescence assays. Tissue sections were prepared and stained with anti-TSLP (green), anti-CD11c Ab (red), and DAPI (4',6-diamidino-2-phenylindole; blue). Arrows indicate the cells where co-localization of TSLP with CD11c was observed. Data are representative of two independent experiments (n = 2 or 3 per group). (c) TSLP+ CD11c+ cells were counted in the fields of the tissue sections (three fields per section). Data are means ± SEM (n = 5 or 6 per group). **P < 0.01. 150µm X20 150µm 75µm b PspA only PspA+CT 75µm 75µm c ** ** Joo et al. Supplementary Figure 1.

  4. (All live cell+ CD45+ gated) a WT spleen WT lung CD11c+MHCⅡ+ DCs CD19+ B cells CD3e+ T cells isotype TSLPR b spleen (All live cell+ CD45+ gated) macrophages (CD11cintMHCⅡ+CD11b+F4/80+) CD8α+ DCs (CD11chighMHCⅡ+CD11b-CD8α+F4/80-) CD11b+ DCs (CD11chighMHCⅡ+CD11b+CD8α-F4/80-) isotype TSLPR NALT NP lung macrophages (CD11cintMHCⅡ+CD11b+F4/80+) CD103+ DCs (CD11chighMHCⅡ+CD11b-CD103+F4/80-) CD11b+ DCs (CD11chighMHCⅡ+CD11b+CD8α-F4/80-) isotype TSLPR Figure S4. Comparison of TSLPR expression between immune cells. (a) Flow cytometric analysis of TSLPR expression in isotype control, CD3e+ T cells, CD19+ B cells, and CD11c+MHC II+DCs from spleen and lung tissue of mice. (b) Mononuclear cells from spleen, NALT, NP, and lung tissue were isolated for analysis of surface TSLPR expression on each myeloid DC subsets according to the expression of CD8α, CD103, CD11b, and F4/80 on live, CD45+ CD11c+MHC II+cells. Data are representative of three independent experiments. Joo et al. Supplementary Figure 4.

  5. SP NALT NP lung I-Ad Figure S5. Activation status of DCs is not affected by the absence of TSLP-TSLPR signaling in the steady state. Expression of the activation markers I-Ad, CD40, and CD86 was determined by flow cytometry in cells isolated from spleen (SP), NALT, NP, and lung tissue of non-immunized WT and TSLPR-KO mice. Data are representative of two independent experiments. CD40 CD86 WT CD11c+DCs TSLPR-KO CD11c+DCs Joo et al. Supplementary Figure 5

  6. a isotype medium TSLP 20ng/ml counts TSLPR IL-7Rα Figure S6. IL-6 production in DC cells treated with recombinant mouse TSLP. (a) The cell-surface expression of TSLPR and IL-7Rα were analyzed by flow cytometry of DC 2.4 cells. Dotted lines, isotype control; blue line, DC cells cultured with medium alone; red line, DC cells stimulated with 20 ng/ml of rTSLP for 48 h. (b) mRNA levels were normalized to that of β actin for the indicated genes in DC 2.4 cells stimulated with rTSLP (20 ng/ml) for 48 h. Data are expressed relative to the level in cells with medium alone (n = 3 per group). Data are representative of two independent experiments. N.D., not detected. *P < 0.05 versus cells with medium alone. b Joo et al. Supplementary Figure 6.

  7. a CD3e+ gated Spleen NP Lung 9 10 7 PspA only (control) 10 14 7 CT only (control) counts 9 19 9 PspA+CT TSLPR b CD19+ gated Spleen NP Lung 19 12 11 PspA only (control) 17 15 20 counts CT only (control) 17 23 22 PspA+CT TSLPR Figure S7. TSLPR expression in T and B cells of nasally immunized mice. Flow cytometric analysis of TSLPR expression in (a) CD3e+ T cells and (b) CD19+ B cells from spleen, NP, and lung tissue of mice nasally immunized with 5 µg PspA only, 1 µg CT only, or 5 µg PspA plus 1 µg CT (open histogram). Mean fluorescence intensity indicating TSLPR expression is shown. Shaded histograms indicate isotype control. Data are representative of two independent experiments. Joo et al. Supplementary Figure 7.

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