Figure S1 (A) Analysis of purified myeloid DCs and T cells. Freshly isolated mDCs were processed for cytospins (A), stained with haematoxylin-eosin (x400 magnification, scale bar represents 50  m). The purity of DCs (B and C) and T cells (D) has been checked by HLA-DR, CD1c, CD11c (not shown), CD3, CD4, CD20 and CD14. Data are representative of 3 experiments. For ICOSL and PD-L1 analysis, mDCs were stained with Lin-1, HLA-DR and CD11c or their isotype controls freshly (E) or after 18hrs culture (F). After washing, mDCs were analyzed by FACS. Lin-1- HLA-DR+ CD11c+ cells were gated for further analysis (F). Data are representative of 8 experiments.

(B) (C) SSC FSC HLA-DR CD1c HLA-DR CD3 control SSC control SSC CD14CD20 Figure S1 DC phenotype (freshly isolated)

control CD3 CD4 HLA-DR CD3CD1c T cell phenotype (freshly isolated) Figure S1 (D) SSC

Figure S1 (E) FSC SSC Lin-1 -1CD11c SSCHLA-DRHLA-DRHLA-DRHLA-DR control DC phenotype (freshly isolated)

FSC Lin-1CD11c SSCHLA-DRHLA-DRSSCHLA-DRHLA-DRSSCHLA-DRHLA-DR (F) control Figure S1 Analysis (ICOSL, PD-L1) DC phenotype (cultured for 18 hrs)

Expression of ICOSL and PD-L1 in freshly isolated and in cultured mDCs. Freshly isolated mDCs or cultured (for 18 hrs) mDCs were stained for ICOSL or PD- L1 (or their isotype controls). Data are from 3 separate experiments. Figure S2 Fresh Cultured (18hrs)

(A) (B) Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  Figure S3 mDC viability in culture, upon LPS stimulation. mDCs were isolated and cultured with LPS at different concentrations and time-periods (medium only as control), and stained with propidium iodide (PI) before FACS analysis (A). In addition, mDCs were counted and percentage is shown as reported to baseline (day 0). Data are from 3 experiments (*P < 0.05, ***P < 0.001).

Expression of B7 co-stimulatory molecules in LPS-activated mDCs. mDCs were cultured overnight with LPS at different concentrations for 18hrs, and then stained with anti-ICOSL (A), anti-PD-L1 (B), anti-CD80 (C), anti-CD86 (D) or their isotype controls. Data are from 3 experiments (*P < 0.05, **P < 0.01, ***P < 0.001). Figure S4 (A) (C) (D) Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  Medium 0.01  g/ml 0.1  g/ml 0.5  g/ml 1  (B)

Expression of ILT3, ILT4 and OX40L in mDCs from allergic rhinitis patients and controls. DCs from allergic rhinitis patients or controls were isolated and stimulated by LPS (1  g/ml). Immunostaining and FACS analysis were performed as described in Figure 1. ILT3 (A), ILT4 (B) and OX40L (C) positive cell percentages are shown. Data are mean values ± SEM from experiments (*P < 0.05, **P < 0.01, ***P < 0.001). Figure S5 (A)(B)(C) Rhinitis Controls Rhinitis Controls Rhinitis Controls

Figure S6 Expression of ICOSL and PD-L1 in LPS-activated mDCs. mDCs were cultured overnight with LPS (1  g/ml), and then stained with anti-ICOSL, anti-PD- L1 or isotype controls. ICOSL (A) and PD-L1 (B) expression is shown for allergic asthma patients (n=9) versus controls (n=8). Data are mean values ± SEM (*P < 0.05, **P < 0.01). Controls Allergic asthma Controls (B) (A)

Figure S7 Cytokine production in co-cultures at different mDC and CD4 + T cell ratios. mDCs were isolated and, after 18hrs, co-cultured for 5 days with allogenic CD4 + T cells. At each indicated ratio, a fixed number of T cells was added to different numbers of mDCs. Supernatants were collected and immuno-assayed for cytokines. Data are from two separate experiments.

Cytokine production by LPS-activated mDCs. mDCs were cultured with LPS at different concentrations and time periods, supernatants were assayed for IL-12p40 (A), TNF (B) and IL-10 (C). Data are mean values ± SEM. Each condition represents 3 experiments (**P < 0.01, ***P < 0.001). (A) (C) (A) Figure S8 (B) 18h42h66h TNF *** pg/ml

Figure S9 Medium TSLP Expression of ICOSL and PD-L1 of mDCs in presence of TSLP. mDCs from control donors were cultured with TSLP or IL-33, or both for 18 hrs, before staining with anti-ICOSL (A), anti-PD-L1 (B), or their isotype controls. Data are representative of 3 experiments.