Anti cd11c

To determine chimerism, splenocytes were stained with anti‐H‐2D^d‐FITC (clone: 34–2–12) and anti‐H‐2D^b‐PE (clone: KH95) (all from Biolegend). Treg cells were determined in the spleens according to manufacturer's instructions using a kit from eBioscience (San Diego, CA). Spleen cells were also characterized using anti‐CD11b (clone: M1/70), anti‐CD11c (clone: N418), anti‐B220/CD45R (clone: RA3‐6B2), anti‐I‐A/I‐E (clone: M5/114.152), anti‐CD4 (clone: RM4‐5 and GK1.5), anti‐pDCA1 (clone: 927), anti‐CD8α (clone: 100712), anti‐TCR‐β (clone: H57‐597), all from Biolegend, and a viability marker (LIVE/DEAD^® Fixable Near‐IR Dead Cell Stain Kit, Life Technologies, Eugene, OR). For MACS cell purification, we used anti‐CD11c (cat. 130‐052‐001), anti‐CD4 (cat. 130‐049‐201), and anti‐CD8 (cat. 130‐049‐301) microbeads (Miltenyi Biotec Bergisch Glabach, Germany). For the in vitro functional assays, DC subpopulations were purified from spleens by MACS. CD11c cells were then sorted by FACS (BD FacsAria III) using APC/Cy7‐labeled anti‐B220, PercP‐labeled anti‐I‐A/I‐E, APC‐labeled anti‐pDCA1, PE‐labeled anti‐CD11b and PE/Cy7‐CD8a. We obtained 90.9 ± 1.35% purity for CD8αcDCs, 95.1 ± 1% for CD11b cDCs and 94.± 0.3% for pDCs. All cells were acquired using a FACS‐LSRFortessa according to BD bioscience protocols and analyzed by FlowJo software version 9.8.1.

C57BL/6 mice were vaccinated in both inguinal regions subcutaneously with PBS, PE, PE + MnJ, or PE + aluminum. Inguinal dLNs were collected 12 or 24 h after immunization. dLN cells were labeled with DAPI and anti-CD11c (Biolegend, Cat# 117325), anti-F4/80 (Biolegend, Cat# 123115), anti-CD8a (Biolegend, Cat# 100721), and anti-CD64 antibodies (Biolegend, Cat# 139315) to identify APCs. The ratio of PE⁺ APCs (CD11c⁺ or F4/80⁺ cells) from dLN cells and the ratio of Mo-DCs (CD64⁺ CD8a⁺) in APCs (CD11c⁺ or F4/80⁺ cells) were analyzed by FACS.

Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1: Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).

After measurements of AHR, the trachea was cannulated and the bronchial alveolar lavage (BAL) fluid was collected as described before^{39 (link)}. Briefly, we tracheostomized and intubated the mice and then washed the airways three times with 1 mL of ice-cold PBS each time, followed by centrifuging at 400 × g for 7 min and harvesting the cells. Data were analyzed with FlowJo software (TreeStar, Ashland, Ore). The absolute cell numbers in BAL fluid were calculated by means of flow cytometry by staining the cells with phycoerythrin (PE)–anti-Siglec-F (E50-2440; BD Biosciences, San Jose, Calif; 1/1000), fluorescein isothiocyanate(FITC)–anti-CD19 (6D5; 1/400), peridinin-chlorophyll-protein complex (PerCP)/Cy5.5–anti-CD3ε (17A2; 1/200), allophycocyanin (APC)–anti–Gr-1 (RB6-8C5; 1/1000), PE/Cy7–anti-CD45 (30-F11; 1/500), APC/Cy7–anti-CD11c (N418; BioLegend, San Diego, Calif; 1/400), and eFluor450–anti-CD11b (M1/70; eBioscience, San Diego, Calif; 1/500) in the presence of anti-mouse FC-block (2.4G2; BioXcell, West Lebanon, NH; 1/200). We used CountBright Absolute Count Beads (Thermo Fisher Scientific, Waltham, Mass), according to the manufacturer’s instructions. At least 10⁵ CD45⁺ cells were acquired on a BD FACSCanto II (BD Biosciences) using the BD FACSDiva software v8.0.1.