Mhcii a700

C57BL/6 mice (Jackson) were infected with 10⁶ Uvitex 2B-labeled (10 µg/ml; 5 min) yeast cells administered intratracheally. Single cell suspensions were made from harvested lungs with a 70-µm cell strainer and treatment with collagenase D (1 mg/ml; Roche) and DNase (50 U/ml; Roche). Leukocytes were enriched by density sedimentation (60%/40% Percoll, 20 min, relative centrifugal force of 600) and collection of cells at the interface. Approximately 10⁶ cells were stained for cellular markers and fixed with 4% paraformaldehyde (30 min). The markers included CD64-fluorescein isothiocyanate, CD45-peridinin chlorophyll protein-Cy5.5, SiglecF-phycoerythrin-Cy7, Ly6C-BV650, CD11c-BV786, CD90.2-allophycocyanin (APC), B220-APC, MHCII-A700, Ly6G-BUV395 (all from BioLegend), and Near IR live/dead stain (Thermo, Fisher). Cells were analyzed with an LSRII flow cytometer (BD Biosciences), and data were processed with FlowJo software (version 10.1).

Cells were harvested and stained for 20 min at 4°C with the antibody mix. After a wash in PBS with 2% of FCS, cells were stained with Fixable Viability Dye eFluor 506 (eBiosciences, 65–0866-14) for 10 min at room temperature. Cells were then fixed for 20 min in 3.2% PFA at room temperature. mAbs in the staining mix for BMDCs or splenocytes were the following: CD11c PeCy7 (N418), MHC II PE (M5/11.15.2), CD86 FITC (GL-1), CD86 PE (GL-1), CD80 PeCy5 (16–10A1), CD40 APC (HM40–3), CD24 FITC (M1/69), NK1.1 APC-Cy7 (PK136), PDL-1 BV605 (10F.9G2) from Biolegend; MHCII A700 (M5/11.15.2), CD11b eF450 (M1/70), Bst-2 EF610 (eBio927), DC-SIGN APC (eBio22D1) from eBiosciences; CD40 PeCy5 (3/23), B220 PETXR (RA3–6B2), CD3 APC-Cy7 (145–2C11), CD19 APC-Cy7 (1D3), CD64 PE (X54–5/7.1), CD8α BV711 (53–6.7) from BD Biosciences. Flow cytometry was performed using a FACS LSRII-UV (BD Biosciences) and data were analysed with the FlowJo_v9.9.4 software.

Flow cytometry data were acquired using a Cytek Aurora equipped with violet (405 nm), blue (488 nm), and red (640 nm) lasers. The following antibodies and stains were used: CCR2-BV421 (clone 475301, 747963; BD), Ly6C-BV510 (clone HK1.4, 128033; Biolegend), B220-BV605 (clone RA3-6B2, 103243; Biolegend), Ly6G-BV711 (clone 1A8, 127643; Biolegend), CD11c-BV785 (clone N418, 117336; Biolegend), CX3CR1-A488 (clone SA011F11, 149021; Biolegend), CX3CR1-BV785 (clone SA011F11, 149029; Biolegend), CD163-PE (clone TNKUPJ, 12-1631-80; Thermo Fisher Scientific), CD64-PE/Cy7 (clone X54-5/7.1, 139313; Biolegend), MRC1-PCP5.5 (clone C068C2, 141716; Biolegend), CD11b-PEFire640 (clone M1/70, 101279; Biolegend) TCRb-APC (clone H57-597, 109212; Biolegend), MHCII-A700 (clone M5/114.15.2, 107622; Biolegend), XCR1-APC/Cy7 (clone ZET, 148223; Biolegend), CD45-APC-Fire810 (clone 30F11, 103173; Biolegend), CD45.1-A647 (clone A20, 110720; Biolegend), CD45.2- BV785 (clone 104, 109839; Biolegend), and LIVE/DEAD Violet dead cell stain kit (L34955; Thermo Fisher Scientific). Flow cytometry data was analyzed using Flowjo 10 software. Dimensionality reduction and cluster identification were performed using the UMAP and Phenograph packages, respectively.