Facsymphony machine

An aliquot (1x10⁶) of total PECs were stained using a viability assay (Zombie UV, Biolegend) and blocked with 5 μg/mL anti-CD16/32 (clone 93; Biolegend) and heat inactivated normal mouse serum (1:10, Sigma-Aldrich) in flow cytometry buffer (0.5% BSA and 2 mM EDTA in Dulbecco’s PBS) before surface staining on ice with antibodies listed in Supplementary Tables 2, 3. For intracellular staining, samples were fixed and permeabilized using eBioscience Foxp3 Staining Buffer kit (Fisher Scientific) following the manufacturer’s instructions before adding antibodies in 1x eBioscience™ Permeabilization Buffer (Thermo Fisher Scientific). All antibodies were purchased from Biolegend unless stated otherwise. PECs were analyzed on a BD FACSymphony machine using BD FACSDiva software (BD Biosciences) and post-acquisition analysis performed using FlowJo v10 software (BD Biosciences). The number of cells per cavity for each population (e.g. F4/80^high resident macrophages) was calculated by determining the percentage of single live cells for each population using FlowJo multiplied by the total number of PECs divided by 100. The gating strategy for flow cytometry analysis is shown in Supplementary Figures 2, 3.

Blood samples collected from the immunized mice were incubated at room temperature for 1 h, followed by centrifugation at 5000 rpm for 10 min at 4 °C. Twenty-five microliters of serum were subjected to bead-based cytokine and chemokine assay (LEGENDplex mouse inflammation panel, BioLegend, San Diego, CA, USA) according to the manufacturer’s recommendations. The analysis was performed on a BD FACSymphony machine (BD Biosciences, Milpitas, CA, USA) using the LEGENDplex data analysis software (BioLegend online software).