Fluorophore labeled antibodies

To analyze the expression of M1 or M2-phenotype markers and cytokines, the macrophages were stained with appropriate Fluorophore-labeled antibodies according to the manufacturer’s instructions via fluorescence-activated cell sorting (FACS). Fluorophore-labeled antibodies against the following proteins, along with matched isotype controls, were purchased from BioLegend (San Diego, CA, USA): TNF-α (502912), TGF-β (349610), ARG-1 (17-3697-82), HLA-DR (307616), and CD163 (333606). All data were collected and analyzed using a CytExpert flow cytometer (BD Biosciences, USA).

BMDCs were harvested as described above. Ten thousand cells were seeded in each well of a 96-well plate. First, BMDC transfection was studied using as assay protocol identical to that described for Caco-2 cells. In a separate assay, nanoparticle-mediated immune stimulation was measured. Here, nanoparticles were prepared with ovalbumin encoding mRNA. Nanoparticles were incubated with the BMDCs for 48 h. BMDCs were collected, rinsed, and stained with fluorophore-labeled antibodies (Biolegend, USA). Specifically, CD11c + BMDCs were gated and surface expression of co-stimulatory molecules (CD40, CD80, CD86) and antigen presentation (SIINFEKL:MHCi) were measured using flow cytometry. A schematic of flow cytometry gating is described in Figure S2. Single-cell flow scatter plots of all treatment groups are included in Fig. 3b. The following antibodies were used for flow cytometry: CD11c-APC-Cy7 (clone N418), CD40-FITC (clone 3/23), CD80-BV421 (clone 16–10A1), CD86-PE (clone GL-1), and SIINFEKL:MHCi-APC (clone 25-D1.16).

To analyze recruited inflammatory cells the air-pouches were rinsed with 3 mL of ice-cold PBS, softly kneaded, and exudates were collected. The liquids obtained were centrifuged (5 min, 350 ×g, 4°C). Staining with the Zombie Red Fixable Viability Kit (1:2000, BioLegend) was performed to exclude dead cells. After washing (3% FBS/PBS), cells were incubated (20 min, 4°C) with the following fluorophore-labeled antibodies (BioLegend, if not stated otherwise) to identify major leukocyte populations: anti-CD16/CD32 (1:100, clone 93), anti-CD45-APC-Cy7 (1:150, 30-F11), anti-CD11b-APC (1:800, M1/70), anti-CD11c-AF488 (1:400, N418), anti-Ly6G-APC-Cy7 (1:150, 1A8), anti-SiglecF-PerCP-Cy5.5 (1:100, E50-2440, BD Biosciences), anti-F4/80-PE-Cy7 (1:100, BM8), anti-I-A/I-E (MHC II)-BV421 (1:120, M5/114.15.2), anti-CD19-AF488 (1:150, 6D5), anti-CD3ε-APC (1:120, 145-2C11), anti-CD4-PerCP-Cy5.5 (1:200, RM4-4), anti-CD8a-PE-Cy7 (1:600, 53-6.7), anti-CD152 (CTLA-4)-PE (1:100, UC10-4B9). Fluorescence minus one control was used for CTLA-4, CD11c, and MHC II. After staining, the cells were fixed with 2% formaldehyde (10 min, 4°C), washed, and measured by FACSAria IIIu (BD). The data was analyzed in FlowJo v.10.7 (BD). For further information on the antibodies and their targets, see Table 1.