Lymphocytes isolation and Flow Cytometry analysis

The PPs from the intestine were collected, grounded and rinsed through 40 μm mesh cell strainers filled with cell culture medium for harvesting cell suspension. The cells were counted by using a blood counting chamber and used in approximately 1x10⁶ cells/ml concentrations. For the monocyte/macrophages panel, the cells were stained with antibodies against surface markers of interests (anti-mouse CD45R-BUV496, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c-AF700). For the B cells panel, the cells were stained with antibodies against surface markers of interests (anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, and CD3-FITC). For the T cells panel, incubation with the following antibodies against surface markers of interests was done (anti-mouse CD45-APC-cy7, CD3-FITC, CD4-V450 and CD8-BV510). For measurement of intracellular markers (anti-mouse IL-4-PE-Cy7, IL-17A-BV650 and IFN-γ-PE), the cells were fixed, permeabilized with 1×Perm Buffer IV and subsequently stained with the above-mentioned antibodies. The staining procedure was performed in the dark. For T cell stimulation, the cells were additionally incubated with or without RMA/Iono for 6 h. After washing with the Flow Cytometry (FACS) buffer, the cells were then resuspended in 500 μl FACS buffer and measured with BD FACS Fortessa. The gating strategies in detail were present in Figure S1 and Figure S2.