Isolation and Analysis of Mucosal Immune Cells

Cells from the colon and small intestine lamina propria were isolated as previously described^{8 (link)}. The cells were stimulated and stained as previously described^{8 (link)}. The following antibodies were used for surface staining of: CD3 (145-2C11, eBioscience); CD4 (L3T4, BD Difco); CD11b (M1/70, eBioscience); CD11c (N418, eBioscience); F4/80 (BM8, eBioscience); CD103 (M290, BD Difco); major histocompatibility complex (MHC) II (M5/114.15.2, BD Dico); TCR-γδ (eBioGL3, eBioscience); and NKp46 (29A1.4, eBioscience). Intracellular cytokine staining was performed using IL-17A (TC11-18H10, BD Difco) and IL-22 (IL-22JOP, eBioscience) antibodies. All antibodies were used at final concentration of 1 μg/ml. The cells were analyzed using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Leukocytes were gated using forward scatter (FSC) and side scatter (SSC), and within the leukocyte gates, the innate immune cells were identified as macrophages (MHCII⁺F4/80⁺CD103⁻CD11b⁺CD11c⁻) or dendritic cells (MHCII⁺F4/80⁻ CD103^+/−CD11b⁻CD11c⁺). For the lymphoid compartment, the leukocytes were gated using FSC and SSC. Within the lymphocyte gate, the populations were identified as T_H17 cells (CD3⁺CD4⁺IL-17⁺IL-22⁺), T_H22 cells (CD3⁺CD4⁺ IL-17⁻IL-22⁺), NKp46⁺ ILCs (including ILC3 and NK cells; CD3⁻CD4⁻NKp46⁺), LTi cells (CD3⁻CD4⁺NKp46⁻), γδ T cells (CD3⁺CD4⁻TCRγδ⁺) or CD3⁻CD4⁻NKp46⁻ cells.