Il22jop

Flow cytometry for surface and intracellular staining was performed using standard protocols [52 (link)]. Cells were stained with: CD3 (SP34), CD4 (L200), CCR5 (3A9), Epithelial antigen (Ber-EP4, Dako), TNF-α (MAB11), IFN-γ (B27), Granzyme B (GB11), IL-2 (MA1–17H12), IL-17 (eBio64CAP17, eBioscience), IL-21 (3A3-N2.1) and IL-22 (IL22JOP, eBioscience), Ki67 (B56), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Grand Island, NY), All antibodies and reagents were purchased from BD Biosciences Pharmingen (San Diego, CA) unless otherwise noted. Samples were resuspended in BD Stabilizing Fixative (BD Biosciences) and acquired on a FACS Calibur or LSRII flow cytometer (Becton Dickinson, San Jose, CA). Data was analyzed with Flow Jo software (Tree star, Ashland, OR).

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.

Cells were pelleted, blocked with anti-mouse CD16/32 (1:500, clone 93; 14-0161-85, eBioscience) and stained with CD3-FITC (1:400, 145-2C11, eBioscience). Intracellular cytokine staining was performed using RORγt-PE (1:200, B2D, eBioscience) and IL22-APC (1:100, IL22JOP, eBioscience) antibodies. Intracellular staining was done using the Foxp3/Transcription factor staining buffer set (eBioscience). Cells were recorded using a FACSCanto II flow cytometer. Live single cells were gated using forward scatter (FSC) and side scatter (SSC), and innate immune cells were identified as T-cells (CD3⁺) or innate lymphoid cells (CD3-RORγt⁺) in which IL22 expression was assessed. Recorded data was analyzed with FlowJo v10 (Treestar Inc.).