Anti il 22

Samples were stained with fluorescence-conjugated antibodies and analyzed using FACS Calibur and Cellquest software or using BD LSR Fortessa and FACS Diva software (Becton Dickinson). All data acquired from flow cytometry was analyzed using FlowJo software (TreeStar). The following antibodies were purchased from eBioscience and BioLegend: anti-CD45 (30-F11), anti-TCR-β (H57-597), anti-TCRγδ (GL3), anti-CD8α (53–6.7), anti-CD8β (eBioH35-17.2), anti-CD4 (RM4-5), anti-CD103 (2E7), anti-Foxp3 (FJK-16s), anti-T-bet (eBio4B10), anti-IL-17A (TC11-18H10.1), anti-IL-22 (IL22JOP), anti-IFN-γ (XMG1.2), anti-IL-10 (JES5-16E3), anti-IL-9 (RM9a4), anti-IL-4 (11B11), and viability stain kit (Zombie Yellow™ Fixable Viability Kit, BioLegend). Cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer (eBioscience) prior to transcription factor staining and intra-nuclear staining. Intracellular cytokine staining was performed after 3.5 h stimulation using 25 ng/ml of Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 3 μM of Ionomycin (Sigma-Aldrich), and 1 μg/ml of Brefeldin A (GolgiPlug™ BD Bioscience). Stimulated cells were permeabilized using Cell Fixation/Permeabilization Kits (BD Bioscience) following the instruction from manufactures.

We used the following antibodies: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE (all from Biolegend, USA), anti-Active Caspase-3 (clone: C92-605)-FITC or -PE (from BD Pharmingen™, USA), and anti-CD69 (clone: H1.2F3)-PE (from eBiosciences, USA).

Flow cytometric analysis was used to measure the number of CCR1-, IFN-γ-, T-bet-, IL-17A-, RORγt, IL-22-, and TNF-α-expressing CD40⁺ cells in the PMBCs. Briefly, PBMCs were stimulated with PMA/ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of a Golgi stop (BD Bioscience, San Jose, USA), as previously described [12 (link),98 (link)]. PBMCs were washed and surface-stained with anti-CCR1 and anti-CD40 antibodies (BioLegend, San Diego, CA, USA). For the staining of cytokines and transcription factors, the cells were fixed, permeabilised, and stained for anti-IFN-γ, anti-T-bet, anti-IL-17A, anti-RORγt, anti-IL-22, and anti-TNF-α (BioLegend, San Diego, CA, USA). Human lymphocytes were isolated from the other immune cells (monocytes and granulocytes) using a conventional gating strategy based on their physical properties (forward and side scatter) to determine different immune markers in the lymphocytes. Chemokine receptors, inflammatory mediators, and transcription factors were identified based on the immunofluorescence characteristics of the antibody-labelled cells in the lymphocyte gate. The proportions of CD40⁺CCR1⁺, CD40⁺IFN-γ⁺, CD40⁺T-bet⁺, CD40⁺IL-17A⁺, CD40⁺RORγt⁺, CD40⁺IL-22⁺, and CD40⁺TNF-α⁺, cells were determined in the lymphocyte gate. Flow cytometry was conducted using an FC500 flow cytometer with CXP software (Beckman Coulter, Brea, CA, USA).