Il 23 il 2

Draining lymph nodes from DED mice were harvested and pooled, and CD4⁺ T cells were isolated via negative selection with a CD4⁺ T cell isolation kit (Miltenyi Biotec Inc.). The isolated cells were >98% CD4⁺ as confirmed by flow cytometry, and they were stained with FITC-anti-CD44 (clone IM7) and PE-anti-CD62L (clone MEL-14, BioLegend) antibodies, and sorted for CD44^loCD62L^- effector subpopulation using a MoFlo^® FACS sorter (Dako Cytomation). The sorted CD44^loCD62L^-CD4⁺ cells were treated with IL-23 (10 ng/ml, eBioscience), IL-2 (50 IU/ml, Pepro Tech), IL-23+IL-2, monoclonal CD25 blocking Ab (CD25 mAb, 10 μg/ml, clone PC61.5, eBioscience), or IL-23+CD25 mAb for 48 hours. Memory Th17 cells, as well as expression levels of IL-7R, IL-15R, and Annexin V were then examined by flow cytometry.