At day 14 and 28 after the corneal NC, the cornea/conjunctiva, draining lymph nodes, and spleens were extracted and collected. The proportion of mature dendritic cells (DCs) was determined by measuring the expression of the costimulatory molecule (CD86) or major histocompatibility complex (MHC) class II on CD11b- or CD11c-expressing cells, using flow cytometry. To determine the subset of the effector T cells, the proportion of IFN-γ, IL-17A, CD103, and the CD69 expression on CD4 or CD8 T cells were assessed. For regulatory T cells, simultaneous expression of CD25 and Forkhead box protein 3 (Foxp3) on CD4 or CD8 T cells were measured.
To get the cell suspensions, the collected lymph nodes and spleens were minced between the frosted ends of two glass slides in RPMI media (WelGENE, Daegu, Korea). The media contained 10% fetal bovine serum and 1% penicillin-streptomycin. The extracted cornea and conjunctiva tissues were cut into small pieces by microscissors and lysed in the RPMI media. Cell suspensions were collected and immunostained with following fluorescence-conjugated anti-mouse antibodies: CD11b (DCs: #11-0112-82; eBioscience, San Diego, CA, USA), CD11c (DCs: #11-0114-82; eBioscience), CD86 and MHC class II (mature DCs: #11-0862-82 and #11-5321-82; eBioscience), CD3 (#11-0032-82; eBioscience), CD4 (#11-0042-82; eBioscience), CD8 (#25-0081-82; eBioscience), CD69 (#17-0691-82; eBioscience), CD 103 (#11-1031-82; eBioscience), IFN-γ (#11-7311-41; eBioscience), IL17A (#559502; BD Pharmingen, San Diego, CA, USA), CD25 and FoxP3 (#17-0251-82 and #12-4771-82; eBioscience). The cells were stimulated for 4 hours with 50 ng/mL phorbol myristate acetate and 1 g/mL ionomycin in the presence of protein transport inhibitor (GolgiPlug; BD Pharmingen) for intracellular staining. Using a flow cytometer (FACSCanto; BD BioSciences, Mountain View, CA, USA), fluorescence assays of the cells were performed. Data were analyzed with software (FlowJo; Tree Star, Inc., Ashland, OR, USA).