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A single-cell suspension of co-culture cells and grafted corneas was prepared as previously described^{7 (link),8 (link),48 (link)}. To avoid non-specific staining, the cells were blocked with an anti-FcR-blocking antibody (eBioscience, San Diego, CA, USA). The isolated cells were stained with the following antibodies: FITC-anti-CD4 (RM4-5), and anti-IFN-γ (XMG1.2) antibodies (BioLegend, San Diego, CA, USA). The stained cells were examined using flow cytometry and the data were analyzed using FlowJo software X 10.5.3 (FlowJo LLC, Ashland, OR, USA).

Corneas and ipsilateral dLNs were harvested, and single-cell suspensions were prepared as described previously^{53 (link),59 (link)}. To avoid non-specific staining, cells were blocked with an anti-FcR blocking antibody (eBioscience, San Diego, CA, USA). The isolated cells were stained with the respective antibodies. Mature DCs were stained with anti-CD11c Alexa488 (N418, BioLegend, CA, USA), anti-CD45 PE (30-F11, eBioscience), and anti-I-A/I-E PeCy7 (M5/114.15.2, BioLegend). For intracellular IFNγ and IL-17 staining, the cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate, and 500 ng/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 6 h at 37 °C in a 5% CO₂ incubator in the presence of GolgiStop (0.7 μL per 100 μL cell culture; BD Biosciences, San Jose, CA, USA) to inhibit cytokine secretion. The cells were then stained with anti-CD4 FITC, anti-IFNγ APC (XMG1.2), and anti-IL-17 PECy7 (TC11-18H10.1) (BioLegend) antibodies. All antibodies and their matched isotype controls, and the fixation and permeabilization buffers were purchased from eBioscience. The stained cells were examined using LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo software X 10.5.3. (FlowJo LLC, Ashland, OR, USA; purchased from https://www.flowjo.com).

Corneas were harvested, and single-cell suspensions were prepared as described previously^{52 ,53 (link)}. To avoid non-specific staining, cells were blocked with an anti-FcR blocking antibody (eBioscience, San Diego, CA, USA). Isolated cells were stained with the respective antibodies. For intracellular IFN-γ staining, cells were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate and 500 ng/mL inomycin (Sigma-Aldrich, St. Louis, MO, USA) for 6 h at 37 °C in the atmosphere of 95% air and 5% CO₂ in the presence of GolgiStop (0.7 μL per 100 μL cell culture; BD Biosciences, San Jose, CA) to inhibit cytokine secretion. The cells were then stained with the following antibodies: anti-CD4 (GK1.5), anti-CD45 (30-F11), anti-CD11b (M1/70), anti-Ly6G (1A8), anti-F4/80 (BM8), and anti-IFN-γ (XMG1.2) (Biolegend, San Diego, CA, USA). All antibodies, their matched isotype controls as well as fixation and permeabilization buffers were purchased from eBioscience. Stained cells were examined by using an LSR Fortessa cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by using FlowJo software X 10.5.3. (FlowJo LLC, Ashland, OR, USA; purchased from https://www.flowjo.com).