Anti ifnγ apc xmg1.2

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).

Single cell suspensions prepared from lungs, BLNs, spleens and bronchoalveolar lavage were stained with the indicated combinations of Alexa Fluor 780-conjugated anti-CD4 (RM4–5, eBioscience), anti-CD8-PEcy7 (53-6.7, BioLegend), biotinylated anti-ICOS (7E.17G9, BioLegend), biotinylated anti-CD43 (1B11, BioLegend) and APC/cy7streptavidin (BioLegend). For intracellular cytokine staining, cells were stimulated for 2 hours with phorbol 12-myristate 13-acetate (PMA) (0.1 µg/ml, Cat#p1585, Sigma) and ionomycin (1 µg/ml, Cat#I0634, Sigma) and with golgi stop (Brefeldin) (5 µl/ml, Cat#B7651, Sigma) for 2 hours, followed by permeabilization with NP-40 (igepal CA-630, Sigma) and staining with anti-IFNγ-APC (XMG1.2, BioLegend). Intracellular FoxP3 expression was determined using the FoxP3 staining buffer kit (FJK-16s, eBioscience) according to the manufacturer’s recommendations. For apoptosis assay, Annexin/7AAD staining was performed as described previously [35] (link). Flow cytometric analysis was performed using a FACSCanto or LSRFortessa (BD Biosciences) running with THE FACSDiva software (version 6.1.3) (BD Biosciences). Analysis was performed using FlowJo version 9.5.3 (Tree star, Inc.).