Live dead dye

Mice of both sexes were euthanized, and spleen cell subsets were analyzed as previously described (51 (link), 76 (link), 77 (link)). After exclusion of doublets and dead cells, CD19⁺ B cells subsets were identified as transitional T1 (CD21^hiCD23^–IgM^hiIgD^lo), transitional T2 (CD21⁺CD23⁺IgM^hiIgD^lo), follicular (FO, CD21⁺CD23⁺IgM⁺IgD⁺), marginal zone (MZ, CD21^hiCD23^–IgM⁺IgD⁺), and GC (CD95⁺GL7⁺). GC B cells were further divided into DZ (CXCR4^hiCD86^lo) and LZ (CXCR4^loCD86^hi) phenotypes. PCs were identified as CD138⁺IgD^–. Memory cells were defined as CCR6⁺CD38⁺ (78 (link)). Subsets of CD3⁺CD4⁺ T cells were identified as activated (CD4⁺CD44⁺PD1^hiPSGL1^–), TFH (CD4⁺CD44⁺PD1^hiPSGL1^–Bcl6⁺FoxP3^–), and TFR (CD4⁺CD44⁺PD1^hiPSGL1^–Bcl6⁺FoxP3⁺) phenotypes. Other T cell subsets were examined as indicated in Table 1. For IL-17A staining, spleen cells were stimulated with or without PMA/ionomycin (1 μg/mL) in the presence of GolgiStop Protein Transport Inhibitor (BD Biosciences) for 4 hours before being loaded with Live/Dead dye (BioLegend), fixed, permeabilized, and stained. Antibodies and their sources are listed in Supplemental Table 1.

Cells were isolated from the retina and CDLNs on day 14 after immunization. Dead cells were excluded using live/dead dye (#423105, BioLegend, San Diego, CA, USA). Then they were stained with the following antibodies: the surface markers included: CD4 Percp-Cy5.5 (#100434), CD45 Brilliant Violet 605 (#103155) (BioLegend), TGFBR2 PE (#FAB532P, R&D Systems). For intracellular cytokine staining, the cells were stimulated with 5 ng/mL of phorbol myristate acetate, 500 ng/mL ionomycin, and 1 mg/mL brefeldin A (Sigma) at 37 °C for 5 h, following by fixation and permeabilization for 30 min. Then, cells were stained with antibodies detecting: IFN-γ PE (#505808), IL-17A Alexa Fluor 647 (#506912), FOXP3 FITC (#11-5773-82), Id2 PE-Cy7 (#25-9475-82, Invitrogen). For the Pim1 staining, cells were stained with surface antibodies, fixed, permeabilized, stained with Pim1 antibody (#3247S), then stained with Alexa Fluor 488-labeled antibody (#4412S) (Cell Signaling Technology, Danvers, USA). Finally, the cells were kept overnight at 4 ^◦C and measured by flow cytometry. The flow cytometer (BD LSRFortessa, USA) was used for analysis and the results were analyzed with FlowJo software (version 10.0.7, Tree Star, Ashland, OR, USA).