Comprehensive Immunophenotyping of Murine Splenocytes

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.