A85 1

Multi-colour flow cytometry analysis was performed as described previously [19 (link)]. Briefly, cells were incubated with a cocktail of monoclonal antibodies against rabbit CD4 (IgG2a, KEN-4), CD8 (IgG1, 12C.7) and IgM (IgG1, NRBM) on ice for 10 min. Cells were washed and further incubated for 10 min on ice with isotype-specific phycoerythrin (PE)-conjugated rat anti-mouse IgG1 (A85-1, BD) and biotinylated rat anti-mouse IgG2a (R19-15, BD) antibodies. After a third wash, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit T cells (KEN-5), washed with allophycocyanin (APC)-conjugated streptavidin (BD), and suspended in 7-AAD. Antibodies were from AbD-Serotec. Bovine CD8⁺ T cells were identified as previously described [18 (link)], and dead cells detected with BD Horizon Fixable Viability Stain 450 (BD biosciences). Data were acquired using a Fortessa X20 flow cytometer (BD) and analyzed using Flowjo v10.0.7 (Treestar).

Single-cell suspensions were prepared from lymphocytes of thymus (T), bone marrow (BM), spleen (Spl), and lymph node (LN) from mice of the described genotypes and ~1 × 10⁵ cells in 1× PBS were stained for 15 min at RT using fluorescence-conjugated antibodies and analyzed by flow cytometry^{60 (link)}. The following antibodies were used: FITC-Cd11b (1:200, M1/70, BD Pharmingen, 553310), APC-Ly-6G/Ly-6C (Gr-1) (1:200, RB6-8C5, Biolegend), PE-IgM (1:400, SouthernBiotech, 1020-09), PE-CD4 (1:200, GK1.5, BD Pharmingen, 553730), FITC-CD8a (1:200, 53-6.7, Biolegend), PE-Cyanine5-CD3e (1:200, 145-2C11, Invitrogen, 15-0031-63).
For CSR assay, single-cell suspensions of spleen cells were sorted with CD43 magnetic beads (MACS, Miltenyi), and CD43- B cells were cultured at a density of 5 × 10⁵ cells per ml in RPMI medium supplemented with 15% FBS and 25 ng ml⁻¹ of IL-4 (R&D) and anti-CD40 (BD Biosciences). Cultured cells were maintained daily at a density of 1 × 10⁶ cells per ml. Cells were collected every day up to day 4.5 and were stained with FITC-conjugated IgG1 (1:200, A85-1, BD Pharmingen, 553443) and PE-Cyanine5-conjugated B220 (1:200, RA3-6B2, BD Pharmingen, 553091). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using FlowJo software package.