FIG. 4.

Peptides SAP-4 and SAP-16 contain, respectively, the immunodominant B-cell epitopes ²¹SKQPTIDIDL³⁰ and ⁷⁶QQEVDYIIDH⁸⁵. They were conjugated with keyhole limpet hemocyanin, emulsified in Freund's adjuvant, and injected separately into two mice. Splenocytes were isolated from each immunized animal and cultured in triplicate at 10⁶ cells/well in 200 μl of RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and stimulated with 5 μg/ml of each peptide. Cells were incubated for 48 h at 37°C under 5% CO₂ atmosphere. After incubation, cells were centrifuged, washed with PBS, stained with anti-CD19-FITC or anti-CD69-PE-Cy7 (BD Pharmigen, San Diego, CA) monoclonal antibodies, and analyzed by fluorescence-activated cell sorting using a FACSort (Becton and Dickinson, San Jose, CA) instrument. Splenocytes were identified by their characteristic appearance on a dot plot of forward scatter versus side scatter. Splenocytes stained with FITC- or PE-Cy7-labeled antibodies were detected by FL1 or FL3, respectively. B-cells were gated on the basis of CD19 staining. The results were reported as the percentage of positive cells within this gate that appear activated.