Phenotype of PBMC stimulated for 6 days with PWM, CD40 mAb, IL-2 and IL-10. (A) 20 × 10⁶ bovine PBMC were cultured for 6 days in 20 mL complete medium alone (media) or in the presence of various combinations of PWM, CD40 mAb, IL-2 and IL-10. The number of total IgG-secreting cells generated from 10⁶ cultured PBMC (mean of duplicate determinations ± SD) was determined for each culture condition. Representative data from 2 independent experiments are shown. (B) 20 × 10⁶ PBMC were cultured for various periods of time with PWM, CD40 mAb, IL-2 and IL-10. The number of total IgG-secreting cells generated from 10⁶ cultured PBMC (mean of duplicate determinations ± SD) was determined for each time point. Representative data from 2 independent experiments are shown. (C) Cytospin slides from fresh (day 0) and cultured (day 6) PBMC were stained with May-Grunwald-Giemsa (arrows indicate cells with plasma cell morphology. (D-E) PBMC were stimulated for 6 days with PWM, CD40 mAb, IL-2 and IL-10. At the end of the culture period, the phenotype of the cells in the resulting population was analysed: flow cytometry was used to determine the percentage of CD4 ⁺ , CD8 ⁺  and γδ⁺ T cells (D) and immunohistochemistry to detect intracellular Ig (in cytospin slides) using an anti-bovine light chain mAb and counter-staining with May-Grunwald-Giemsa (E). Representative data from 3 independent experiments are shown. Original magnification: × 40. ASC: Ab-secreting cells.