Changes in Surface Immunoglobulin Isotypes on Purified Antigen-Binding Cells after Antigenic Stimulation

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Abstract
Antigen-binding cells (ABC) were isolated from 5-day immune and nonimmune mice via a two-step centrifugation procedure. Their surface immunoglobulins (sIg) were then stained with fluorescein-conjugated anti-Ig, anti-µ, and anti-γ-specific reagents and analyzed on the fluorescence-activated cell sorter (FACS), or labeled by lactoperoxidase-catalyzed surface iodination and examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Nonimmune ABC, unfractionated and ABC-depleted lymphocytes had identical FACS profiles after anti-µ and anti-Ig staining. Surface radioiodination and SDS-PAGE analysis showed that these nonimmune ABC exhibited a greater amount of δ than µ on their surface with only a trace of γ. In contrast, immune ABC bore more µ than δ and 30 to 50% of these ABC also bore surface γ. Immune ABC also showed a reduced amount of total sIg compared to Fl-anti-Ig stained unfractionated or ABC-depleted lymphocytes. Since the intensity of staining with Fl anti-µ did not change after immunization, the reduced total sIg and increased µ/δ on the immune ABC were attributable to loss of sIgD after antigenic stimulation of the ABC. This antigen-induced loss of sIgD could be simulated on non-immune ABC and on unfractionated spleen cells by proteolytic removal of the sIgD.