Immunoglobulin (Ig) Allotype Markers on Rabbit Lymphocytes: Separation of Cells Bearing Different Allotypes and Demonstration of the Binding of Ig to Lymphoid Cell Membranes

Abstract

Lymphocytes from peripheral blood and Peyer's patches of b⁵b⁹ and b⁴b⁵ rabbits were membrane stained with rhodamine- and fluorescein-labeled anti-allotype reagents. Up to 63% of peripheral blood lymphocytes and 15% of Peyer's patch lymphocytes were found to double stain for both allotypes on their membranes. With a fluorescence-activated electronic cell sorter, cell populations were fractionated according to membrane allotype. A double pass separation procedure after staining yielded four distinct cell populations: one each of cells single-stained for one of the two allotypes, one containing cells double-stained for both allotypes, and one of unstained cells. Properties of the cells bearing two allotypes were examined. Cells put into culture after they were stripped of their membrane immunoglobulin (Ig) with Pronase regenerated their membrane Ig; however, the proportion of cells bearing both allotypes was greatly reduced. This suggested that the lymphocytes were restricted to the synthesis of membrane Ig molecules of a single allotype at a given time. It was found that a high proportion of rabbit lymphocytes could bind exogenous Ig from serum in vitro and probably also in vivo, explaining the presence of molecules of both allotypes on individual cells.