DIFFERENTIATION OF ANTIBODY-FORMING CELLS IN TOAD SPLEEN A Study Using Density and Sedimentation Velocity Cell Separation

Antibody-forming cells (AFC), developing in toad spleen after stimulation with polylnerized flagellin, were studied with an immune adherence assay. Differentiation was followed by several parameters : thymidine uptake to monitor dividing cells ; equilibrium density centrifugation in albumin gradients to monitor cell density ; microscopic measurements and sedimentation velocity separation to monitor cell size; stained preparations to follow cell morphology. Almost all AFC observed early in the response were dividing cells ; the proportion of dividing AFC dropped to 4% 2 wk after stimulation . The earliest AFC detected (3 days) formed a relatively homogeneous light density population, and were purified 17-fold by equilibrium density centrifugation . As the response developed, additional denser peaks were found, so that late in the response dense AFC predominated . Dividing AFC were confined to the light density region throughout the response . Cell diameter measurements revealed that the earliest AFC were all very large cells . In a manner analogous to the density changes, smaller AFC appeared as the response developed until they finally comprised the majority of the AFC population . Dividing AFC were always relatively large, but encompassed a wide range of sizes . Sedimentation velocity separation was employed in a closer study of the immature AFC ; they were purified 140-fold by this procedure. The earliest AFC consisted of several readily separable size populations in the range 9-18 u diameter . The presence of separate peaks related by factors of two in volume suggested that the largest cells undergo a series of halving divisions before entering a division growth cycle. The results suggest an AFC differentiation sequence from a very large, light density, dividing "blast" cell to a nondividing cell with the size, density, and morphological appearance of a small lymphocyte . Stages of this sequence can be defined and selected out for investigation, using sedimentation velocity and equilibrium density centrifugation as complementary techniques.