Analysis of the cell populations capable of initiating a response to sheep and horse erythrocyte antigens has been carried out by means of equilibrium density gradient centrifugation. The results indicate that there are at least six distinguishable AS cell populations for sheep erythrocytes, but only three for horse erythrocytes in the spleen of the Lewis rat. Evidence is presented for the existence of metabolic, physiological, and immunological differences among these populations. It is suggested that at least one population of AS cells responds only to the specific antigen and at least one other population is sensitive to stimulation by a broad range of antigens. It is assumed that the difference between these two AS cells results from a process of differentiation of AS cells primed into DNA synthesis by antigen stimulation.

Density distribution analysis is a convenient objective means of characterizing different cell populations (1)(2)(3)(4)(5)28). With most populations a prominent feature of this technique is a reproducible heterogeneity of components which is apparently not an artifact (3, 5). Attempts to understand this heterogeneity in terms of functional differences have been only partially successful; preliminary results have indicated that differences in density of 19S hemolysin-forming ceils reflect differences in physiological and metabolic properties (1, 5).Accordingly, it became of some interest to carry out density distribution analysis on the precursor cells of the 19S hemolytic antibody-forming cell (AS1 cell). I t was hoped that, aside from possible metabolic and physiological dif ferences, it might be possible to detect AS cells differing in immunological func tion. Preliminary accounts of this work have been published elsewhere (2, 4)- Materials and MethodsAnimals.--Male Lewis rats (9-12 wk) bred in the Hall Institute were used as cell donors in all experiments except those involving newborn or very young animals, in which case rats of both sexes were employed. Male and female Lewis rats (5--9 wk) were used as irradiated recipients.Irradiation of Animals.--Rats were irradiated, six at a time, in a Perspex box (18 X 17.5 X 10 cm.), external dimensions, designed to give no more than 2% variation from animal to animal under conditions of maximum backscatter. A Phillips 250 kv X-ray unit was used employing a 0.2 mm Cu filtration (half value layer [HVL] ~ 0.8 mm Cu). The dose of 800-850 fads was given in two units from opposite sides of the box. Sources of Cells.--(a) Spleen:Suspensions of single cells were obtained by teasing freshly excised spleens in balanced salt solution (BSS) containing 10% fetal calf serum (6). This procedure was carried