Spleen cells from unimmunized adult CBA mice were subjected to fractionation procedures dependent on their capacity to adhere to monolayers or erythrocytes on which the haptenic determinant NIP was exposed. Subsequent to removal of adherent antigen, in vitro analysis for anti-NIP precursors was performed by culture at limiting dilution in a microculture system with thymus cells as a “filler” population and by using the “T-independent” antigen NIP-POL to induce antibody formation. Cultures were harvested and assayed for anti-NIP-specific PFC after 3 to 7 days and the frequency of clonable anti-NIP precursor B cells determined by Poisson analysis.

Whereas one cycle of NIP-gelatin fractionation produced a population in which the average frequency of anti-NIP precursors was about 0.04, three successive cycles (two of NIP-gelatin fractionation and one of NIP-SRBC rosetting) raised this frequency to 0.28. The fact that one cell in three and one-half from a “virgin” population could make an antibody-forming clone in vitro with specificity for the hapten used in preparing the fractionated population should be helpful for many detailed studies of B cell interactions with antigen. Bearing in mind the technical limitations of lymphocyte culture and the possibility of functional heterogeneity among B lymphocytes of a given specificity, one may assume that it may not be easy to surpass this cloning efficiency by antigen fractionation alone.

Cells obtained from one cycle NIP-gelatin fractionation of neonatal germfree or SPF spleen, or adult bone marrow, behaved in vitro essentially as cells obtained by one cycle fractionation of adult spleen, strongly suggesting that the technique is applicable to virgin cell populations. Some cultures harvested later than the standard 3 to 4 day peak of direct plaque formation showed clones consisting chiefly or solely of enhancable plaques. These may have been IgG-producing clones, raising the possibility that T helper cells had been activated in vitro by the carrier POL.

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