Clonal analysis of B-lymphocyte subpopulations separated on the basis of Lyb surface antigens

The aim of these experiments was to see whether antisera of the Lyb series could be used to identify B cells capable of responding differentially in T-dependent and T-independent systems. The antisera tested were against the alloantigens Lyb 1.1,2.1,3,4.1,5.2, and LyM 1. A polyvalent sheep anti-mouse immunoglobulin (Ig) antibody acted as a positive control for the identification of B cells. As a first step, all spleen B cells were treated to remove this surface Ig by a capping procedure. They were then washed, reacted with a mouse alloantiserum, and allowed to form rosettes with sheep erythrocytes to which a sheep anti-mouse IgG had been coupled. Rosetted fractions were prepared on a Percoll density gradient. After removal of erythrocytes by osmotic shock, the cells were tested for their capacity to respond to antigenic stimulation. To allow accurate estimation of functional potential, two B-cell cloning assays were used. To enumerate T-dependent B cells, the Klinman splenic microfocus assay was employed using haptenated KLH⁴ as antigen. To study T-independent cells, a limiting-dilution liquid microculture method employing hapten-polymerized flagellin as antigen was used. The results showed that none of the Lyb antigens clearly demarcated T-dependent from T-independent B cells. Rosetted fractions consisting of Lyb 1.1-, 2.1-, 3-, or 4.1-positive cells responded well in both assays. Fractions enriched for LyM 1-positive cells behaved like unfractionated spleen cells. Only the Lyb 5.2-rosetted fraction showed any discordance between the two assays, the fraction being enriched for cells responding in the T-dependent system and slightly depleted of cells responding in the T-independent system. Taken as a whole, the results suggest that these alloantigens will not serve as useful markers to characterize T-dependent and T-independent B-cell subsets. In fact, the experiments cast further doubt on whether such a distinction is valid.