Clonal anergy: inhibition of antigen-driven proliferation among single B lymphocytes from tolerant animals, and partial breakage of anergy by mitogens.

Mice were injected with fluoresceinated human gamma globulin (FLU-HGG) either at 2-3 days of age or as pregnant females. At 2 weeks of age, the spleen cells of the injected suckling mice or offspring were fractionated on FLU-gelatin dishes to yield FLU-binding B cells. These B cells were then cloned in microcultures using one of two recently described systems in which single B cells grow in the absence of feeder or filler cells, namely following stimulation with FLU-polymerized flagellin (FLU-POL) and conditioned media containing B cell growth and differentiation factor(s); or mitogenic activation by a mixture of E. coli lipopolysaccharide (LPS) and dextran sulfate (DxS). As such cultures permit visualization of clonal proliferation as well as ultimate harvesting of cultures for assay of hemolytic plaque-forming cells, it was possible to ask whether the lesion in the tolerant state affected the B cell's capacity to divide, to differentiate to antibody secretion, or both. The results indicated that, when stimulated with antigen, the anergic cells could neither divide nor differentiate. However, when the strong mitogen mixture was used, clonal anergy was partially broken. The cells proliferated, and a small proportion of them differentiated into anti-FLU antibody-forming cells. A marked variation in antigen-binding avidity of the FLU-binding cells made it difficult to quantitate the degree of uncoupling of proliferation and differentiation among tolerant, LPS plus DxS-stimulated cells. Nevertheless, a partial reversibility of clonal anergy must affect views on mechanisms of self-tolerance.