Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin.

We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.