FLOW CYTOMETRIC ANALYSIS OF T-INDEPENDENT ANTIGEN-BINDING TO DINITROPHENYL-SPECIFIC CELLS

Binding of Ag to membrane Ig (mig) can lead to either activation or de sensitization of the B cell. For thymus-independent (TI) Ags the natur e and concentration of the Ag determines what type of signal is delive red to the cell. These Ags are capable of directly activating B lympho cytes and are an important model system for the study of mechanisms in volved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, conc entration, and epitope density. Various epitope densities of two struc turally different TI Ags, DNP-polymerized flagellin (pot) and DNP-dext ran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRIT C) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell Ii ne. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytome try. For each Ag and dose, FITC-labeled DNP-L-papain was used to quant itate receptor sites not occupied by Ag. Approximately 5% receptor occ upancy was observed for immunogenic doses of Ag. Higher Ag concentrati ons that can induce tolerance caused a substantial increase in the fra ction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activ ity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation.