Sl. Woodard et al., FLOW CYTOMETRIC ANALYSIS OF T-INDEPENDENT ANTIGEN-BINDING TO DINITROPHENYL-SPECIFIC CELLS, The Journal of immunology, 155(1), 1995, pp. 163-171
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.