PMID: 7541414Jul 1, 1995Paper

Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells

The Journal of Immunology : Official Journal of the American Association of Immunologists
S L WoodardB G Barisas

Abstract

Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP-dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogeni...Continue Reading

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