We have previously demonstrated that anti-immunoglobulin antibody conjugated to high molecular weight dextran stimulates increases in expression of MHC class II molecules, cell size and DNA synthesis in B cells at concentrations which are 10,000 fold lower than that required for unconjugated anti-Ig. We therefore felt that these conjugates would provide a physiologically relevant model to study sIg mediated B cell activation. We propose first to explore the parameters which influence the mitogenicity and immunogenicity of these conjugates by varying the chemical nature and molecular weight of the carrier molecule, the epitope density of anti-Ig antibodies per carrier molecule and by conjugating to it monoclonal anti-delta or anti-mu antibodies of varying but defined specificity and avidity. Since antigen binds to more than one determinant on the B cell, we will construct dextran conjugates which contain combinations of antibodies with specificity to B cell surface determinants including IgM, IgD, Fc-gamma receptor, Ia and Lyb2. using these conjugates, we will determine whether simultaneous interaction with both IgM and IgD or Ig in combination with other determinants activates B cells differently than when these determinants are used independently. After defining the B cell stimulatory properties of the various conjugates, we will employ fluorescence digital analysis to study alterations that are induced in concentrations of intracellular ionized calcium [Ca2+] i early after cell activation. This recently developed technology will enable us to study how oscillation frequency, amplitude or pattern of changes in [Ca2+] i correlates with various levels of B cell activation and how protein kinase C activation or depletion influences the agonist stimulated alteration in [Ca2+]i. The experiments will employ B cells at various stages of maturation and activation to determine to what degree the maturational state of the cell influences the intracellular events stimulated by the various agonists. These approaches will clarify the extent to which various agonists. These approaches will clarify the extent to which various patterns of change in [Ca2+]1 at the single cell level influences B cell activation.
Showing the most recent 10 out of 21 publications
