Antibody production of the immunoglobulin E (IgE) class is causally related to the allergic disorder in both human and experimental animals. Our understanding of this class of immunity has been greatly expanded in the past two decades through the elucidation of structure and function of IgE molecules. Recently, we have discovered a novel model system to induce polyclonal IgE tolerance in mice neonatally treated with IgE ligands. However, much remains to be learned about how regulatory cells exert control over the stage-specific differentiation of B-cells of the EgE lineage (BEpsilon-cells). This particular difficulty is largely due to the lack of a reproducible in vitro IgE antibody system. The objective of this research proposal is to determine the fine tolerogenic epitope inherent in the IgE molecule and examine how class-specific negative cells influence the stage-specific differentiation of the immortalized BEpsilon in vitro. Specific Aim 1: Resolution of the tolerogenic IgE epitope: To stepwise (i) examine the molecular conservation of this epitope among different IgE molecules, (ii) localize the tolerogenic domain by enzymatic cleavage, and constructing this epitope through synthetic peptide techniques. Specific Aim 2: Construction of immortal BEpsilon-cell lines: This aim is to be achieved by applying our expertise in the growth of B-cell lines utilizing B-cell growth factors (BCGF) and our newly developed DNA transfection technique for inserting transforming genes into lymphoid cells. The primary BEpsilon-cell candidates for transfection are from IgE-defective SJA/20 and normal BALB/c mice. Specific Aim 3: Elucidation of control mechanism of clonal negative regulatory T-cells on the stage-specific differentiation of BEpsilon-cell lines. To demonstrate a forward differentiation of BEpsilon-cells at different maturation stages and to modulate these processes by regulatory T-cells and factors prepared from IgE tolerant mice. Subsequently clonal class-specific regulatory T-cells are examined for their direct or indirect effects on BEpsilon-cells. Contributions from this project will be made to two areas. First, an in vitro IgE antibody system will serve as a paradigm for class-specific regulation of other isotypes. Second, the identification of the minimum tolerogenic epitope in IgE will enable the preparation of a safe and efficient vaccine for IgE-mediated disorders provoked by diverse allergens.