This project will evaluate the impact of membrane IgM:ligand affinity on the mechanism by which human s cells undergo clonal expansion. The studies will assess the affinity requisites for mIgM to function in three capacities: (1) transducer of mitogenic signals for B cell S phase entry in the absence of T cell accessory signals, (2) transducer of signals for the partial activation of B cells, and (3) mediator of ligand internalization for processing and presentation to T cells. The research will directly test the hypothesis that the amount of T cell help required for expansion of ligand-specific B cell clones is indirectly related to the affinity of the ligand:mIgM interaction. The studies should enhance our understanding of the affinity constraints determining the immunoregulatory potential of foreign ligands, autologous rheumatoid factors, and anti-idiotype Abs under varying physiological conditions, and in so doing, should aid in the design of more optimal vaccines for B cell clonal expansion. The studies will utilize a large group of mouse anti-human IgM MoAbs that bind to bivalently expressed epitopes on mIgM with a wide range of affinities (Ka = 2x105 to 6x108). The work will have four major aims: (1) Establish that the pronounced T cell and T cell factor independent (TI) signaling potential of certain anti- IgM MoAb mixtures is due to an enhanced functional affinity for mIgM, and assess whether different phases of the prolonged signaling period needed for TI ligand-induced B cell DNA synthesis have distinct affinity requirements. This will be addressed by in vitro culture experiments, immunoelectron microscopy, and cellular equilibrium binding analyses. (2) Evaluate the minimal binding affinity for ligand induction of certain pre-S phase phenomena. This will involve Indo 1 analysis of intracellular free Ca2+ and FACS analysis of the membrane expression of class II MHC and activation-associated molecules. (3) Assess the minimal ligand affinity requirement for signaling B cell S phase entry in the presence of IFN-gamma, IL4, or low MW BCGF. (4) Evaluate the minimal ligand affinity for B cell proliferation with linked, cognate T cell help. This will involve B cell culture with MoAb Fab fragments and mouse Ig specific human T cell clones. Results from these in vitro studies with polyclonally-reactive, affinity diverse ligands should provide a unifying explanation for the apparently diverse functions attributed to mIgM in facilitating B cell clonal expansion.
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