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The role of germ-line immunoglobulins in autoimmunity and B cell activation Cho, Chin-wen C.

Abstract

The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies that are all derived from the same V H and V K germline genes. Our laboratory has previously reconstructed the putative germ-line-based ancestor of a somatically hypermutated antibody (8F9) against the AD-2S1 and characterized and compared the binding of the germline- based ancestral immunoglobulin and 8F9 (Igs) to the AD-2SI epitope. IgG1 versions of these antibodies bound to AD-2S1 by ELISA and glycoprotein B (gB) by flow cytometry. Here, I show that 8F9 neutralizes viral infectivity whereas the germ-line Igs do not. However, the germ-line Igs bind to an unidentified, phylogenetically conserved auto-antigen. Small changes in the binding site of the germ-line Igs abolished binding to the auto-antigen. Even though mechanisms do exist during B cell development to eliminate self-reactive B cells, these observations suggest that autoreactivity is not necessarily a barrier for immature B cells to further develop into follicular B cells that subsequently undergo antigen-driven affinity maturation. Our observations that the hypermutated high affinity 8F9 antibody was derived from a primary immunoglobulin that was reactive with both an auto-antigen and AD-2S1 raise important questions including 1) the threshold of affinity required for triggering the clonal expansion and somatic mutation of new B cells, and, 2) the role of low-affinity cross-reactivity with ubiquitous antigens (eg self-antigens), in rescuing B lymphocytes with primary receptors for important pathogens from "death by neglect". To further understand the initial primary B cell receptor (BCR) response to AD-2S1, we expressed the membrane IgM forms of the 8F9 and primary germ-line Igs. In our experiments, the human germ-line Igs and 8F9 were expressed in a mouse cell-line as membrane IgM. I showed that cross-linking this human IgM triggered calcium release and tyrosine phosphorylation of intra-cellular signalling molecules. Morever, the membrane IgM bound AD2S1. Thus, I demonstrated that the human IgM expressed in a murine cell line is capable of activating many B cell responses and can serve as an in vitro model to look at affinity threshold. I also constructed lentiviral vectors to enable infection of RAG-1 knockout stem cells and reconstitution of RAG-1 knockout mice with B cells expressing human germ-line IgMs. The in vitro and in vivo systems will be useful in studying the affinity threshold needed to initiate B cell activation and other questions that are important in B cell immunity.

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