Jeffrey E. Berman

Expression of the Immunoglobulin Heavy-Chain Variable Gene Repertoire

A major challenge in immunology is the elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire. A large body of knowledge concerning murine antibody gene organization and diversity has been accumulated. However, the underlying processes governing usage of these genes remains in question. Knowledge of the mechanisms which regulate immunoglobulin variable (V) gene rearrangement and the relative expression of the various V gene families in developing B–cell populations may be crucial to an understanding of generation of the antibody repertoire in normal as well as disease states. Current investigation of human V H gene organization and diversity will evwntually allow extension of information gained about the murine repertoire to studies of V H gene usage in humans.

Immunoglobulin heavy chain junctional diversity in young and aged humans.

The causes of observed deficiencies to the humoral immune response in aged humans are unknown. Since a major source of antibody diversity is generated at the VH-D-JH junctional regions of the immunoglobulin heavy chain, we determined whether differences in junctional diversity are manifested with aging. We compared the CDR3 regions of IgM heavy chain transcripts isolated from young adult and aged humans. A PCR assay that measures CDR3 length in the majority of mu-heavy chains showed the same average size and normal range of CDR3 length in aged individuals as observed in young adults. To characterize the features of junctional diversity of aged adults in more detail, we determined the CDR3 sequences of a subset of the mu-heavy chain repertoire that utilizes members of the VH 5 family. In general CDR3 length, D family usage, and JH gene usage were similar in aged compared to young adults. Thus, in contrast to dramatic changes in heavy chain junctional diversity associated with fetal to adult development, no major differences were found between young and aged adults. Since the CDR3 repertoire generated in aged individuals appears to be as diverse as that observed in younger adults, the decline in humoral immunocompetence with aging cannot be attributed to a restriction in heavy chain junctional diversification processes.

Frequency of VH-gene utilization in human EBV-transformed B-cell lines: The most JH-proximal VH segment encodes autoantibodies

We have studied VH-gene utilization in a collection of 187 IgM-secreting EBV-transformed cell lines and have begun to correlate VH-gene family expression with binding properties of the secreted immunoglobulins. The results of these studies demonstrate that (1) frequency of VH-gene utilization in fetal and adult tissue-derived cell lines correlates with the complexity of the family and (2) the single-membered most JH-proximal VH-6 family encodes autoantibodies reminiscent of autoantibodies found in the sera of patients with systemic lupus erythematosus. Nucleotide sequence analysis of VH-6-expressing clones revealed that each clone utilizes a short DH segment, resulting in a CDR3 region of conserved length. Our data suggest that EBV does not selectively transform human B cells on the basis of VH-gene family expression and that the VH-6 family encodes polyspecific autoantibodies that may serve an important regulatory function in the immune system.

Human Heavy Chain Variable Region Gene Diversity, Organization, and Expression

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (VH) gene locus. In this review we present the current knowledge of VH gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.

Control of Recombination Events During Lymphocyte Differentiation: Heavy Chain Variable Region Gene Assembly and Heavy Chain Class Switching

Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene

ABSTRACT The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA forPb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of differentPb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Organization and Expression of Human VH Genes

The variable regions of immunoglobulin heavy chains are encoded by the three germline gene segments: V(ariable), D(iversity) and J(oining). These segments are joined during precursor B cell differentiation to form a functional VHDJH variable region gene. In the mouse there are hundreds of different VH gene segments that can be subdivided into families based on nucleotide sequence homology (reviewed in Alt, et al., 1987). Various studies of transformed and normal murine B lineage cells have shown that chromosomal position of VH segments is a major determinant of their rearrangement frequency, resulting in a preferential rearrangement of VH segments proximal to the cluster of JH elements (Yancopoulos, et al., 1984, 1988; Reth, et al., 1986; Perlmutter, et al., 1985). This preferential rearrangement phenomenon leads to the biased expression of JH-proximal VH segments in primary B cell repertoires; for example, these gene segments are the major contributors to the Ig heavy chain mRNA produced by B lineage cells of the fetal liver (Yancopoulos, et al., 1988). In contrast, B cells in peripheral lymphoid organs of adult mice appear to utilize most VH segments at equal frequency; that is the representation of different families in the peripheral Ig heavy chain mRNA repertoire correlates with the complexity of each family and is not related to chromosomal position (Yancopoulos, et al., 1988; Dildrop, et al., 1985). These findings led to the proposal that an initially biased repertoire is randomized, probably by cellular mechanisms, in the transition from primary to peripheral lymphoid tissues (Yancopoulos, et al., 1988).

B Cell Differentiation: Development of Antibody Repertoires

In mammals, primary B cell differentiation occurs as an antigen-independent process in the liver of the fetus and in the bone marrow of the adult. During these stages, stem cells give rise to precursor B lymphocytes — the cells in which antibody variable region genes are assembled from component germline gene segments (reviewed by Alt et al., 1987). The genes that encode Ig heavy and light chain variable regions are assembled at distinct stages of pre-B cell differentiation. First, heavy chain variable region gene is assembled from component VH, D and JH segments and subsequently light chain variable region genes are assembled. Functional heavy and light chains associate to form a complete Ig molecule; the expression of this molecule on the cell surface defines the next major stage of B cell differentiation, the B lymphocyte. Only a single heavy chain and a single light chain are expressed on the surface of any particular clonal population of B cells; this ensures B lymphocyte clones will express a single species of Ig with a unique set of binding specificities. Following generation in primary differentiation organs, B cells migrate to peripheral lymphoid organs such as the spleen. Upon appropriate interaction with a cognate antigen, peripheral B lymphocytes are stimulated to further mature into antibody-secreting cells. During this antigen-driven clonal selection process, additional types of B lineage cells may be generated including cells that have undergone heavy chain class-switching or somatic mutational events.