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The development of antigen-specific cells of the immune system, the T and B lymphocytes, creates a dilemma. On the one hand, survival of the organism depends upon the generation of a nearly limitless repertoire of potential antigen-binding specificities so that cells able to respond to pathogens are present prior to contact. However, by devising genetic strategies to maximize receptor diversity, the generation of T and B cells with autoreactive receptors is inevitable. B cells have an even greater opportunity than T cells to become autoreactive, since they may randomly alter the amino acid sequence and hence the specificity of their receptors during an immune response. Observing the system, one might wonder not why autoimmune diseases occasionally develop, but rather why they are not more frequent or even unavoidable. In this review, we examine the generation of B cells and their repertoire of antigen receptors, describe mechanisms that have evolved to prevent self-reactive B cells from causing autoimmune diseases, and discuss scenarios that may lead to a breakdown of self tolerance.

The onset of IgH transcription and rearrangement is a defining characteristic of the progenitor population in which B-lineage commitment occurs. These features were used to better define the earliest stage of B-cell commitment in humans and to determine if these stages differ as a function of human ontogeny. Fetal and adult bone marrow mononuclear cells were sorted into B-lineage subpopulations on the basis of surface expression of the stem cell marker CD34, the pan-B-cell marker CD19, and IgM and analyzed for transcription and rearrangement of the IgH locus. The locus was found to be transcriptionally active before surface expression of CD19, as indicated by the presence of germline I mu, C mu, and D(H)Q52 transcripts in the CD34+ CD19- subpopulation. Transcripts from IgH alleles that had undergone DJC mu rearrangements were also detected in the CD34+ CD19- subpopulation. Within this subpopulation, low levels of DXP-containing DJC mu transcripts were detected in both fetal and adult cells. Although D(H)Q52 DJC mu transcripts were abundant in fetal CD34+ CD19- cells, they were not detected in cells of the same phenotype derived from adult bone marrow. In both fetus and adult, V(H)3-and V(H)6-containing VDJC mu transcripts were detected only in the CD19+ subpopulations. These data indicate that transcription of D(H)Q52-J(H) and DXP-J(H) rearrangements differs during fetal and adult B lymphopoiesis. Moreover, in both fetus and adult, transcription of unrearranged components of the IgH locus and DJ rearrangements can proceed before the surface expression of CD19.

This analysis of B cell development as a function of age reveals a relatively widespread distribution of progenitor B (pro-B), pre-B, and B cells in fetal tissues, and thus supports the idea of a multifocal origin of B lineage cells during embryonic development. From mid-gestation onward, the bone marrow is the major site of B cell generation in humans. A relatively constant ratio of bone marrow precursors to B cells of immature phenotype (CD24highCD10+CD20lowIgD-) is maintained from mid-gestation through the eighth decade of life. The persistence of recombinase gene activity in pro-B cells further attests the sustained production of B cells in bone marrow. Interestingly, a subpopulation of B cells with mature phenotype (CD24lowCD10-CD20highIgD+) accumulates in the bone marrow during childhood, and this becomes the predominant B cell subpopulation in adult bone marrow. This mature population of bone marrow B cells may represent a subpopulation of recirculating B cells that have undergone selection in the periphery.

The Ag receptor on B lymphocytes is a multimeric complex that is composed of an Ag-specific component, surface Ig, which is noncovalently associated with at least two other proteins, Ig alpha and Ig beta. These are the glycoprotein products of the B lineage-restricted mb-1 and B29 genes and are crucial for the cell surface expression and function of the Ag receptor on B lymphocytes. To better understand the regulation of mb-1, we have cloned and sequenced a 5.7-kb genomic DNA fragment that contained the human gene. The overall structure of human mb-1 is very similar to that of the murine gene, including the number and approximate size of exons. The promoter region lacks a TATA element, but contains two copies of an early B cell factor-binding motif, which previously has been shown to be important for murine mb-1 expression. Other structural features include two nuclear factor-kappa B binding sites at the 5' end of the gene and a long stretch of AG rich-sequence between exons 3 and 4, downstream of an Alu repeat sequence that contains a potential stem-loop structure. The mb-1 gene was localized to chromosome 19q13.2-13.3 by a combination of two methods, PCR amplification of DNA from a somatic cell hybrid-mapping panel and fluorescence in situ hybridization. An examination of the methylation pattern revealed a striking correlation between demethylation in the 5' region of the gene and expression of mb-1. The demethylated HpaII/MspI sites are adjacent to the nuclear factor-kappa B-binding motifs, which suggests a role for this transcription factor in the regulation of human mb-1 gene expression.

Normal differentiation of B lineage cells has been the subject of intensive investigation over the past three decades. Current models of this process in humans are melded from the results of studies in a variety of organisms, including humans, mice and birds. Several recent developments have significantly reshaped and refined these models. The technique of homologous recombination in embryonic stem cells has allowed the production of mice with selectively disrupted genes that are important for B cell development in mice. At the same time, functional studies of human B cell differentiation, together with analysis of naturally occurring mutations that disrupt this process, have progressed rapidly. This has provided insight into the pathogenesis of lymphoproliferative and immunodeficiency diseases as well as a clearer view of normal developmental events. In this chapter we have reviewed human B cell differentiation with particular emphasis on newly emerging concepts. We also discussed CD5, a pan-T cell antigen that is expressed in low levels on a subpopulation of B cells implicated in the pathogenesis of chronic lymphocytic leukaemia (CLL). Finally, we discussed the issue of restricted variable region gene usage during B cell ontogeny and in CLL.

Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa. We have isolated a full-length cDNA encoding a human porin. This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots. The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL). The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus. The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons. The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro. Taken together, these results suggest that multiple porin proteins can be expressed in humans. Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man.

Previous studies have suggested that transcription of germline heavy chain constant region (CH) genes in murine B cells may determine the potential of their different CH regions to undergo isotype switch recombination. We have examined the transcriptional activity across the immunoglobulin heavy chain (IgH) locus in human B lineage cells. Transcription of germline C gamma and C alpha was observed in every surface IgM+ or surface IgM+/IgD+ B cell stage cell line and malignancy. In contrast, such transcription could not be detected in pre-B cells and only low levels of C alpha but not C gamma transcription were evident in IgM-secreting plasmablast cells. Transcriptional activity of germline IgH C epsilon was singularly absent at all stages of B cell development. Our results suggest that germline transcription of the C gamma and C alpha regions may be a constitutive feature of the human B cell differentiation program. Because this transcriptional activity is limited primarily to the B cell stage and occurs prior to the actual isotype switch, the induction of C gamma and C alpha transcription may represent preparation of the downstream IgH chromatin for potential switch recombination.

The progression of lymphocytes along the B cell developmental pathway is marked by the regulated appearance and disappearance of a variety of cell surface markers including immunoglobulin. In this review, several of these antigens are discussed in the context of their possible functions during normal B cell differentiation.

This article reviews the normal differentiation of B-lineage cells. The scheme of normal differentiation of the B lineage provides a context in which to analyze patients with immunodeficiencies and B-lymphoproliferative disorders.

The lineage and stage specificity of human isotype switch recombination was investigated by examining the IgH gene configuration in 61 hemopoietic malignancies representing different stages of B and T cell development. An unexpectedly high frequency (20%) of IgM-producing B cell leukemias and lymphomas had undergone CH gene rearrangements and deletions consistent with attempted switch recombination. These CH gene alterations were found on productive, non-productive, and 14q+ chromosomes. These data support the concept of a non-specific (common) switch recombinase activity that is often ineffective. No evidence of such switch recombination was found in 25 mu- or mu+ pre-B cell leukemias with the single exception of a mu- pre-B leukemia in which subsets of the cells were producing gamma- or alpha-H chains. The switch recombinase activity gamma- or alpha-H chains. The switch recombinase activity may be restricted to the B cell lineage, inasmuch as CH gene deletions were not observed in T lineage malignancies.

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.