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  B-CELL DIFFERENTIATION AND RISKS FOR NEOPLASTIC TRANSFORMATION   SR BAUER   INTRODUCTION  
    Among the various differentiated lineages of cells found in animals, the B lymphoid cells undergo an unique differentiation process that generates their essential ability to detect antigens and then to respond by antibody production.  The process of antibody formation involves several steps in which deliberate alterations of the genome are induced in individual B-cells. These deliberate alterations can be considered a type of mutation since the resultant genome in a particular B-cell is different from germline in DNA content and sequence. The B-cell’s inherent capacity to rearrange and mutate its immunoglobulin genes is critical for generation of specific antibody-mediated immunity.  However, this capacity also introduces risk since the processes of immunoglobulin gene rearrangement and mutation can go awry, resulting in unintentional rearrangement or mutation of genes involved in control of cell growth. In addition to this high mutation risk, B-cells have an innate capability to undergo high rates of proliferation at various stages of differentiation or in response to antigen. Thus mutations which accumulate can be unintentionally selected when an altered B-cell is undergoing the high natural proliferation in response to antigen or during a normal phase of differentiation. This selection then sets the stage for further accumulation of mutation and consequent B-cell neoplasia.
  CONTROLLED GENETIC ALTERATION IN B-CELLS  
    A unique capacity of B-cells is to initiate rearrangement of dispersed gene segments in order to assemble a new, intact immunoglobulin gene. The V, D, and J segments rearrange to form the antigen binding variable domain of the final immunoglobulin molecule. The V (variable) gene segments encode the bulk of the variable region while the D (diversity) and J (joining) segments
form a link to the C (constant) region and form a hypervariable region which contributes to antigen specificity of the antibody molecule. When successfully rearranged, the segments encode a novel antibody molecule. The rearrangement takes place between V, D and J segments in the heavy chain locus and V and J in the light chain locus. These rearrangements are catalyzed by a multi-subunit enzymatic complex called V(D)J recombinase. This enzyme complex randomly initiates specific cleavage of double stranded DNA at conserved sites surrounding single V, D, and J gene segments. Cleavage is followed by rejoining of a D to J segment or a V to the rearranged DJ segment. Intervening DNA is excised or inverted and moved out of the immunoglobulin gene coding regions. The rearrangements of these gene segments is a major cause of the diversity of antibody molecules since each immunoglobulin is the result of random assembly of a large pool of gene segments. In mouse there are approximately a thousand V region segments, 12 D region segments, and 4 J regions segments. In the human, there are approximately 90 V H gene segments grouped into seven families; 30 functional DH gene segments and six J H gene segments (Schroeder and Dighiero, 1994).
 
    The critical alterations of immunoglobulin gene loci are controlled by a variety of mechanisms and take place at different anatomical sites. The sequence in which various antibody gene fragments are rearranged is ordered and the stages of B-cell differentiation are largely defined by the status of antibody gene rearrangement. B-cells undergo stages of both antigen dependent and antigen independent differentiation, proliferation, and quiescence (reviewed in Rolink and Melchers, 1991).
 
    The molecular events which influence transition between various stages of B-cell maturation also expose these cells to the risk of deleterious mutations. These mutations result from normal processes which operate in abnormal fashion. Figure 1 diagrams the stages of B-cell differentiation with respect to the gene rearrangements or mutations that take place in both the antigen independent and antigen dependent phases of B-cell differentiation.
    CONTROLLED IMMUNOGLOBULIN GENE REARRANGEMENTS   Locus Exclusion  
    Following commitment of a hematopoeitic cell to the B lymphoid lineage, the immunoglobulin V(D)J recombinase enzymatic machinery is activated.  This enzyme complex has the capacity to rearrange V, D and J elements of both the T-cell receptor gene loci and the immunoglobulin gene loci. However, precursor cells in the T- or B-cell lineage undergo rearrangement of only the
appropriate gene loci; the T cell receptor genes rearrange in T-cell precursors and the immunoglobulin genes rearrange in B-cell precursors. The signal sequences recognized by the recombinase are the same in both loci but the inappropriate substrates are rendered inaccessible or unrecognizable by unknown mechanisms.
  Ordered Rearrangement  
    The process of V(D)J recombination is highly controlled. As V(D)J recombinase activity develops, the order in which gene segments are rearranged seems to be orchestrated so that D to J joining in the heavy chain locus occurs first. Then V to DJ rearrangement on one allele of the two heavy chain loci occurs. If this V to DJ rearrangement is productive, heavy chain rearrangement ceases. If not, rearrangement of V to DJ is attempted at the second heavy chain locus.
  Light Chain Rearrangement Order  
    Following productive heavy chain rearrangement, a V to J join occurs on one of two kappa chain loci. As in the heavy chain, only one of the two kappa loci rearranges to produce immunoglobulin light chain protein. In contrast to the heavy chain locus, there are two separate light chain loci, each having two alleles. Light chain rearrangement starts at the kappa locus. If recombination at the first allele is not productive, the second kappa allele can undergo V to J recombination. Upon productive kappa chain rearrangement, V(D)J recombinase activity ceases. However, if neither kappa rearrangement is successful, the lambda locus begins to recombine and has two alleles to attempt rearrangement.  Following successful recombination, V(D)J recombination activity ceases and a surface immunoglobulin positive B-cell has been produced and is ready to enter circulation. This ordered rearrangement assures that a particular B-cell only produces one light chain isotype (reviewed in Tonegawa, 1983).
  Allelic Exclusion  
    Although there are two chromosomal copies of the immunoglobulin heavy chain genes, B-cells produce only one heavy chain protein. Successful rearrangement of the immunoglobulin VDJ segments on one of the two alleles results in production of heavy chain polypeptide. Through unknown mechanisms, this event signals cessation of rearrangement of the other heavy chain
gene segments, thus precluding development of a cell producing more than one antibody species. This is known as allelic exclusion and requires expression of the membrane associated form of the mu heavy chain. In addition to mediating allelic exclusion, productive rearrangement of the heavy chain also stimulates onset of rearrangement of the light chain kappa locus.
  Switch Recombination  
    Another deliberate mutation induced in the immunoglobulin locus is that of immunoglobulin heavy chain isotype switching. This process involves intrachromosomal deletion of constant region genes between switch recombination signal sequences downstream of the intronic enhancer and switch recombination sequences upstream of the target constant region segment.  Intervening constant regions are deleted and the target constant region gene is brought into the transcriptional unit of the VDJ promoter and the intronic heavy chain enhancer. This process allows B-cells that express the same VDJ and light chain VJ rearrangements to develop clonal progeny that make different heavy chain isotypes but recognize the same epitopes. The various heavy chain isotypes mediate different effector functions such as complement fixation and ability to cross the placental barrier. Thus, antigen stimulation followed by clonal selection of an IgM expressing cell can lead to expansion of related subclones expressing the same antigen specificity but different effector functions.
 
    The process of switch recombination is also highly controlled and can be specifically directed in response to various cytokines from accessory cells.  With the exception of B-cells simultaneously co-expressing the same VDJ region on mu and delta isotypes, B-cells that have undergone switch recombination generally express only one heavy chain isotype.
  SOMATIC MUTATION  
    Another mutational mechanism employed by B-cells is that of somatic mutation (Berek, 1993). Somatic mutation occurs in germinal centers in response to antigenic challenge and involves specific hypermutation of the rearranged immunoglobulin gene segments encoding the antigen binding portions of the immunoglobulin molecules. This process generates mutations randomly but results in antibodies of higher affinity due to selection processes in the germinal center. B-cells which have undergone mutations that allow a higher affinity antibody molecule to be made are preferentially selected and go on to expand and secrete antibody specific to the antigenic stimulus.
  MECHANISMS CONTROLLING ALLELIC EXCLUSION  
    It has been demonstrated that the surface bound form of the mu heavy chain is necessary for allelic exclusion to occur (Kitamura and Rajewsky, 1992). However, it is paradoxical that the surface form of mu heavy chain is required for allelic exclusion since B-cells rearrange the light chain only after heavy chain rearrangement ceases. This means that the process of allelic exclusion has already occurred at a stage of B-cell development in which no light chains are available to form surface immunoglobulin. How can the mu heavy chain appear on the cell surface and mediate allelic exclusion before light chains are available?
  Surrogate Immunoglobulin Light Chains in V(D)J Recombination  
Recently, it was found that preB-cells express two surrogate light chain proteins which can associate with the mu heavy chain (reviewed in Melchers et al., 1993). One of the genes, l5, encodes a protein homologous to the constant and joining region portions of authentic lambda light chain protein.  However, the amino-terminal end of the l5 molecule is not a variable region nor is it related to other immunoglobulin gene family members. The second gene, VpreB, encodes a protein resembling the leader and variable regions of an immunoglobulin molecule. However, the carboxy-terminal end of the protein is not linked to a joining region nor is it related to immunoglobulin family members. Both proteins associate with the mu heavy chain. The l5 protein is covalently linked while the VpreB protein is very tightly but non-covalently linked to the mu heavy chain. Figure 2 summarizes characteristics of the genes and proteins and shows a hypothetical structure for the m/l5 /VpreB surrogate immunoglobulin molecule. RNA expression of the human VpreB gene has been shown to be useful as a diagnostic marker for preB ALL and could be used to differentiate acute undifferentiated leukemia (AUL) subtypes that had undergone some commitment to the B-cell lineage (Bauer et al., 1991).
 
    The role of these proteins in B-cell development remains a key question.  The importance of these proteins in B-cell development has been demonstrated since knock out mice which lack the l5 gene have severely impaired B-cell development.
 
    One potential clue to the function of this complex has been suggested by molecular studies of B-cell lineage tumors arising in transgenic mice (Scheuermann and Bauer, 1990). Among the various tumors observed, tumors that expressed the surrogate immunoglobulin molecule on the cell surface did not express RAG gene RNAs nor did they have V(D)J-recombinase activity.  Tumors from stages preceding and following this stage of B-cell development expressed the RAG genes. This observation led to the hypothesis that the function of the surrogate light chain proteins is to signal successful rearrangement of a heavy chain allele and then to inhibit rearrangement at the other heavy chain locus. This would provide a molecular mechanism explaining allelic exclusion and would account for the necessity of membrane bound mu heavy chain to mediate allelic exclusion. Figure 2 is a diagram illustrating this hypothesis (Bauer and Scheuermann, 1993).
    CONTROLLED PROLIFERATION IN THE B-CELL LINEAGE  
    B-cells go through two stages of differentiation, antigen independent and antigen dependent (Figure 1).
 
    Antigen independent differentiation commences with commitment of a stem cell to the B-cell lineage. During precursor B-cell differentiation there are several stages characterized by differing rates of cellular proliferation (reviewed in Osmond, 1990). Proliferating large preB-cells can be differentiated from small resting preB-cells by markers such as TdT and by cell density.  The antigen independent phase of differentiation ends when a B-cell reaches the stage of surface immunoglobulin expression and is prepared to respond to antigen.
 
    Antigen dependent differentiation occurs upon encounter of a B-cell with its antigen. This encounter can lead to tolerance induction (anergy), immunoglobulin heavy chain isotype switching, plasma cell differentiation leading to antibody secretion, and memory B-cell formation. The anergic B-cell and the memory B-cell populations do not undergo large bursts of proliferation or clonal expansion (Nossal, 1994). In contrast, the pathway of plasma cell differentiation involves large scale clonal expansion followed by terminal differentiation into the plasma cell. Plasma cells produce and secrete large amounts of antigen specific antibody but are not long lived nor do they divide.
 
    A second lineage of cells, the CD5 B-cells, are formed during fetal development and have the capacity to self renew throughout the life of a mouse.  This self-renewing capacity is unique to the CD5 lineage and proceeds with no obvious large scale clonal proliferation but rather with an ongoing, life-long homeostatic production of these cells.
  B-CELLS AS SENTINELS FOR TOXIC EXPOSURE  
    B-cells may serve as uniquely sensitive indicators of environmental exposure to mutagenic agents due to their capacity to undergo developmental steps in which deliberate genetic alterations are induced by endogenous cellular mechanisms. These genetic changes are then tolerated by the mechanisms that normally repair altered DNA. This inherent capacity to tolerate genetic alterations may permit environmental agents to mutagenize B-cells during developmental stages in which genetic alterations are ongoing and tolerated as part of the normal differentiation process. Accumulation of unintended mutations in non-immunoglobulin genes may put B-cells at risk for growth dysregulation or transformation. Indeed, neoplastic transformation is generally regarded as a process involving multiple mutations. Compared to other cell lineages, B-cells may be more permissive for transformation since they may be able to tolerate mutations that other cells would repair. An environmentally induced mutation in a B-cell could have several consequences. Such a mutation could be inert, immediately lethal, or could be dysregulatory and allow abnormal growth of the affected B-cell. Such a mutation would be inherited by all progeny of an affected B-cell and could thus cause abnormal expansion of a B-cell clone or set the stage for acceleration of further mutation by affecting a tumor suppressor gene such as p53. B-cells with dysregulated growth would potentially manifest as clonal populations identifiable by uniform cell surface marker profiles and identical immunoglobulin gene rearrangements.  Thus, detection of an abnormally expanded, clonal B-cell population could serve to indicate previous exposure to a mutagenic agent.
 
    The potentially deleterious aspect of the normal process of V(D)J recombination has recently been illustrated by studies which showed that aberrant V(D)J recombinase activity can occur in normal and genetically susceptible individuals. Aberrant V(D)J rearrangements can occur between immune receptor loci, resulting in hybrid antigen-receptor genes and the formation of several different lymphocyte-specific chromosomal aberrations. These aberrant V(D)J recombinase mediated events occur at a low frequency in the peripheral blood lymphocytes (PBL) of normal individuals but show an increased incidence in the PBL of individuals with the autosomal recessive disease ataxia-telangiectasia (AT) (Lipkowitz et al., 1990). The increase in aberrant V(D)J recombinase activity in AT patients is paralleled by an increase in lymphogenesis in these patients. Several chromosomal translocations are associated with B-cell neoplasia and may indeed involve aberrant V(D)J or switch recombination mechanisms.
 
    It has recently been shown that environmental exposure to pesticides can transiently increase the frequency of aberrant V(D)J recombinase activity (Lipkowitz et al., 1992). Thus, the increase in aberrant V(D)J recombinase activity can be correlated with both an increased risk of lymphomagenesis and with environmental exposure to pesticides. These observation lend credence to the idea that B-cells can serve as indicators of environmental exposure by displaying an abnormal pattern of clonal expansion following exposure.
 
    The concept of B-cells as sentinels for environmental mutagen exposure is somewhat analogous to the usefulness of the canary in the cage traditionally used by coal miners to detect the presence of harmful gases in their dangerous environment. The properties of B-cells could allow them to be sensitive sentinels of environmental exposure to mutagenic agents. Acute or chronic exposure to environmental mutagens may result in abnormal rates of B-cell dysregulation in populations of exposed individuals. Detection of abnormal B-cell expansions could thus be used to indicate that environmental mutagenic agents were present in a suspect location.
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