CHAPTER 83 FUNCTIONS OF B LYMPHOCYTES AND PLASMA CELLS IN IMMUNOGLOBULIN PRODUCTION
CHAPTER 83 FUNCTIONS OF B LYMPHOCYTES AND PLASMA CELLS IN IMMUNOGLOBULIN PRODUCTION
THOMAS J. KIPPS
Immunoglobulin Structure and Function
Basic Immunoglobulin Structure
Genetics of Immunoglobulins
Immunoglobulin Gene Complexes
Immunoglobulin Gene Rearrangement and Expression During B-Cell Development
Mechanisms for Generating Antibody Diversity
Immunoglobulin Variable Region Structure
Immunoglobulin Variable Region Subgroups
Genetic Markers on Immunoglobulin Constant Regions
Lambda Light-Chain Isotypes
Immunoglobulin Synthesis and Secretion
Regulation of Immunoglobulin Synthesis
Much of our immune defense against invading organisms is predicated upon the tremendous diversity of immunoglobulin molecules. Immunoglobulins are glycoproteins produced by B lymphocytes and plasma cells. These molecules may be considered receptors, in that the primary function of the immunoglobulin molecule is to bind antigen. A single person can synthesize 10 to 100 million different immunoglobulin molecules, each having a distinct antigen-binding specificity. This great diversity in the so-called humoral immune system allows us to generate antibodies specific for a variety of substances, including synthetic molecules not naturally present in our environment. Despite the diversity in the specificities of antibody molecules, the binding of antibody to antigen initiates a limited series of biologically important effector functions, such as complement activation and/or adherence of the immune complex to receptors on leukocytes. The eventual outcome is the clearance and degradation of the foreign substance. This chapter describes the structure of immunoglobulins and outlines the mechanisms by which B cells can produce molecules of such tremendous diversity with defined effector functions.
Acronyms and abbreviations that appear in this chapter include: ADCC, antibody-dependent cell-mediated cytotoxicity; BiP, immunoglobulin “binding protein”; C, constant; CDR, complementarity-determining region; CRIs, cross-reactive idiotypes; D, diversity; FR, framework region; GM-CSF, granulocyte-macrophage colony stimulating factor; H, heavy; INF-gc, interferon gamma; Kde, kappa-deleting element; L, light; NK or K, natural killer cells; RAG-1 or RAG-2, recombination activating genes 1 or 2; RSS, recombination signal sequences; SCID, severe-combined immunodeficient; TdT, terminal deoxynucleotidyl transferase; TNF-a, tumor necrosis factor alpha (or cachectin); V, variable.
IMMUNOGLOBULIN STRUCTURE AND FUNCTION
BASIC IMMUNOGLOBULIN STRUCTURE
All naturally occurring immunoglobulin molecules are composed of one or several basic units consisting of two identical heavy (H) chains and two identical light (L) chains (Fig. 83-1). The four polypeptides are held in a bilaterally symmetrical, Y-shaped structure by disulfide bonds and noncovalent interactions.1,2 The internal disulfide bonds of the heavy and light chains cause the polypeptides to fold into compact globe-shaped regions, called domains, each containing about 110 to 120 amino acid residues.3 Each domain forms a common fold of a type of protein structure known as beta-pleated sheets and is stabilized by a conserved disulfide bond (Fig. 83-1). The light chains have two domains; the heavy chains have four or five domains. The amino-terminal domains of the heavy and light chains are designated the variable (V) regions, because their primary structure varies markedly among different immunoglobulin molecules.4 The carboxy-terminal domains, however, are referred to as constant (C) regions, because their primary structure is the same among immunoglobulins of the same class or subclass. The amino acids in the light- and heavy-chain variable regions interact to form an antigen-binding site.1,5 Each four-chain immunoglobulin basic unit has two identical binding sites. The constant region domains of the heavy and light chains provide stability for the immunoglobulin molecule. The heavy-chain constant regions also mediate the specific effector functions of the different immunoglobulin classes (Table 83-1).6
FIGURE 83-1 Schematic model of an IgG molecule of the IgG1 subclass. The sites of proteolytic cleavage by papain and by pepsin are indicated. The papain-generated fragments Fab and Fc are indicated to the right of the schematic drawing. Thin lines indicate the intrachain disulfide bonds of the variable region domains (VH and VL) and constant region domains and the interchain disulfide bonds near the antibody hinge region (labeled Hinge). NH3 or COO– indicate the amino terminus or carboxyl terminus of each polypeptide respectively. Key functional sites of the antibody responsible for antigen binding, complement fixation, or Fc receptor binding are as indicated by the brackets. The glycosylation sites on the constant region carbohydrate groups are indicated by filled circles.
TABLE 83-1 PHYSICAL PROPERTIES OF HUMAN IMMUNOGLOBULINS
Immunoglobulin light chains have an approximate Mw of 23,000. They are divided into two types, kappa (k) and lambda (l), based upon multiple amino acid sequence differences in the single constant region domain.4 The l chains are divided further into subclasses. The proportion of k to l chains in adult human plasma is about 2:1. The immunoglobulin-light chain-constant region has no known effector function. Its main purpose may be to allow for proper assembly and release of an intact immunoglobulin molecule. Soon after synthesis, the antibody light chain constant region associates with the nascent immunoglobulin heavy chain (Fig. 83-1), releasing the latter from the immunoglobulin “binding protein,” or BiP. BiP is a heat-shock protein that, in the absence of antibody light chain, binds the first constant region domain of the newly synthesized heavy chain, thereby retaining the heavy chain polypeptide in the cell’s endoplasmic reticulum.7
Immunoglobulin heavy chains have a Mw of 50,000 to 70,000, depending upon the number and length of the constant region domains. The five major isotypes of heavy chains, gamma, alpha, mu, delta, and epsilon, determine the five corresponding classes of immunoglobulin: IgG, IgA, IgM, IgD, and IgE. The individual immunoglobulin molecules of each isotype may contain either k or l light chains, but not both. The distinct physical and functional properties of the human immunoglobulin classes are summarized in Table 83-1 and Table 83-2.
TABLE 83-2 BIOLOGIC PROPERTIES OF HUMAN IMMUNOGLOBULINS
Approximately 80 percent of the immunoglobulins in adult plasma are IgG. The IgG molecule is composed of the basic 150,000-dalton immunoglobulin four-chain structure, plus about 3 percent carbohydrate. IgG is the predominant antibody produced during the secondary immune response. IgG molecules effectively penetrate extravascular spaces and readily cross the placental barrier to provide passive immunity to the newborn.
Near the junction of the two arms of the Y-shaped immunoglobulin molecule, the two heavy chains interact to form a flexible “hinge” region (Fig. 83-1). Exposed between constant region globular domains, the hinge region is attacked readily by the proteolytic enzyme papain or pepsin. The cleavage sites are shown in Fig. 83-1. Digestion of IgG with papain yields three fragments. The single Fc piece contains the carboxy-terminal region of both heavy chains. The two identical F(ab) pieces contain the entire light chain and the amino-terminal portion of the heavy chain.
Within the IgG class are four major subclasses, designated IgG1, IgG2, IgG3, IgG4. Each subclass has a distinct heavy-chain constant region and mediates different effector functions (Table 83-3).6 The average half-life of circulating IgG molecules is approximately 21 days, although the exact value varies among the IgG subclasses (Table 83-3). The most abundant subclass is IgG1, which constitutes 65 percent of the total IgG in plasma. Whereas IgG1 and IgG3 proteins activate complement via the classical pathway, IgG2 molecules fix complement poorly and IgG4 proteins not at all. IgG3 myeloma protein may aggregate spontaneously to produce a hyperviscosity syndrome.
TABLE 83-3 CHARACTERISTICS OF MAJOR IgG SUBCLASSES
Either aggregated IgG or antigen-antibody complexes may bind to specific receptors for the Fc fragment, designated FcRI (CD64), FcRII (CD32), and FcRIII (CD16). Of the IgG subclasses, IgG1 binds best to FcRI (CD64) and FcRII (CD32), with affinities (Kd) of 1 × 10–8 M and 5 × 10–7 M respectively (Table 83-3). IgG1 and IgG3 bind equally well to FcRIII (CD16), with an affinity (Kd) of 2 × 10–6 M (Table 83-3). This is the Fc receptor expressed by natural killer cells (NK cells or K cells) that mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Proteins of the IgG4 or IgG2 subclass bind poorly to FcRI (CD64) or FcRII (CD32), and to FcRIII (CD16) not at all (Table 83-3).
IgA comprises about 13 percent of plasma immunoglobulins (Table 83-1). Specific IgA antibodies are synthesized during secondary immune responses. IgA circulates as a monomer, dimer, or higher polymer containing approximately 8 percent carbohydrate. Within the IgA class there are two major subclasses, designated IgA1 and IgA2. The most abundant subclass is IgA1, which constitutes approximately 85 percent of the total IgA in plasma. The half-life of circulating IgA of either subclass is approximately 6 days.
The primary role for IgA is in mucosal immunity.8,9 A modified form of IgA is the principal antibody in saliva, tears, colostrum, and the fluids of the gastrointestinal, respiratory, and urinary tracts. These secreted immunoglobulins consist of an IgA dimer bound to the J- (or joining) chain polypeptide and a secretory protein of 70,000 daltons. The J chain is required for proper hepatic transport of IgA.10 The secretory component actually is part of a Fc receptor for dimeric IgA that is not synthesized by B cells but rather by epithelial cells of organs such as the intestine. This protein facilitates the transport of the IgA protein across the epithelial cell and may protect the secreted IgA molecule from proteolytic digestion by enzymes in the intestinal lumen. IgA antibodies do not cross the placenta, fix complement via the classical pathway, or bind efficiently to cell surfaces. Indeed, their main function may be to prevent foreign substances from adhering to mucosal surfaces and entering the blood.
In a normal adult, approximately 6 percent of the total plasma immunoglobulins belong to the IgM class (Table 83-1 and Table 83-2). IgM molecules classically are termed macroglobulins because of their large molecular weight. Circulating IgM molecules contain 12 percent carbohydrate and are formed through the linkage of five identical immunoglobulin units by disulfide bonds and by a J chain10 (Fig. 83-2). IgM represents the predominant immunoglobulin class formed during a primary immune response. IgM macroglobulins do not penetrate easily into extravascular spaces or readily cross the placenta. Compared to monomeric IgG antibodies, pentavalent IgM antibodies fix complement more efficiently. A single IgM molecule on the surface of a red blood cell can initiate complement-mediated hemolysis. IgM is catabolized rapidly, with a plasma half-life of only 6 days. The monomeric form of IgM, with only two heavy and two light chains, is the major immunoglobulin expressed on the B-cell surface (Fig. 83-3).
FIGURE 83-2 Schematic model of an IgM pentamer. This diagram shows the positions of the heavy (H) chain, light (L) chains, and the single J chain. Intrachain and interchain disulfide bonds are indicated by the thin lines.
FIGURE 83-3 Schematic of membrane IgM (mIgM) and associated membrane proteins Ig-a and Ig-b or Ig-a and Ig-g. Intrachain and interchain disulfide bonds are indicated by the thin lines. Filled circles depict the lipid bilayer. The immunoglobulin variable and constant region domains are indicated as in Fig. 83-1.
Although a trace serum protein that comprises less than 1 percent of plasma immunoglobulins, IgD is expressed on most peripheral B cells along with IgM. The molecule has the basic four-chain constant region and contains 11 percent carbohydrate (Table 83-1 and Table 83-2). Sensitive to proteolytic degradation, IgD antibodies do not penetrate extravascular spaces efficiently, cross the placental barrier, or fix complement via the classical pathway. Rather, IgD functions primarily as a B-cell membrane receptor for antigen that facilitates recruitment of B cells into specific antigen-driven responses.11
Although four human IgE isoforms can be produced by alternative splicing of the epsilon primary transcript,12 each isoform appears to have similar function. IgE has been called reaginic antibody to denote its association with immediate hypersensitivity. It normally constitutes only 0.004 percent of total plasma immunoglobulin (Table 83-1 and Table 83-2). In patients with parasitic infestation, and in some children with atopic diseases, plasma IgE levels may rise to 5 to 20 times normal. The IgE molecule consists of a four-chain basic unit, plus 12 percent carbohydrate. Monomeric IgE binds via the Fc region to high-affinity receptors on the surface membranes of basophils and mast cells. When bound to tissue mast cells, IgE has a much longer half-life than in plasma, in which its half-life is only about 2 days (Table 83-2). Cross-linking of cell-bound IgE antibody by antigen induces the release of vasoactive amines, lipid-derived inflammatory mediators, proteases, proteoglycans, and cytokines, such as tumor necrosis factor alpha (TNF-a or cachectin), interferon gamma (INF-g), granulocyte-macrophage colony stimulating factor (GM-CSF), or interleukins 1, 3, 4, 5, and 6. These substances act on adjacent cells and may regulate the metabolism of the connective tissue extracellular matrix. These lipid mediators and biogenic amines may produce the rapid components of immediate hypersensitivity, such as vascular leakage, vasodilatation, and bronchoconstriction. The released cytokines, on the other hand, are responsible for the late phase of the immediate hypersensitivity response. The physiologic function of this response is not clear. Instead, the immediate hypersensitivity response actually may represent a pathologic systemic exaggeration of a local physiologic process that may potentiate the inflammatory response to invading organisms.
Any one of the immunoglobulin isotypes may serve as a B-cell membrane receptor for antigen. However, most B cells express surface IgM with or without IgD. Each immunoglobulin is expressed on the surface membrane as a monomer complexed noncovalently with disulfide-linked heterodimeric glycoproteins that, together with surface immunoglobulin, form the B-cell antigen-receptor complex13 (Fig. 83-3). For surface IgM, each heterodimer is composed of CD79a, an IgM-a chain of 33 kDa, complexed with CD79b, an Ig-b chain of 37 kDa (see Chap. 13). CD79a interacts with the CH4 domain and the transmembrane domain of the surface IgM molecule (Fig. 83-3). This chain is a product of the human mb-1 gene located at 19q13.2, while CD79b is the product of another gene located on a different chromosome at 17q23.13 B cells that lack expression of CD79a or CD79b cannot express surface immunoglobulin.
CD79a/CD79b are necessary, not only for transport of the assembled immunoglobulin to the cell surface, but also for signal transduction following surface immunoglobulin-receptor cross-linking by antigen. The cytoplasmic tails of these accessory molecules each contain immunoreceptor tyrosine activation motifs, or ITAMs. Such motifs are found in the cytoplasmic domains of several immune system signaling molecules, including those of the T-cell receptor complex (see Chap. 84). Upon surface immunoglobulin cross-linking, the tyrosine residues in these motifs are phosphorylated, allowing cytoplasmic-signaling molecules to recognize and bind to the activated B-cell receptor complex. These signaling molecules in turn activate a variety of other intracellular signaling molecules (see Chap. 15). As such, tyrosine phosphorylation is the first step in the intracellular cascade triggered by antigen binding to specific surface immunoglobulin.14
There are three major tyrosine kinases associated with the surface immunoglobulin receptor complex. These tyrosine kinases Fyn, Blk, and Lyn can phosphorylate tyrosine residues in the ITAMs of CD79a and CD79b. The phosphorylated ITAMs are then recognized and bound by another tyrosine kinase, called Syk, that in turns triggers activation of a cascade of intracellular signaling molecules (see Chap. 15).
To mitigate the accidental initiation of signal transduction, the signaling cascade is subject to negative controls.15,16 One of these acts directly on the receptor-associated kinases that are activated by phosphorylation at one site but are inhibited by phosphorylation at another. Activation of such kinases thus requires dephosphorylation of the inhibitory site and phosphorylation of this activating site. Dephosphorylation of the inhibitory site is mediated by CD45, a membrane phosphatase that also is known as leukocyte common antigen. As such, CD45 contributes to the activation of B lymphocytes by removing the inhibitory phosphates from the receptor-associated kinases.17
Another protein, called SH2-domain-containing-phosphotyrosine phosphatase-1 (SHP-1) or protein-tyrosine phosphatase 1c (PTP1c), is responsible for turning off the activated tyrosine kinases, thereby limiting the response of the activated B cell.17,18 SHP is a cytoplasmic phosphatase that acts by removing the activating phosphate groups from such kinases. The importance of this enzyme is demonstrated in mutant mice that lack this enzyme.19 The B lymphocytes of such animals are stimulated by much lower concentrations of antigen than the lymphocytes of normal mice. Because of this, these mice have abnormal B-cell proliferation, develop autoimmune disease, and die within a few weeks of birth.
GENETICS OF IMMUNOGLOBULINS
IMMUNOGLOBULIN GENE COMPLEXES
Immunoglobulin genes are inherited in three unlinked gene complexes: one for the heavy-chain classes, one for k light chains, and one for l light chains. The immunoglobulin heavy-chain gene complex is located at band q32 of the long arm of chromosome 14. This complex is composed of 51 functional heavy-chain variable region genes (VH genes), 25 functional 27 diversity (D) segments, 6 functional JH minigenes, and exons encoding the constant regions for each of the immunoglobulin heavy-chain isotypes (Fig. 83-4).20,21 and 22 The k light chain gene complex is contained within band p12 on the short arm of chromosome 2. This gene complex consists of approximately 40 functional kappa light-chain variable region genes (Vk genes), 5 Jk segments, 1 constant region exon, and 1 kappa-deleting element (Kde) (Fig. 83-5).23,24 Many of the Vk genes in the region most proximal to the Jk segments are in the opposite orientation of the Jk segments, thus requiring that the Vk exons in the proximal region undergo inversion during immunoglobulin gene rearrangement (Fig. 83-5). The l light chain gene complex is located at band q11.12 on the long arm of chromosome 22. This gene complex consists of approximately 39 functional lambda light-chain variable region genes (Vl genes) along with 4 functional l constant region genes (Cl1, Cl2, Cl3, and Cl7) and 3 l constant-region pseudogenes (Cl4, Cl5, and Cl6), each associated with 1 Jl segment (Fig. 83-5).25 The constant region elements of the heavy-chain gene complex are proximal to variable region segments on chromosome 14, while the constant region segments of the two light chains are in the opposite orientation, telomeric to the variable region genes.
FIGURE 83-4 The human heavy chain immunoglobulin gene complex on chromosome 14q32. The heavy chain exons encoding the constant regions are represented by black boxes, and the associated intronic switch regions (S) are each depicted as a line. These exons are labeled to the right of these symbols. A Y next to the heavy chain isotype designation indicates that the gene is a pseudogene. JH segments and D segments are indicated by lines. Each VH gene locus is labeled on the right of each symbol. By convention, the loci encoding each of the various VH genes are assigned a number corresponding to the VH gene subgroup followed by a hyphen and then the rank order distance from the heavy chain D segments. Listed in parentheses are the alternative names that have been used to designate each locus. Identified polymorphic insertions and/or duplications are indicated with brackets. Black squares represent VH gene loci that are known to be functional. Open circles represent VH pseudogenes. The open boxes depict VH exons that appear functional, but rarely are found to encode a functional heavy chain gene rearrangement. At the end of the line containing the symbols are arrows that indicate the direction to the centromere or the telomere. (For updates see: http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html).
FIGURE 83-5 The immunoglobulin light chain gene complexes. The figure on the left depicts the kappa light-chain gene complex on chromosome 2p11-12. The black boxes in this figure represent the Kde element or the Ck constant region exon as indicated to the right of each box. Positioned between the Ck constant region exon and the Jk segments is the kappa light chain enhancer (E). The Jk segments are indicated by lines. The Vk genes are clustered in two regions centromeric to the Jk and Ck exons, each region spanning approximately 500 kb. Approximately 800 kb separate the two regions. The region proximal to Jk and Ck, designated p, contains 40 Vk genes (B3®B1, L13®L1, A30®A15, and O18®O11), while the distal region (d contains 36 gene segments (O1®O10, A1®A14, L14®L25). Vk genes that can encode functional kappa light chains are represented by black boxes and are labeled to the right of each symbol. Thirty-two of the 76 Vk genes are pseudogenes (open boxes). The d region apparently arose through duplication of a large portion of the p region. Consequently, there are 33 pairs of Vk genes that share 95–100% nucleic acid sequence homology, accounting for 66 of the 76 Vk genes in the k light chain complex. There Vk genes can be grouped further into four clusters, A, B, L, and O, three of which (A, L, and O) are duplicated and found in both the Jk-proximal p region and the Jk-distal d region. The B cluster, containing Vk genes B1, B2 (EV15), and B3 (DPK26), is found only in the Jk-proximal p region. Each Vk gene can be assigned to one of three main subgroups, I-III, and several smaller subgroups (IV, V, VI, and VII) based on nucleotide sequence homology. The largest subgroup is VkI, with 21 functional genes, depicted as black boxes. The next largest subgroups are Vk2, with 11 functional genes, and Vk3, with 7. There are 3 functional genes in the Vk6 subgroup, and one each for the Vk4 and Vk5 subgroups. The Vk7 subgroup consists of one of the non-functional pseudogene, which are depicted as open squares. Open squares depict Vk pseudogenes. Black arrows indicate the transcriptional orientation of the V genes in the complex. At the end of the line connecting the symbols are arrows that indicate the direction to the centromere or the telomore.
The figure on the right depicts the lambda-light-chain gene complex on chromosome 22q11.2. The black boxes represent functional Jl-Cl exons, whereas open boxes represent Jl-Cl pseudogenes. The Jl-Cl exon pairs are labeled to the right of each symbol. The figure also depicts as black boxes the 39 functional Vl genes centromeric to the lambda constant regions. These Vl genes are arranged into 10 subgroups, each comprised of Vl genes sharing greater than 75% nucleotide sequence homology. The first number in the labels to the right of each Vl gene provides the number of the subgroup, followed by a hyphen, and then the relative rank order of the V gene from the constant region exons. Note that the Vl genes have been mapped into three clusters within 860 kb of the Jl and Cl genes that are each separated from one another in the figure by double lines. The cluster most proximal to the Jl-Cl exons, designated “A”, is comprised of 18 functional Vl genes mostly belonging to the Vl2 and Vl3 gene subgroups. The next cluster, “B”, contains 15 functional Vl genes of the Vl1, Vl5, Vl7, Vl9 gene subgroups. The third cluster, “C”, contains 6 functional Vl genes of the Vl4, Vl6, Vl8, Vl10, and Vl11 gene subgroups along with exon encoding VpreB. Some individuals have an insertion of a functional Vl gene, 5-39 (5a), marked as “polymorphic insertion”. Black arrows indicate the transcriptional orientation of the V genes in the complex.
Each germ line V gene, D element, and J segment is flanked by recognition sequences that are necessary to direct site-specific recombination. Such sequences consist of a highly conserved palindromic heptamer (5′ CACAGTG 3′), a nonconserved spacer of 12 or 23 bp, and a conserved nonamer (5′ ACAAAAACC 3′). Joining usually occurs only between segments flanked by recognition sequences with unequal spacers.26,27 and 28 This is referred to as the 12/23 joining rule. Each spacer varies in sequence, but its length is conserved and corresponds to one or two turns of the DNA double helix. Each spacer serves to bring the heptamer and nonamer sequences to one side of the DNA helix, where they can be bound by the protein complex that catalyzes recombination. Similar recognition sequences flank the elements that rearrange to form the T-cell antigen receptor (see Chap. 84). Such heptamer-spacer-nonamer sequences are targets of lymphocyte-specific recombinases and are often called recombination signal sequences, or RSS.27,28
IMMUNOGLOBULIN GENE REARRANGEMENT AND EXPRESSION DURING B-CELL DEVELOPMENT
IMMUNOGLOBULIN GENE REARRANGEMENT
During B-cell ontogeny, the first immunoglobulin gene rearrangements generally occur within the heavy-chain gene complex (Fig. 83-6A).26 One or more D segments may rearrange and become juxtaposed with a single JH element, generating a DJH complex that then may rearrange with one of the 51 functional VH genes. Subsequently, gene rearrangements occur in the light-chain loci (Fig. 83-6B). One of the 40 functional Vk genes can rearrange with any one of five Jk segments. Should these gene rearrangements fail to generate a functional Vk Jk exon, the kappa-deleting element may rearrange to a site in or immediately downstream of the Vk Jk exon, thus deleting the kappa light-chain constant region exon.29 Subsequent to kappa light-chain gene rearrangement, one of over 30 functional Vl exons can rearrange with any one of the 4 functional Jl-Cl exons to generate a gene that can encode a lambda light chain (Fig. 83-6).25
FIGURE 83-6 Immunoglobulin gene complexes and rearrangement. Diagonal double lines indicate that there is a large DNA distance between the flanking genes depicted as rectangular boxes (not drawn to scale). The upper diagram in A, B, or C shows the germ line DNA configuration of the immunoglobulin heavy-chain genes, kappa light-chain genes, or lambda light-chain genes respectively. Depicted on the left side of each immunoglobulin gene complex are exemplary immunoglobulin heavy-chain variable region genes (VH’, VH”, VH”), immunoglobulin kappa light-chain genes (Vk’, Vk’, Vk’), or immunoglobulin lambda light-chain variable region genes (Vl’, Vl’, Vl’). D denotes the diversity gene segments of the antibody heavy-chain locus. JH, Jk, or Jl indicates the joining gene segments of the antibody heavy chain, k light chain, or l light chain respectively. Cµ or Cd denotes the constant region exons of the µ or d heavy chain, respectively. Below each is a possible immunoglobulin gene rearrangement comprised of a VHDJH, for the antibody heavy chain, or a VkJk or a VlJl for the k or l light-chain genes respectively. Below the representative l constant region loci in row C are listed the names of the l nonallelic genetic markers, Mcg, Ke– Oz–, Ke– Oz+, and Ke+ Oz– on Cl1, Cl2, Cl3, or Cl7 respectively. As indicated, Cl4, Cl5, Cl6 are psuedogenes (Y gene) that do not encode protein.
The commonest mode of recombination involves the looping-out and deletion of the DNA intervening between two gene segments on the same chromosome. The 12-mer-spaced and 23-mer-spaced recombination signal sequences are brought together by interactions between proteins that specifically recognize the length of spacer between the heptamer and nonamer signals, thus accounting for the 12/23 joining rule.27,28 The two DNA molecules then are broken and religated.30 The ends of the heptamer sequences are joined precisely in a head-to-head configuration to form a signal joint in a circular piece of DNA that then is lost from the genome when the cell divides. The termini of gene segments that subsequently give rise to the coding joint form short DNA hairpins that subsequently are cleaved at a random site by an endonuclease. Depending on the site of cleavage, the single-stranded DNA may contain nucleotides that originally were complementary in the double-stranded DNA and therefore form short DNA palindromes, also known as P-nucleotides. If the cell also expresses the enzyme terminal deoxynucleotidyl transferase (TdT), then nucleotides are added at random to the ends of the single-stranded segments. These nucleotides are called non-template-encoded nucleotides, or N-nucleotides. The randomness of insertion of P-nucleotides and N-nucleotides at the junction of rearranged gene segments provides an important mechanism with which to generate diversity in the functionally rearranged immunoglobulin genes. A similar process accounts for generation of diversity in the rearrangement of T-cell receptor gene segments (see Chap. 84).
A complex of several enzymes, called V(D)J recombinase, acts in concert to mediate somatic V-region gene recombination. This complex mostly is comprised of cleavage and repair enzymes that are present in all cells and that are required for the normal maintenance of genomic DNA integrity. The first cleavage step, however, requires an additional specialized heterodimeric endonuclease encoded by two genes, called RAG-1 and RAG-2 for recombination activating genes 1 and 2.31 RAG-1 and RAG-2 are adjacent genes located on the short arm of chromosome 11 (11p13-p12)32 that were isolated based upon their ability to enable fibroblasts to catalyze V(D)J recombination of nonrearranged immunoglobulin genes that were cointroduced via gene transfer. RAG-1 has sequence similarities to bacterial topoisomerases that catalyze the breakage and rejoining of DNA. RAG-1 and RAG-2 are coexpressed normally only in developing lymphocytes that are undergoing receptor gene rearrangement. Mice with either RAG gene knocked out cannot undergo immunoglobulin or T-cell receptor gene rearrangements and consequently fail to produce mature B or T lymphocytes.33 Mutations that impair, but do not completely abolish, the function of RAG-1 or RAG-2 in humans result in a form of combined immune deficiency called Omenn syndrome.34
The other components of the V(D)J recombinase consist of enzymes that normally work to repair double-stranded breaks in genomic DNA. These include an autoantigen, called Ku, and DNA-dependent protein kinase. The latter enzyme is required to repair the junctions between the gene segments encoding the coding joints of rearranged gene segments. Consequently, mice that are deficient in this enzyme can make only trivial amounts of immunoglobulin or T-cell receptors and are called severe-combined immunodeficient mice, or SCID mice.
SURROGATE LAMBDA LIGHT CHAINS
Precursor B cells that only have rearranged D and JH elements are referred to as progenitor B cells, or “pro-B cells.” The term pre-B cells is reserved for precursor B cells that have completed immunoglobulin heavy-chain-gene rearrangement and have a functional VHDJH complex. Both pro-B cells and pre-B cells have immunoglobulin light-chain loci in germ line configuration.
Despite this, pre-B cells express some immunoglobulin µ chains in association with “surrogate” l light chains. One of these proteins, called l5, has similarity with known Cl light-chain domains.35 Another protein is called VpreB, because it resembles a V domain but bears an extra N-terminal protein sequence. Both proteins are encoded by genes located on chromosome 22. The l5 gene is situated within a l-like locus that is telomeric to the true l light-chain locus. The VpreB gene is located within the cluster of immunoglobulin Vl genes (Fig. 83-5), defined by breakpoints of chromosomal translocations found in a few leukemias and lymphomas.35 Together, VpreB and l5 pair with the µ heavy chains to form a primitive immunoglobulin receptor that, together with CD79a and CD79b, may be expressed on the surface membrane of the developing pre-B cell.36 Monoclonal antibodies that recognize l5 or VpreB specifically bind to pre-B cells and can react with B-lineage acute lymphocytic leukemias.37
The pre-B cell receptor complex is expressed only transiently, as production of l5 ceases as soon as it is formed. Nevertheless, this protein plays an important role in normal B-cell development. In normal mice, the appearance of the pre-B cell receptor coincides with inactivation of the RAG-2 protein by phosphorylation, and degradation of RAG-1 and RAG-2 mRNA, suggesting that this receptor plays a role in suppressing further immunoglobulin gene rearrangement. However, expression of the pre-B cell receptor on the surface membrane is associated with cell activation and proliferation, leading to generation of small, resting pre-B daughter cells that again express RAG-1 and RAG-2. This leads to subsequent light-chain gene rearrangement. As such, expression of the pre-B cell receptor appears to signal that a complete µ heavy-chain gene has been formed, that further rearrangements at this locus should be suppressed, and that development to the next stage can proceed. Therefore, the surrogate light chains play a critical role in normal B-cell development. This is underscored by studies on transgenic mice that lack functional l5 genes.38 In these mice, B-cell development in the marrow is blocked at the pre-B cell stage, thereby markedly reducing in the numbers of functional mature B lymphocytes in the blood and lymphoid tissues.39 Similarly, humans that have inactivating mutations in the l5 genes on both alleles of chromosome 22 have agammaglobulinemia and markedly reduced numbers of B cells.40
HEAVY-CHAIN CLASS SWITCHING
During differentiation, a single B lymphocyte can synthesize heavy chains with different constant regions coupled to the same variable region.41 As pre-B cells develop into mature B cells, intact IgM monomers are inserted into the plasma membrane, followed by IgD molecules with the same antigen-binding specificity. The IgM and IgD constant region genes are closely linked in embryonic DNA (see Fig. 83-4) and may be transcribed together. The differential splicing of the transcript allows the simultaneous synthesis of the two immunoglobulin heavy chains from a single species of messenger RNA.
The secretion of IgM, and the switch from IgM to IgG, IgA, or IgE synthesis, generally requires the prior interaction of lymphocytes with antigen or mitogen. Interleukins provided by antigen-reactive T lymphocytes strongly influence (1) which B cells differentiate into IgM-secreting plasma cells and (2) which B cells switch to synthesizing the heavy chain of another immunoglobulin isotype, such as IgG and IgA.41,42
Isotype switch recombination occurs in or near the switch region located in the intron between the rearranged VDJH sequence and the µ gene and any one of similar regions located upstream of the C genes encoding each of the other heavy-chain isotypes, with the exception of the d gene (Fig. 83-4). The µ switch region, designated as Sµ, consists of approximately 150 repeats of the sequence (GAGCT)n(GGGGGT), where n is generally three but can be as many as seven. The sequences of the other switch regions (Sl, Sd, and Se) are similar in that they also contain repeats of the GAGCT and GGGGGT sequences. The switch in heavy-chain classes results from DNA recombination between Sµ and Sl, Sd, or Se accompanied by the deletion of intervening DNA segments and the apposition of the previously rearranged variable region gene next to the new constant region gene. Switch recombination events produce genes that can encode a functional protein because they involve switch sequences within the introns and therefore cannot cause frame shift mutations in the exons encoding the immunoglobulin molecule.
MECHANISMS FOR GENERATING ANTIBODY DIVERSITY
Several mechanisms contribute to the generation of diversity among immunoglobulin polypeptide variable regions.26 These are (1) the presence in the germ line of multiple different V, J, and D gene segments; (2) the random joining of these DNA segments to produce a complete variable region exon; (3) uncorrected errors made during the recombination process; (4) the coming together of the heavy- and light-chain polypeptides to produce a complete immunoglobulin monomer capable of binding antigen; and (5) somatic mutations within the rearranged DNA segments themselves. The latter occurs through a process called somatic hypermutation.43
Somatic hypermutation is not active in all B cells and cannot be triggered merely by mitogen-induced B cell activation. However, during discrete stages of B-cell differentiation, expressed immunoglobulin V genes may incur new mutations at rates as high as 10–3 base substitutions per base pair per generation over several cell divisions, particularly during the secondary humoral immune response to antigen. The pattern of somatic mutations in rearranged variable genes differs from that of meiotic mutations, indicating that a different mechanism generates somatic hypermutation than that responsible for spontaneous mutation.44 Hypermutations begin on the 5′ end of rearranged V genes downstream of the transcription initiation site and continue through the V gene and into the 3′-flanking region before tapering off. As such, the mutations are clustered in the region spanning from 300 bp 5′ of the rearranged variable region exon to approximately 1 kb 3′ of the rearranged minigene J segment. Subsequent selection of the immunoglobulin encoded by such mutated immunoglobulin V genes may enhance the frequency of nonconservative base substitutions in the DNA sequences encoding the combining site for antigen.45
Under normal conditions, a B lymphocyte or plasma cell synthesizes only one species of light chain and heavy chain, even though the cell has two different sets of each of the immunoglobulin gene complexes that initially undergo seemingly independent immunoglobulin gene rearrangements. Indeed, the specificity of the humoral immune response depends on antigenic selection of unique clones of B cells, each clone expressing a homogeneous set of immunoglobulin receptors. Such restriction is achieved by limiting a given B cell to functional rearrangement and expression of only a single heavy-chain allele and a single light-chain allele. This phenomenon is called allelic exclusion. Although occasional neoplastic B cell populations may lack allelic exclusion and express both immunoglobulin alleles, this phenomenon generally is observed with most B-cell tumors.46
IMMUNOGLOBULIN VARIABLE REGION STRUCTURE
IMMUNOGLOBULIN VARIABLE REGION SUBGROUPS
Despite the large number of different immunoglobulin variable regions that can be generated through the above mechanisms, each antibody polypeptide may be assigned to one of a relatively small number of variable region subgroups.4 Comparisons of the amino acid sequences of a large number of different monoclonal immunoglobulin proteins reveal four segments of limited amino acid sequence diversity between different antibody heavy- or light-chain variable regions. These segments are designated the immunoglobulin variable region frameworks (FR) (Fig. 83-7). Each immunoglobulin polypeptide may be assigned to one of a relatively small number of variable region subgroups based upon the primary structure of its first three frameworks. Moreover, each subgroup has characteristic framework sequences that serve to distinguish it from other variable region subgroups.
FIGURE 83-7 Schematic diagrams depicting each FR and CDR of the immunoglobulin heavy chain (VH) or light chain (VL). The first, second, third, and fourth framework regions are labeled FR1, FR2, FR3, and FR4 respectively. Similarly, the first through third complementarity-determining regions are labeled CDR1, CDR2, and CDR3 respectively. The numbers beneath each diagram indicate the numbers of the amino acid residues that define the borders between these regions according to Kabat.4
Satisfying expectations that immunoglobulin subgroups defined families of highly related antibody V genes, variable region amino acid subgroup homologies are found to extend to the nucleic acid sequence level.47,48 and 49 Cloned immunoglobulin V genes whose deduced amino acid sequences belong to a given subgroup generally share greater than 80 percent nucleic acid sequence homology. The human heavy-chain variable regions may be grouped into seven subgroups, while kappa or lambda light chains may be divided into 6 or 11 subgroups, respectively.
Crystallographic data of immunoglobulin variable regions indicate that amino acids within the first and third FR regions of either the light or heavy chain form beta bonds on the external surface of the molecule.5,50 These regions form relatively compact structures on the external solvent-accessible face of the antibody molecule that are not adjacent to the classic antibody combining site for antigen. Accordingly, amino acid differences noted between the different variable region subgroups are amenable to recognition by antisubgroup antibodies.51
Antisubgroup antibodies, however, need to be distinguished from anti-idiotypic antibodies. Positioned between the FR regions are three segments of extreme hypervariability in both light and heavy chain sequences.4 The third hypervariable region is generated through the recombinatorial process that joins the antibody light-chain V gene with the J segment, in the case of the light chain, or the VH gene with the somatically generated DJH segment of the antibody heavy chain.52,53 and 54 The diversity in first and second hypervariable regions in part reflects germ line DNA-encoded differences between disparate antibody V genes, a diversity often noted even between V genes of the same subgroup.4,55,56 During an immune response, somatic hypermutation subsequent to V gene rearrangement also may play an important role in increasing the amino acid sequence diversity noted within these regions (discussed above). These hypervariable regions on both chains fold together to form the antigen combining site.3,50 Hence, each of these regions of hypervariability is designated as being a complementarity-determining region, or CDR (Fig. 83-7).
During secondary immune responses, extensive amino acid substitutions may occur in the complementarity-determining regions. In contrast, amino acid replacement mutations are noted to be much less frequent in the framework regions than would be anticipated if the nucleic acid substitutions were occurring randomly. As a consequence, the subgroup determinants that characterize an entire variable region subgroup may be relatively resilient to the process of somatic hypermutation. On the other hand, the complementarity-determining regions may form determinants of unique specificity that contribute to the epitopes recognized by anti-idiotypic antibodies.
Despite the tremendous potential for diversity in Ig V gene expression and genetic polymorphism, antibodies produced by B-cell malignancies or normal B cells of unrelated persons may share common idiotypic determinants.57 These common idiotypes, designated cross-reactive idiotypes or CRIs, were defined initially on IgM autoantibodies, such as rheumatoid factors. However, cross-reactive idiotypes may be found on antibodies that do not have anti-self-reactivity. Molecular studies have demonstrated several of these cross-reactive idiotypes to represent serologic markers for expression of conserved immunoglobulin variable region genes with little or no somatic mutation.58
GENETIC MARKERS ON IMMUNOGLOBULIN CONSTANT REGIONS
Human immunoglobulins have inherited differences in structure, termed allotypes. These genetic markers usually are detected with agglutinating sera from individuals naturally immunized through transfusion or pregnancy. These antibodies recognize minor amino acid sequence variations in the constant regions of g, a, and k chains.59,60 No definite allotypic differences have been detected on µ or d chains. On e chains, a monoclonal antibody to IgE defined an allotype that was common to persons of all races except for a few individuals of Asian or Melanesian background.
The k light-chain allotypes are designated Km allotypes (formerly called inv). There exist at least three major Km allotypes, designated Km(1), Km(1,2), and Km(3), that may be recognized serologically or, more recently, via the polymerase chain reaction.61 The a chain allotypes, designated Am allotypes, are on the heavy chains of the IgA2 subclass.60 The g chain allotypes are on the heavy chains of the IgG1, IgG2, or IgG3 subclasses and are designated G1m, G2m, and G3m respectively. Over 24 Gm allotypic markers have been identified serologically.60 As discussed earlier, all the heavy-chain constant region genes reside on chromosome 14. Therefore, different combinations of heavy-chain allotype markers are inherited as haplotypic units, in an autosomal codominant manner. The frequency of the various allelic markers differs among ethnic groups.
LAMBDA LIGHT-CHAIN ISOTYPES
The l light chains have four isotypes, termed Mcg+, Ke– Oz–, Ke– Oz+, and Ke+ Oz–, that were defined on the basis of their reactivity with the Oz, Kern, and Mcg antisera raised against l Bence Jones proteins.62 These isotypes reflect minor nonallelic amino acid differences in the l light-chain constant regions that are each encoded by one of the multiple constant region genes in the lambda light-chain complex.25 Mcg+, Ke– Oz–, Ke– Oz+, and Ke+ Oz– isotypes are each associated with the Cl1, Cl2, Cl3, or Cl7 l light-chain constant regions respectively (Fig. 83-6).
IMMUNOGLOBULIN SYNTHESIS AND SECRETION
The total IgG content of the adult human body is about 75 g, of which 2.2 g is synthesized each day. Most immunoglobulin is produced by mature plasma cells, which have abundant rough endoplasmic reticulum and a well-developed Golgi apparatus.
The final messenger RNA for immunoglobulin light and heavy chains are derived by the processing of large nuclear RNA transcripts. In plasma cells, the rearranged and spliced mRNA molecules for the heavy- and light-chain polypeptides are translated on separate ribosomal complexes. An amino-terminal leader peptide approximately 18 to 30 residues long is cleaved prior to the release of the completed light and heavy chains in the cisternae of the endoplasmic reticulum. There the two polypeptides spontaneously combine to form immunoglobulin half molecules that are stabilized by disulfide bonds. The joining of two identical half molecules by disulfide bonds yields a basic four-chain immunoglobulin unit.
Glycosyltransferase enzymes add a defined sequence of sugars to the assembled immunoglobulin unit to form branched-chain oligosaccharides composed of N-acetyl-glucosamine, mannose, galactose, fructose, and sialic acid. The oligosaccharides are attached covalently to the immunoglobulin heavy chain at several sites. The carbohydrate facilitates the transport of the antibody molecule across the plasma membrane and into the extracellular space and increases the solubility of the secreted protein.
Five monomeric units of IgM combine to form a pentameric macroglobulin linked by disulfide bonds and a single J-chain polypeptide. Usually polymerization immediately precedes or occurs simultaneously with IgM secretion. Similarly, IgA molecules form dimers and polymers linked by the J chain just prior to secretion from the plasma cell.
REGULATION OF IMMUNOGLOBULIN SYNTHESIS
A normal adult has preexisting B lymphocytes that can interact with almost any foreign antigen. In the presence of accessory T lymphocytes and macrophages, an antigen-binding clone of B lymphocytes may transform into antibody-secreting plasma cells and memory B cells.63 Most plasma cells are terminally differentiated and do not divide. Therefore, the continued production of antibody depends upon the rate of plasma cell generation, the functional life span of the plasma cell, and the half-life of the immunoglobulin in the body.64
B lymphocytes are produced throughout life by differentiation of hematopoietic cells in the marrow and proliferation of B lymphocytes in secondary lymphoid tissues.65 Many B lymphocytes survive for but a few weeks without stimulation by antigen and activated accessory T lymphocytes.66 Without antigen to cross-link their surface immunoglobulin receptors, B lymphocytes that home to the germinal centers of secondary lymphoid tissues will undergo apoptosis, or programmed cell death, within a matter of hours in vitro.67,68 and 69 Not only does this select for B lymphocytes that have surface immunoglobulin with high affinity for antigen,70 the requirement for antigen-directed surface immunoglobulin cross-linking also allows for secreted specific antibody to regulate its own production. Under normal short-term exposure to antigen, newly formed B lymphocytes must compete with secreted antibody and other antigen-specific B lymphocytes for ever-decreasing amounts of circulating antigen. By preventing the interaction of antigen with immunoglobulin receptors on B lymphocytes, secreted antibody may inhibit the generation of more plasma cells.
Edelman GM: Antibody structure and molecular immunology Scand J Immunol 34:1, 1991.
Virella G, Wang AC: Immunoglobulin structure. Immunol Ser 58:75, 1993.
Alzari PM, Lascombe MB, Poljak RJ: Three-dimensional structure of antibodies. Annu Rev Immunol 6:555, 1988.
Kabat E, Wu TT, Perry HM, et al: Sequences of Proteins of Immunological Interest, 5th edition. U.S. Department of Health and Human Services, Bethesda, MD, 1991.
Harris LJ, Larson SB, Hasel KW, et al: The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 360:369, 1992.
Jefferis R, Lund J, Goodall M: Recognition sites on human IgG for Fc gamma receptors: the role of glycosylation. Immunol Lett 44:111, 1995.
Lee YK, Brewer JW, Hellman R, Hendershot LM: BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Mol Biol Cell 10:2209, 1999.
Lamm ME, Nedrud JG, Kaetzel CS, Mazanec MB: IgA and mucosal defense. Apmis 103:241, 1995.
Corthesy B, Kraehenbuhl JP: Antibody-mediated protection of mucosal surfaces. Curr Top Microbiol Immunol 236:93, 1999.
Niles MJ, Matsuuchi L, Koshland ME: Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis. Proc Natl Acad Sci USA 92:2884, 1995.
Roes J, Rajewsky K: Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells. J Exp Med 177:45, 1993.
Lyczak JB, Zhang K, Saxon A, Morrison SL: Expression of novel secreted isoforms of human immunoglobulin E proteins. J Biol Chem 271:3428, 1996.
Cambier JC, Campbell KS: Membrane immunoglobulin and its accomplices: new lessons from an old receptor. FASEB J 6:3207, 1992.
Kurosaki T: Genetic analysis of B cell antigen receptor signaling. Annu Rev Immunol 17:555, 1999.
Healy JI, Goodnow CC: Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol 16:645, 1998.
Cornall RJ, Goodnow CC, Cyster JG: Regulation of B cell antigen receptor signaling by the Lyn/CD22/SHP1 pathway. Curr Top Micobiol Immunol 244:57, 1999.
Plas DR, Thomas ML: Negative regulation of antigen receptor signaling in lymphocytes. J Mol Med 76:589, 1998.
Siminovitch KA, Neel BG: Regulation of B cell signal transduction by SH2-containing protein-tyrosine phosphatases. Semin Immunol 10:329, 1998.
Shultz LD, Rajan TV, Greiner DL: Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. Trends Biotechnol 15:302, 1997.
Matsuda F, Honjo T: Organization of the human immunoglobulin heavy-chain locus. Adv Immunol 62:1, 1996.
Tomlinson IM, Cook GP, Walter G, et al: A complete map of the human immunoglobulin VH locus. Ann NY Acad Sci 764:43, 1995.
Kipps TJ: Human B cell biology. Int Rev Immunol 15:243, 1997.
Zachau HG: The immunoglobulin kappa locus or what has been learned from looking closely at one-tenth of a percent of the human genome. Gene 135:167, 1993.
Schäble K, Thiebe R, Flügel A, et al: The human immunoglobulin kappa locus: pseudogenes, unique and repetitive sequences. Biol Chem Hoppe-Seyler 375:189, 1994.
Pallarès N, Frippiat JP, Giudicelli V, Lefranc MP: The human immunoglobulin lambda variable (IgLV) genes and joining (IgLJ) segments. Exp Clin Immunogenet 15:8, 1998.
Tonegawa S: The Nobel lectures in immunology. The Nobel Prize for physiology or medicine, 1987. Somatic generation of immune diversity. Scand J Immunol 38:303, 1993.
Gellert M: Recent advances in understanding V(D)J recombination. Adv Immunol 64:39, 1997.
Steen SB, Gomelsky L, Speidel SL, Roth DB: Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks. EMBO J 16:2656, 1997.
Graninger WB, Goldman PL, Morton CC, et al: The kappa-deleting element. Germline and rearranged, duplicated and dispersed forms. J Exp Med 167:488, 1988.
Grawunder U, West RB, Lieber MR: Antigen receptor gene rearrangement. Curr Opin Immunol 10:172, 1998.
Oettinger MA, Schatz DG, Gorka C, Baltimore D: RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248:1517, 1990.
Oettinger MA, Stanger B, Schatz DG, et al: The recombination activating genes, RAG 1 and RAG 2, are on chromosome 11p in humans and chromosome 2p in mice. Immunogenetics 35:97, 1992.
Shinkai Y, Rathbun G, Lam KP, et al: RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855, 1992.
Villa A, Santagata S, Bozzi F, et al: Omenn syndrome: a disorder of Rag1 and Rag2 genes. J Clin Immunol 19:87, 1999.
Melchers F, Karasuyama H, Haasner D, et al: The surrogate light chain in B-cell development. Immunol Today 14:60, 1993.
Ten Boekel E, Yamagami T, Andersson J, et al: The formation and selection of cells expressing preB cell receptors and B cell receptors. Curr Top Micobiol Immunol 246:3, 1999.
Tsuganezawa K, Kiyokawa N, Matsuo Y, et al: Flow cytometric diagnosis of the cell lineage and developmental stage of acute lymphoblastic leukemia by novel monoclonal antibodies specific to human pre-B-cell receptor. Blood 92:4317, 1998.
Kitamura D, Kudo A, Schaal S, et al: A critical role of lambda 5 protein in B cell development. Cell 69:823, 1992.
Corcos D, Dunda O, Butor C, et al: Pre-B-cell development in the absence of lambda 5 in transgenic mice expressing a heavy-chain disease protein. Curr Biol 5:1140, 1995.
Minegishi Y, Coustan-Smith E, Wang YH, et al: Mutations in the human lambda 5/14.1 gene result in B cell deficiency and agammaglobulinemia. J Exp Med 187:71, 1998.
Stavnezer J: Immunoglobulin class switching. Curr Opin Immunol 8:199, 1996.
Lorenz MG, Radbruch A: Insights into the control of immunoglobulin class switch recombination from analysis of targeted mice. Res Immunol 148:460, 1997.
Wabl M, Cascalho M, Steinberg C: Hypermutation in antibody affinity maturation. Curr Opin Immunol 11:186, 1999.
Winter DB, Gearhart PJ: Dual enigma of somatic hypermutation of immunoglobulin variable genes: targeting and mechanism. Immunol Rev 162:89, 1998.
Dörner T, Foster SJ, Brezinschek HP, Lipsky PE: Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human VHDJH rearrangements. Immunol Rev 162:161, 1998.
Rassenti LZ, Kipps TJ: Lack of allelic exclusion in B cell chronic lymphocytic leukemia. J Exp Med 185:1435, 1997.
Cook GP, Tomlinson IM: The human immunoglobulin VH repertoire. Immunol Today 16:237, 1995.
Kipps TJ: Human B cell biology. Int Rev Immunol 15:243, 1997.
Frippiat JP, Williams SC, Tomlinson IM, et al: Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum Mol Genet 4:983, 1995.
Poljak RJ: Structure of antibodies and their complexes with antigens. Mol Immunol 28:1341, 1991.
Jefferis R: Nomenclature of V region markers. Immunol Today 16:207, 1995.
Tonegawa S: Somatic generation of antibody diversity. Nature 302:575, 1983.
Sakano H, Kurosawa Y, Weigert M, Tonegawa S: Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes. Nature 290:562, 1981.
Early P, Huang H, Davis M, et al: An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell 19:981, 1980.
Berman JE, Mellis SJ, Pollock R, et al: Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus. EMBO J 7:727, 1988.
Zachau HG: The human immunoglobulin kappa locus and some of its acrobatics. Biol Chem Hoppe-Seyler 371:1, 1990.
Kipps TJ, Carson DA: Autoantibodies in chronic lymphocytic leukemia and related systemic autoimmune diseases. Blood 81:2475, 1993.
Kipps TJ: Immunologic and therapeutic implications of anti-idiotype antibodies, in Chronic Lymphocytic Leukemia, edited by BD Cheson, p 123. Marcel Dekker, New York, 1992.
Williams RC Jr, Malone CC, Solomon A: Conformational dependency of human IgG heavy chain-associated Gm allotypes. Mol Immunol 30:341, 1993.
Schanfield MS, van Loghem E: Human immunoglobulin allotypes, in Handbook of Experimental Immunology—Genetics and Molecular Immunology, 4th ed, edited by DM Weir, LA Herzenberg, C Blackwell, L Herzenberg, p 941. Blackwell, Oxford, 1986.
Moxley G, Gibbs RS: Polymerase chain reaction-based genotyping for allotypic markers of immunoglobulin kappa shows allelic association of Km with kappa variable segment. Genomics 13:104, 1992.
Hess M, Hilschmann N, Rivat L, et al: Isotypes in human immunoglobulin lambda-chains. Nature New Biol 234:58, 1971.
Gray D: Immunologic memory. Annu Rev Immunol 11:49, 1993.
Virella G, Wang AC: Biosynthesis, metabolism and biological properties of immunoglobulins. Immunol Ser 58:91, 1993.
Nunez C, Nishimoto N, Gartland GL, et al: B cells are generated throughout life in humans. J Immunol 156:866, 1996.
Benschop RJ, Cambier JC: B cell development: signal transduction by antigen receptors and their surrogates. Curr Opin Immunol 11:143, 1999.
Choi YS: Differentiation and apoptosis of human germinal center B-lymphocytes. Immunol Res 16:161, 1997.
Liu YJ, de Bouteiller O, Fugier-Vivier I: Mechanisms of selection and differentiation in germinal centers. Curr Opin Immunol 9:256, 1997.
Hollowood K, Goodlad JR: Germinal centre cell kinetics. J Pathol 185:229, 1998.
Przylepa J, Himes C, Kelsoe G: Lymphocyte development and selection in germinal centers. Curr Top Micobiol Immunol 229:85, 1998.
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn