Structure and function of immunoglobulins

Immunoglobulins are heterodimeric proteins composed of 2 heavy and 2 light chains. They can be separated functionally into variable domains that bind antigens and constant domains that specify effector functions, such as activation of complement or binding to Fc receptors. The variable domains are created by means of a complex series of gene rearrangement events and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation. Each variable domain can be split into 3 regions of sequence variability termed the complementarity-determining regions (CDRs) and 4 regions of relatively constant sequence termed the framework regions. The 3 CDRs of the heavy chain are paired with the 3 CDRs of the light chain to form the antigen-binding site, as classically defined. The constant domains of the heavy chain can be switched to allow altered effector function while maintaining antigen specificity. There are 5 main classes of heavy chain constant domains. Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into 4 subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biologic properties, and IgA can similarly be split into IgA1 and IgA2. Immunoglobulins are heterodimeric proteins composed of 2 heavy and 2 light chains. They can be separated functionally into variable domains that bind antigens and constant domains that specify effector functions, such as activation of complement or binding to Fc receptors. The variable domains are created by means of a complex series of gene rearrangement events and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation. Each variable domain can be split into 3 regions of sequence variability termed the complementarity-determining regions (CDRs) and 4 regions of relatively constant sequence termed the framework regions. The 3 CDRs of the heavy chain are paired with the 3 CDRs of the light chain to form the antigen-binding site, as classically defined. The constant domains of the heavy chain can be switched to allow altered effector function while maintaining antigen specificity. There are 5 main classes of heavy chain constant domains. Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into 4 subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biologic properties, and IgA can similarly be split into IgA1 and IgA2. In 1890, von Behring and Kitasato reported the existence of an agent in the blood that could neutralize diphtheria toxin. The following year, reference was made to “Antikörper,” or antibodies, in studies describing the ability of the agent to discriminate between 2 immune substances. Subsequently, the substance that induces the production of an antibody was referred to as the “Antisomatogen + Immunkörperbildner,” or the agent that induces the antibody. The term “antigen” is a contraction of this term. Thus an antibody and its antigen represent a classic tautology. In 1939, Tiselius and Kabat used electrophoresis to separate immunized serum into albumin, α-globulin, β-globulin, and γ-globulin fractions. Absorption of the serum against the antigen depleted the γ-globulin fraction, yielding the terms γ-globulin, immunoglobulin, and IgG. “Sizing” columns were then used to separate immunoglobulins into those that were “heavy” (IgM), “regular” (IgA, IgE, IgD, and IgG), and “light” (light chain dimers). More than 100 years of investigation into the structure and function of immunoglobulin has only served to emphasize the complex nature of this protein. Typically, receptors bind to a limited and defined set of ligands. However, although individual immunoglobulin also bind a limited and defined set of ligands, immunoglobulins as a population can bind to a virtually unlimited array of antigens sharing little or no similarity. This property of adjustable binding depends on a complex array of mechanisms that alter the DNA of individual B cells. Immunoglobulins also serve 2 purposes: that of cell-surface receptors for antigen, which permit cell signaling and cell activation, and that of soluble effector molecules, which can individually bind and neutralize antigens at a distance. The molecular mechanisms that permit these many and varied functions are the focus of this chapter. Immunoglobulins belong to the eponymous immunoglobulin superfamily (IgSF).1Williams A.F. Barclay A.N. The immunoglobulin superfamily—domains for cell surface recognition.Annu Rev Immunol. 1988; 6: 381-405Crossref PubMed Scopus (1798) Google Scholar, 2Harpaz Y. Chothia C. Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains.J Mol Biol. 1994; 238: 528-539Crossref PubMed Scopus (377) Google Scholar, 3Torres R.M. Imboden J. Schroeder Jr., H.W. Antigen receptor genes, gene products, and co-receptors.in: Rich R.R. Fleisher T.A. Shearer W.T. Schroeder Jr., H.W. Frew A.J. Weyand C.M. Clinical immunology: principles and practice. 3rd ed. Mosby Elsevier, London2008: 53-77Crossref Scopus (2) Google Scholar They consist of 2 heavy (H) and 2 light (L) chains (Fig 1), where the L chain can consist of either a κ or a λ chain. Each component chain contains one NH2-terminal variable (V) IgSF domain and 1 or more COOH-terminal constant (C) IgSF domains, each of which consists of 2 sandwiched β-pleated sheets pinned together by a disulfide bridge between 2 conserved cysteine residues.1Williams A.F. Barclay A.N. The immunoglobulin superfamily—domains for cell surface recognition.Annu Rev Immunol. 1988; 6: 381-405Crossref PubMed Scopus (1798) Google Scholar Each V or C domain consists of approximately 110 to 130 amino acids, averaging 12,000 to 13,000 kd. Both immunoglobulin L chains contain only 1 C domain, whereas immunoglobulin H chains contain either 3 or 4 such domains. H chains with 3 C domains tend to include a spacer hinge region between the first (CH1) and second (CH2) domains. A typical L chain will thus mass approximately 25 kd, and a 3 C domain Cγ H chain with its hinge will mass approximately 55 kd. Considerable variability is allowed to the amino acids that populate the external surface of the IgSF domain and to the loops that link the β strands. These solvent-exposed surfaces offer multiple targets for docking with other molecules. Early studies of immunoglobulin structure were facilitated by the use of enzymes to fragment IgG molecules. Papain digests IgG into 2 Fab fragments, each of which can bind antigen, and a single Fc fragment. Pepsin splits IgG into an Fc fragment and a single dimeric F(ab)2 that can cross-link, as well as bind, antigens. The Fab contains 1 complete L chain in its entirety and the V and CH1 portion of 1 H chain (Fig 1). The Fab can be further divided into a variable fragment (Fv) composed of the VH and VL domains, and a constant fragment composed of the CL and CH1 domains. Single Fv fragments can be genetically engineered to recapitulate the monovalent antigen-binding characteristics of the original parent antibody.4Smith K.A. Nelson P.N. Warren P. Astley S.J. Murray P.G. Greenman J. Demystified…recombinant antibodies.J Clin Pathol. 2004; 57: 912-917Crossref PubMed Scopus (38) Google Scholar Intriguingly, a subset of antibodies in a minority of species (camelids5Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. et al.Naturally occurring antibodies devoid of light chains.Nature. 1993; 363: 446-448Crossref PubMed Scopus (2287) Google Scholar and nurse shark6Roux K.H. Greenberg A.S. Greene L. Strelets L. Avila D. McKinney E.C. et al.Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.Proc Natl Acad Sci U S A. 1998; 95: 11804-11809Crossref PubMed Scopus (192) Google Scholar) lack light chains entirely and use only the heavy chain for antigen binding. Although these unusual variants are not found in human subjects, there are a number of ongoing attempts to humanize these types of antibodies for therapeutic and diagnostic purposes.7Vincke C. Loris R. Saerens D. Martinez-Rodriguez S. Muyldermans S. Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold.J Biol Chem. 2009; 284: 3273-3284Crossref PubMed Scopus (367) Google Scholar Immunoglobulin-antigen interactions typically take place between the paratope, the site on the immunoglobulin at which the antigen binds, and the epitope, which is the site on the antigen that is bound. In vivo immunoglobulins tend to be produced against intact antigens in soluble form and thus preferentially identify surface epitopes that can represent conformational structures that are noncontiguous in the antigen's primary sequence. This ability to identify component parts of the antigen independently of the rest makes it possible for the B cell to discriminate between 2 closely related antigens, each of which can be viewed as a collection of epitopes. It also permits the same antibody to bind divergent antigens that share equivalent or similar epitopes, a phenomenon referred to as cross-reactivity. Immunization of heterologous species with mAbs (or a restricted set of immunoglobulins) allowed the identification of both common and individual immunoglobulin antigenic determinants. Individual determinants, termed idiotypes, are contained within V domains. Common determinants, termed isotypes, are specific for the constant portion of the antibody and allow grouping of immunoglobulins into recognized classes, with each class defining an individual type of C domain. Determinants common to subsets of individuals within a species yet differing between other members of that species are termed allotypes and define inherited polymorphisms that result from gene alleles.8Jazwinska E.C. Dunckley H. Propert D.N. Gatenby P.A. Serjeantson S.W. GM typing by immunoglobulin heavy chain gene RFLP analysis.Am J Hum Genet. 1988; 43: 175-181PubMed Google Scholar Immunoglobulin heavy and light chains are each encoded by a separate multigene family,9Leder P. The genetics of antibody diversity.Sci Am. 1982; 246: 102-115Crossref PubMed Scopus (129) Google Scholar, 10Tonegawa S. Somatic generation of antibody diversity.Nature. 1983; 302: 575-581Crossref PubMed Scopus (3200) Google Scholar and the individual V and C domains are each encoded by independent elements: V(D)J gene segments for the V domain and individual exons for the C domains. The primary sequence of the V domain is functionally divided into 3 hypervariable intervals termed complementarity-determining regions (CDRs) that are situated between 4 regions of stable sequence termed framework regions (FRs; Fig 1). Each V gene segment typically contains its own promoter, a leader exon, an intervening intron, an exon that encodes the first 3 framework regions (FRs 1, 2, and 3), CDRs 1 and 2 in their entirety, the amino-terminal portion of CDR3, and a recombination signal sequence (RSS). Each joining (J) gene segment begins with its own recombination signal, the carboxy terminal portion of CDR3, and the complete FR4 (Fig 1, Fig 2). The creation of a V domain is directed by the RSSs that flank the rearranging gene segments. Each RSS contains a strongly conserved 7-bp (or heptamer) sequence (eg, CACAGTG) that is separated from a less well-conserved 9-bp (or nonamer) sequence (eg, ACAAAACCC) by either a 12- or 23-bp spacer. These spacers place the heptamer and nonamer sequences on the same side of the DNA molecule separated by either 1 or 2 turns of the DNA helix. A 1-turn RSS (12-bp spacer) will preferentially recognize a 2-turn signal sequence (23-bp spacer), thereby avoiding wasteful V-V or J-J rearrangements. Initiation of the V(D)J recombination reaction requires recombination-activating genes (RAGs) 1 and 2, which are almost exclusively expressed in developing lymphocytes.11Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv Immunol. 2005; 86: 43-112Crossref PubMed Scopus (225) Google Scholar RAG1 and RAG2 introduce a DNA double-strand break between the terminus of the rearranging gene segment and its adjacent RSS. These breaks are then repaired by ubiquitously expressed components of a DNA repair process, which is known as nonhomologous end-joining (NHEJ), that are common to all cells of the body. Thus although mutations of RAG affect only lymphocytes, loss or alteration-of-function mutations in NHEJ proteins yield susceptibility to DNA damage in all cells of the body. The NHEJ process creates precise joins between the RSS ends and imprecise joins of the coding ends. Terminal deoxynucleotidyl transferase (TdT), which is expressed only in lymphocytes, can variably add non–germline-encoded nucleotides (N nucleotides) to the coding ends of the recombination product. Typically, the initial event in recombination will be recognition of 12-bp spacer RSS by RAG1. RAG2 then associates with RAG1 and the heptamer to form a synaptic complex. Binding of a second RAG1 and RAG2 complex to the 23-bp, 2-turn RSS permits the interaction of the 2 synaptic complexes to form what is known as a paired complex, a process that is facilitated by the actions of the DNA-bending proteins HMG1 and HMG2. After paired complex assembly, the RAG proteins single-strand cut the DNA at the heptamer sequence. The 3′ OH of the coding sequence ligates to 5′ phosphate and creates a hairpin loop. The clean-cut ends of the signal sequences enable formation of precise signal joints. However, the hairpin junction created at the coding ends must be resolved by renicking the DNA, usually within 4 to 5 nucleotides from the end of the hairpin. This forms a 3′ overhang that is amenable to further modification. It can be filled in through DNA polymerases, be nibbled, or serve as a substrate for TdT-catalyzed N addition. DNA polymerase μ, which shares homology with TdT, appears to play a role in maintaining the integrity of the terminus of the coding sequence. The cut ends of the coding sequence are then repaired by the NHEJ proteins. NHEJ proteins involved in V(D)J recombination include Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, and ligase.4Smith K.A. Nelson P.N. Warren P. Astley S.J. Murray P.G. Greenman J. Demystified…recombinant antibodies.J Clin Pathol. 2004; 57: 912-917Crossref PubMed Scopus (38) Google Scholar Ku70 and Ku80 form a heterodimer (Ku) that directly associates with DNA double-strand breaks to protect the DNA ends from degradation, permit juxtaposition of the ends to facilitate coding end ligation, and help recruit other members of the repair complex. DNA-PKcs phosphorylates Artemis, inducing an endonuclease activity that plays a role in the opening of the coding joint hairpin. Finally, XRCC4 and ligase 4 help rejoin the ends of the broken DNA. Deficiency of any of these proteins creates sensitivity to DNA breakage and can lead to a severe combined immunodeficiency phenotype. The κ locus is located on chromosome 2p11.2.12Zachau H.G. The human immunoglobulin kappa genes.in: Honjo T. Alt F.W. Rabbitts P.H. Immunoglobulin genes. 2nd ed. Academic Press, London1995: 173Crossref Google Scholar κ V domains represent the joined product of Vκ and Jκ gene segments (Fig 2), whereas the κ C domains are encoded by a single Cκ exon. The locus contains 5 Jκ and 75 Vκ gene segments upstream of Cκ (Fig 3). One third of the Vκ gene segments contain frameshift mutations or stop codons that preclude them from forming functional protein, and of the remaining sequences, less than 30 of the Vκ gene segments have actually been found in functional immunoglobulins. V gene segments can be grouped into families on the basis of sequence and structural similarity.13Brodeur P.H. Riblet R.J. The immunoglobulin heavy chain variable region (IgH-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes.Eur J Immunol. 1984; 14: 922-930Crossref PubMed Scopus (432) Google Scholar, 14Kirkham P.M. Schroeder Jr., H.W. Antibody structure and the evolution of immunoglobulin V gene segments.Semin Immunol. 1994; 6: 347-360Crossref PubMed Scopus (95) Google Scholar There are 6 such families for Vκ. Each active Vκ gene segment has the potential to rearrange to any of the 5 Jκ elements, generating a potential “combinatorial” repertoire of more than 140 distinct VJ combinations. The Vκ gene segment contains FR1, FR2, and FR3; CDR1 and CDR2; and the amino-terminal portion of CDR3. The Jκ element contains the carboxy terminus of CDR3 and FR4 in its entirety. The terminus of each rearranging gene segment can undergo a loss of 1 to 5 nucleotides during the recombination process, yielding additional junctional diversity. In human subjects TdT can introduce random N nucleotides to either replace some or all of the lost Vκ or Jκ nucleotides or to add to the original germline sequence.15Lee S.K. Bridges Jr., S.L. Koopman W.J. Schroeder Jr., H.W. The immunoglobulin kappa light chain repertoire expressed in the synovium of a patient with rheumatoid arthritis.Arthritis Rheum. 1992; 35: 905-913Crossref PubMed Scopus (52) Google Scholar Each codon created by N addition increases the potential diversity of the repertoire 20-fold. Thus the initial diversification of the κ repertoire is focused at the VJ junction that defines the light chain CDR3, or CDR-L3. The λ locus, which is located on chromosome 22q11.2, contains 4 functional Cλ exons, each of which is associated with its own Jλ (Fig 3). Vλ genes are arranged in 3 distinct clusters, each containing members of different Vλ families.16Kawasaki K. Minoshima S. Nakato E. Shibuya K. Shintani A. Schmeits J.L. et al.One-megabase sequence analysis of the human immunoglobulin lambda gene locus.PCR Methods Appl. 1997; 7: 250-261Crossref Scopus (175) Google Scholar Depending on the individual haplotype, there are approximately 30 to 36 potentially functional Vλ gene segments and an equal number of pseudogenes. During early B-cell development, H chains form a complex with unconventional λ light chains, known as surrogate or pseudo-light chains (ΨLC), to form a pre–B-cell receptor. The genes encoding the ΨLC proteins λ14.1 (λ5) and VpreB are located within the λ light chain locus on chromosome 22. Together, these 2 genes create a product with considerable homology to conventional λ light chains. A critical difference between these unconventional ΨLC genes and other L chains is that λ14.1 and VpreB gene rearrangement is not required for ΨLC expression. The region of the ΨLC gene that corresponds to CDR-L3 covers CDR-H3 in the pre–B-cell receptor, allowing the pre–B cell to avoid antigen-specific selection.17Bankovich A.J. Raunser S. Juo Z.S. Walz T. Davis M.M. Garcia K.C. Structural insight into pre-B cell receptor function.Science. 2007; 316: 291-294Crossref PubMed Scopus (82) Google Scholar The H chain locus, which is located on chromosome 14q32.33, is considerably more complex than the light chain clusters. The approximately 80 VH gene segments near the telomere of the long arm of chromosome 14 can be grouped into 7 different families of related gene segments.18Matsuda F. Ishii K. Bourvagnet P. Kuma K. Hayashida H. Miyata T. et al.The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus.J Exp Med. 1998; 188: 2151-2162Crossref PubMed Scopus (313) Google Scholar Of these, approximately 39 are functional. Adjacent to the most centromeric VH, V6-1, are 27 DH (D for diversity) gene segments (Fig 3)19Corbett S.J. Tomlinson I.M. Sonnhammer E.L. Buck D. Winter G. Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D-D recombination.J Mol Biol. 1997; 270: 587-597Crossref PubMed Scopus (249) Google Scholar and 6 JH gene segments. Each VH and JH gene segment is associated with a 2-turn RSS, which prevents direct V → J joining. A pair of 1-turn RSSs flanks each DH segment. Recombination begins with the joining of a DH to a JH gene segment, followed by the joining of a VH element to the amino-terminal end of the DJ intermediate. The VH gene segment contains FR1, FR2, and FR3; CDR1 and CDR2; and the amino-terminal portion of CDR3. The DH gene segment forms the middle of CDR3, and the JH element contains the carboxy terminus of CDR3 and FR4 in its entirety (Fig 1). Random assortment of one of approximately 39 active VH and one of 27 DH gene segments with one of the 6 JH gene segments can generate more than 104 different VDJ combinations (Fig 4). Although combinatorial joining of individual V, D, and J gene segments maximizes germline-encoded diversity, the junctional diversity created by VDJ joining is the major source of variation in the preimmune repertoire (Fig 4). First, DH gene segments can rearrange by either inversion or deletion, and each DH gene segment can be spliced and translated in each of the 3 potential reading frames. This gives each DH gene segment the potential to encode 6 different peptide fragments. Second, the rearrangement process proceeds through a step that creates a hairpin ligation between the 5′ and 3′ termini of the rearranging gene segment. Nicking to resolve the hairpin structure leaves a 3′ overhang that creates a palindromic extension, termed a P junction, that can add germline-encoded nucleotides. Third, the terminus of each rearranging gene segment can undergo a loss of 1 to several nucleotides during the recombination process. Fourth, TdT can add numerous N nucleotides at random to replace or add to the original germline sequence. N nucleotides can be inserted between the V and D segments, as well as between the D and J segments. The imprecision of the joining process and variation in the extent of N addition permits generation of CDR-H3s of varying length and structure. As a result, more than 107 different H chain VDJ junctions, or CDR-H3s, can be generated at the time of gene segment rearrangement. Taken as a whole, somatic variation in CDR3, combinatorial rearrangement of individual gene segments, and combinatorial association between different L and H chains can yield a potential preimmune antibody repertoire of greater than 1016 different immunoglobulins. Located downstream of the VDJ loci are 9 functional CH genes (Fig 3).20Honjo T. Immunoglobulin genes.Annu Rev Immunol. 1983; 1: 499-528Crossref PubMed Scopus (258) Google Scholar These constant genes consist of a series of exons, each encoding a separate domain, hinge, or terminus. All CH genes can undergo alternative splicing to generate 2 different types of carboxy termini: either a membrane terminus that anchors immunoglobulin on the B-lymphocyte surface or a secreted terminus that occurs in the soluble form of the immunoglobulin. With the exception of CH1δ, each CH1 constant region is preceded by both an exon that cannot be translated (an I exon) and a region of repetitive DNA termed the switch. Cocktails of cytokine signals transmitted by T cells or other extracellular influences variably activate the I exon, initiating transcription and thus activating the gene. Through recombination between the Cμ switch region and one of the switch regions of the 7 other H chain constant regions (a process termed class-switching or class-switch recombination [CSR]), the same VDJ heavy chain variable domain can be juxtaposed to any of the H chain classes.20Honjo T. Immunoglobulin genes.Annu Rev Immunol. 1983; 1: 499-528Crossref PubMed Scopus (258) Google Scholar This enables the B cell to tailor both the receptor and the effector ends of the antibody molecule to meet a specific need. A final mechanism of immunoglobulin diversity is engaged only after exposure to antigen. With T-cell help, the variable domain genes of germinal center lymphocytes undergo somatic hypermutation (SHM) at a rate of up to 10−3 changes per base pair per cell cycle. SHM is correlated with transcription of the locus, and in human subjects 2 separate mechanisms are involved: the first mechanism targets mutation hot spots with the RGYW (purine/G/pyrimidine/A) motif,21Dorner T. Foster S.J. Farner N.L. Lipsky P.E. Somatic hypermutation of human immunoglobulin heavy chain genes: targeting of RGYW motifs on both DNA strands.Eur J Immunol. 1998; 28: 3384-3396Crossref PubMed Scopus (106) Google Scholar and the second mechanism incorporates an error-prone DNA synthesis that can lead to a nucleotide mismatch between the original template and the mutated DNA strand.22Rada C. Ehrenstein M.R. Neuberger M.S. Milstein C. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.Immunity. 1998; 9: 135-141Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar Other species use gene conversion between functional and nonfunctional V sequences to introduce additional somatic diversity. SHM allows affinity maturation of the antibody repertoire in response to repeated immunization or exposure to antigen. Activation-induced cytidine deaminase (AID) plays a key role in both CSR and SHM.11Dudley D.D. Chaudhuri J. Bassing C.H. Alt F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.Adv Immunol. 2005; 86: 43-112Crossref PubMed Scopus (225) Google Scholar, 23Neuberger M.S. Antibody diversification by somatic mutation: from Burnet onwards.Immunol Cell Biol. 2008; 86: 124-132Crossref PubMed Scopus (88) Google Scholar AID is a single-strand DNA cytidine deaminase that can be expressed in activated germinal center B cells.24Muramatsu M. Nagaoka H. Shinkura R. Begum N.A. Honjo T. Discovery of activation-induced cytidine deaminase, the engraver of antibody memory.Adv Immunol. 2007; 94: 1-36Crossref PubMed Scopus (100) Google Scholar Transcription of an immunoglobulin V domain or of the switch region upstream of the CH1 domain opens the DNA helix to generate single-strand DNA that can then be deaminated by AID to form mismatched dU/dG DNA base pairs. The base excision repair protein uracil DNA glycosylase removes the mismatched dU base, creating an abasic site. Differential repair of the lesion leads to either SHM or CSR. The mismatch repair proteins MSH2 and MSH6 can also bind and process the dU:dG mismatch. Deficiencies of AID and uracil DNA glycosylase underlie some forms of the hyper-IgM syndrome. Creation of immunoglobulin diversity is hierarchical. In pro–B cells DH → JH joining precedes VH → DJH rearrangement, and VL → JL joining takes place at the late pre–B-cell stage. Production of a properly functioning B-cell receptor is essential for development beyond the pre–B-cell stage. For example, function-loss mutations in RAG1/2 and DNA-dependent protein kinase (DNA-PKcs and Ku 70/80) preclude B-cell development, as well as T-cell development, leading to severe combined immune deficiency. In frame, functional VDJH rearrangement allows the pro–B cell to produce μ H chains, most of which are retained in the endoplasmic reticulum. The appearance of cytoplasmic μ H chains defines the pre–B cell. Pre–B cells whose μ H chains can associate VpreB and λ14.1 (λ5), which together form the surrogate light chain (ΨLC), begin to express a pre–B-cell receptor. Its appearance turns off RAG1 and RAG2, preventing further H chain rearrangement (allelic exclusion). This is followed by 4 to 6 cycles of cell division.25Matthias P. Rolink A.G. Transcriptional networks in developing and mature B cells.Nat Rev Immunol. 2005; 5: 497-508Crossref PubMed Scopus (189) Google Scholar Late pre–B daughter cells reactivate RAG1 and RAG2 and begin to undergo VL → JL rearrangement. Successful production of a complete κ or λ light chain permits expression of conventional IgM on the cell surface (sIgM), which identifies the immature B cell. Immature B cells that have successfully produced an acceptable IgM B-cell receptor extend transcription of the H chain locus to include the Cδ exons downstream of Cμ. Alternative splicing permits co-production of IgM and IgD. These now newly mature IgM+IgD+ B cells enter the blood and migrate to the periphery, where they form the majority of the B-cell pool in the spleen and the other secondary lymphoid organs. The IgM and IgD on each of these cells share the same variable domains. The lifespan of mature B cells expressing surface IgM and IgD appears entirely dependent on antigen selection. After leaving the bone marrow, unstimulated cells live only days or a few weeks. As originally postulated by Burnet's “clonal selection” theory, B cells are rescued from apoptosis by their response to a cognate antigen. The reaction to antigen leads to activation, which might then be followed by diversification. The nature of the activation process is critical. T cell–independent stimulation of B cells induces differentiation into short-lived plasma cells with limited class switching. T-dependent stimulation adds additional layers of diversification, including SHM of the variable domains, which permits affinity maturation, class-switching to the entire array of classes available, and differentiation into the long-lived memory B-cell pool or into the long-lived plasma cell population. In general, the C domain of the H chain defines effector function, whereas the paired V domains of the antibody confer antigenic specificity. The H chain constant domain is generally defined as CH1-CH2-CH3 (IgG, IgA, and IgD), with an additional domain (CH4) for IgM and IgE. As described above, the CH1 domain is located within the F(ab) region, whereas the remaining CH domains (CH2-CH3 or CH2-CH4) comprise the Fc fragment. This Fc fragment defines the isotype and subclass of the immunoglobulin. Despite amino acid differences