Structure of Immunoglobulin and the B-Cell Receptor for Antigen

Immunoglobulin molecules, or antibodies, are glycoproteins that act as BCRs. These molecules can also be secreted in large quantities by activated B cells and plasma cells. Figure 13-1 shows the basic structure of an Ig molecule. Each Ig molecule is bifunctional—one region (Fab) binds to antigen, and a different region (Fc) mediates various effector functions, such as binding to host tissues (via their cell surface Fc receptors) and binding to and activating the first component of the classic complement system.

The structure of immunoglobulin molecules.

A, Basic immunoglobulin (Ig) structure with two heavy chains and two light chains linked together by disulfide bonds. The Fab region is involved in antigen binding, whereas the Fc region mediates various effector functions. B, Increasing detail of IgG molecule showing domains of both heavy and light chains. Each domain has an internal disulfide bond. The sites of antigen binding involve the NH ₂ -terminal domains of both the heavy and the light chains and are referred to as the “variable regions” (V _H and V _L , respectively). The rest of each chain has a relatively constant structure (C _H and C _L domains). C, Close-up of the antigen-binding variable regions. On the left, the complementarity-determining regions (CDRs) are shaded because they are the most variable components and are most involved in actual antigen binding. The CDRs are separated by intervening segments referred to as “framework regions.” On the right, the Ig polypeptide regions are correlated with the Ig gene segment (exons) that encode the variable region. Note that CDR3 corresponds to the V _H /D _H /J _H junctional region of the rearranged heavy chain gene and the V _L /J _L junctional region of the rearranged light chain gene.

(Adapted from MKSAP in the specialty of rheumatology , Philadelphia, 1993, American College of Physicians, p 6.)

The basic structure of all Ig molecules involves two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds (see Fig. 13-1A ). The isotype (class or subclass) of an Ig molecule is determined by its heavy chain type. There are five Ig classes—IgG, IgM, IgA, IgD, and IgE—corresponding to the γ, µ, α, δ, and ε heavy chain types. In humans, there are four IgG subclasses, IgG1 to IgG4. There are major differences in the structure and main functions of these different Ig classes and subtypes.

In Figure 13-1B , the IgG molecule is shown as an example of basic antibody structure. Each chain is composed of a series of globular regions or domains. Each domain encompasses about 60 to 70 amino acids and has an internal disulfide bond. The site of antigen binding is the amino (NH ₂ )-terminal domain for both the heavy (H) and light (L) chains. This domain is characterized by remarkable sequence variability and is referred to as the “variable region” of the heavy and light chains (V _H and V _L region, respectively). The combination of V _H and V _L forms the antigen-binding site, and there are two such sites per IgG molecule (see Fig. 13-1B ). The rest of each polypeptide has a relatively constant structure. The constant domain of the light chain is termed the “C _L region,” whereas the heavy chain has three constant domains: C _H 1, C _H 2, and C _H 3. The hinge region, located between the C _H 1 and C _H 2 domains, provides flexibility and independence to the two antigen-binding sites. In both µ and ε heavy chains, there is an additional constant domain between C _H 1 and C _H 2, resulting in a total of four constant domains.

The greatest variability in antibody molecules takes place in the V _H and V _L domains, and these domains are responsible for the specificity in antigen binding. Within the variable domains (see Fig. 13-1C ), certain short segments show exceptional variability and are called “hypervariable regions.” These regions are also referred to as complementarity-determining regions (CDRs) because they are directly involved in the binding to antigen. In both the V _H and V _L regions, there are three CDRs (CDR1 to CDR3) with intervening segments referred to as “framework regions” (see Fig. 13-1C ). CDR3 (a component of the polypeptide V region) is formed by parts of the V (variable), D (diversity), and J (joining) gene segments. Variation within the V _H and V _L regions distinguishes one antibody molecule from another and is referred to as “idiotype” or “idiotypic variation . ” Additional information on heavy chain rearrangement is shown in eFig. 13-1 .

Formation of the B-Cell Receptor Repertoire

The BCR repertoire and the Ig molecules are characterized by enormous diversity. The principal genetic mechanisms that are used to generate this diversity include (1) genetic recombination of gene segments to form a functional Ig gene, (2) combinatorial diversity of heavy and light chain matching, and (3) somatic mutation of rearranged genes. Another major force in shaping the antibody repertoire has been termed “receptor editing , ” which allows receptors with self-reactive potential to be modified by additional recombination events during B-cell differentiation.

Figure 13-2A illustrates the principle of genetic recombination for an Ig gene. In this case, one of several variable (V) region gene segments (normally separated upstream from the constant [C] region gene segments on the same chromosome) can be linked to a single C region gene segment by genetic recombination. The combining of gene segments, rather than the existence of a single gene coding for every individual antibody molecule, considerably reduces the amount of genetic information required to encode many different antibody molecules. In Figure 13-2B the different gene segments that encode a κ light polypeptide chain are shown. Note that in this case, a V region rearranges proximal to a J segment, which is linked to the C region segment. Rearrangement of κ or γ light chain genes involves V, J, and C region gene segments (see Fig. 13-2A ; see also Fig. 13-1C ). In the formation of a functional heavy chain gene, a successful rearrangement of gene segments includes one V, one D, and one J region segment linked to a C region segment (compare Figs. 13-1C and 13-2B ).

Principles of genetic recombination.

A, In the germline DNA, the V region segments are located upstream from the D, J, and C segments on the same chromosome. Recombination, which takes place only in lymphocytes, brings the V gene segments in proximity to the downstream segments. B, In the B cell, rearrangement of gene segments separated in the germline DNA allows for the formation of a functional immunoglobulin light chain gene (compare with Fig. 13-1C ). mRNA, messenger RNA.

(Adapted from MKSAP in the specialty of rheumatology , Philadelphia, 1993, American College of Physicians, p 8.)

During genetic recombination, additional variability is generated by a process known as “junctional diversification.” When two gene segments are brought together during rearrangement, the linking is not precise. Instead, some nucleotides are inserted or deleted randomly at the junctional site. This introduces new codons, and therefore new amino acids, into the junctional sequence. If an incorrect number of junctional nucleotides are added or deleted, functional molecules will not be encoded, because the rest of the gene will be “out of frame” or a “stop” sequence will be introduced. Junctional diversification takes place in the hypervariable CDR3 region of the molecule (see Fig. 13-1C ).

In general, a single B cell usually expresses an Ig molecule of only one antigen specificity, composed of one heavy chain molecule and one light chain molecule. Even though there are two chromosomal copies of the heavy chain genes in each cell, only one is usually functionally expressed. This phenomenon of using genes on only one parental chromosome is known as “allelic exclusion.” Once there has been a functional rearrangement of heavy chain genes on one parental chromosome, rearrangement of heavy chain genes on the other chromosome is mostly prevented. If the first rearrangement is not successful, the second one can take place. All of the genetic events described earlier happen before the B cell encounters its antigen. The B-cell (or antibody) response diversifies further after interaction with antigen by a process known as “somatic mutation,” primarily involving point mutations in the hypervariable regions of the V regions. Somatic mutation can be viewed as “fine tuning” of the antibody response, occurring after the primary response to a stimulus and during the development of memory B cells. It is, therefore, mostly observed during a secondary immune response. Somatic mutation allows for the production of antibodies with higher affinity for antigen, that is, the ability to bind more strongly to an antigen. Although the mutations are random, B cells with high affinity are selectively expanded (referred to as “affinity maturation”) because the stronger binding allows preferential stimulation by the target antigen. Somatic mutation is closely tied to isotype switching, and T-cell help is required. Somatic mutation usually takes place at the site of T-cell–B-cell interaction in the germinal centers of lymph node or spleen.

Isotype Switching and Function of the Different Immunoglobulin Classes

One B cell usually makes antibody of a single specificity that is fixed by the nature of V _L J _L and V _H D _H J _H rearrangements. During the lifetime of this cell, however, it can switch from making an IgM molecule to producing a different class of antibody, such as IgG or IgA, while retaining the same antigenic specificity. This phenomenon is known as “class switching” or “isotype switching.”

Figure 13-3 shows the predominant mechanism by which a B cell switches from production of surface IgM to secretion of an IgG, IgA, or IgE molecule. The mechanism involves further DNA rearrangement (a process unique to Ig heavy chain genes), linking rearranged VDJ gene segments with a different heavy chain C region gene segment downstream. The switch (S) region is a stretch of repeating sequences 5′ to the C region that allows a VDJ unit (previously linked to the Cµ region gene segment) to rearrange to another C region downstream. In the process the intervening DNA is deleted. The B cell therefore cannot return to IgM production. Class switching generally follows stimulation of the B cell by antigen and frequently is dependent on factors released by T helper cells.

Genetic mechanisms involved in immunoglobulin (Ig) isotype switching.

Top, The initial rearrangement of Ig heavy chain gene segments has already taken place in the B cell to put the V region proximal to the D, J, and Cµ segments. This latter genetic region contains other constant region gene segments farther downstream, including Cδ, various Cγ and Cα gene segments, and Cε. In the process of T-cell–dependent B-cell activation in the germinal centers, the B cell is signaled to undergo isotype switching to allow high-rate secretion of an IgG1 antibody. Bottom, This is accomplished by additional DNA rearrangements that put the VDJ unit adjacent to the Cγ1 segment downstream. The intervening DNA is deleted.

Each class of secreted Ig has a different set of functions ( Table 13-4 ). IgG is the major antibody of secondary immune responses and is most important for obtaining effective immunization to various toxins, toxoids, and certain extracellular pathogens. IgG accounts for 70% to 75% of the total Ig pool and is the major Ig class in normal human serum. It also crosses the placenta and confers immunity to newborns and neonates for the first few months of life. The interaction of IgG antibodies with antigen can have numerous consequences, including precipitation, agglutination, complement activation, and various cellular effector functions via IgG receptors. There are four IgG subclasses, with IgG1 and IgG3 being most effective at fixing complement and activating complement-mediated effector functions.

IgM is the predominant early-secreted antibody, frequently seen in primary immune responses. IgM is important for effective responses to antigenically complex infectious organisms, especially those with polysaccharide-containing cell walls. IgM antibodies may also be extremely effective at precipitation, agglutination, and complement activation after binding to antigen. Secreted IgM is usually found as a pentamer of the basic (four-domain) Ig unit. Polymerization is facilitated by the binding of the heavy chain tailpieces to a joining (J) peptide.

IgA plays a major role in mucosal immunity and is the predominant Ig in secretions such as saliva and tracheobronchial secretions. Secretory IgA exists mainly in dimeric form and contains a secretory component, which is synthesized by epithelial cells and facilitates transport into secretions as well as protection from proteolysis. Secretory IgA is involved in the prevention of microbial adherence to mucosal cells and in the agglutination of microorganisms. IgA provides the first line of defense against a variety of pathogens.

IgE is scarce in the serum. Its major importance relates to its ability to bind to FcεR1 receptors on mast cells and basophils, and cross-linking of IgE bound to these cells results in cellular activation, degranulation, and release of mediators involved in allergic responses, such as histamine and various leukotrienes. IgE plays a role in immunity to parasites, but in developed countries, it is more commonly associated with allergic responses and allergic diseases, such as hay fever and asthma (see Chapters 41 and 42 ).

B-Cell Development

B cells are produced in the specialized microenvironments of the fetal liver and the adult bone marrow. After migration out of the bone marrow, the life span of mature naive B cells is limited unless there is contact with antigen. B cells interact with foreign antigen in the peripheral lymphoid tissues, particularly in the germinal centers of lymph nodes and spleen. The interaction with antigen, in the setting of T-cell help, results in the generation of memory B cells and cells that secrete a large amount of Ig. This frequently involves class switching and somatic mutation, which allows a secondary antibody response to generate antibodies of a different isotype with higher affinity for the stimulating antigen. An additional view of B-cell development is shown in eFig. 13-2 .

The formation of the B-cell repertoire in the bone marrow is mostly random, and B cells with self-reactivity are generated. Negative selection of cells capable of strongly binding to self-antigens is an important part of B-cell development. This process of B-cell self-tolerance involves both deletion (elimination) and functional inactivation (anergy) of self-reactive cells. In addition, B cells with specificity for self-antigens can modify their receptors through a process called “receptor editing . ” These self-reactive B cells undergo a reversible arrest of development and reinitiate light chain gene rearrangements to alter their BCR. If a B cell fails to edit its BCR, it is destined for cell death (apoptosis). Immature B cells in the bone marrow appear to be capable of receptor editing, whereas mature B cells in the peripheral lymphoid tissues normally lose this ability to initiate a new round of Ig gene rearrangements.

Immunoglobulin Interactions with Antigen

An antigen is a molecule or molecular complex recognized by B cells or T cells. The term immunogen usually refers to a substance capable of eliciting an immune response, and therefore it must also be capable of being recognized as an antigen. An antigen (e.g., a protein molecule) is usually much larger than the small region fitting into the binding site of an Ig molecule or the processed peptide recognized by a TCR. This smaller region is frequently referred to as an “antigenic determinant” or “epitope . ” In a protein a B-cell epitope can theoretically be constructed in two ways—as a continuous or a discontinuous epitope. In a continuous epitope, the amino acid residues are part of a single uninterrupted sequence, whereas in a discontinuous epitope, residues are not contiguous in the primary structure but are brought together by the folding of the polypeptide chain. Because this kind of epitope requires a special conformation of the antigen, it is frequently referred to as a “conformational epitope.”

B cells and T cells usually recognize different parts of an antigen. B cells and their secreted Ig molecules most commonly recognize unprocessed or “native” antigens. These antigens have maintained their native configuration, and most of the epitopes are usually of the discontinuous or conformational type. In general, only a minor component of a B-cell response is directed to small linear peptide regions of the antigen. Studies indicate that epitopes recognized by B cells are not randomly distributed throughout the antigen but rather reside in regions with particular structural features. One important feature is “accessibility,” because epitopes normally must be on the outer surface of a protein and may protrude from an antigen’s globular surface to be able to interact with the BCR.

T-Cell Receptors

The TCR shows important structural similarities with Ig molecules ( Fig. 13-4 ). The αβ TCR is expressed by at least 90% of peripheral blood T cells. Essentially, all CD4 ⁺ T cells and most CD8 ⁺ T cells express this form of the TCR. A small percentage of αβ-expressing T cells have a double-negative (CD4 ⁻ and CD8 ⁻ ) phenotype. As shown in Figure 13-4 , each chain consists of two extracellular Ig-like domains anchored into the plasma membrane by a transmembrane region and a short cytoplasmic tail. Similar to Ig molecules, the outer NH ₂ – terminal domain of each chain constitutes the variable region. Outside of the transmembrane region, the two chains are covalently linked together by disulfide bonds. Due to the short cytoplasmic tail, the αβ heterodimer is not capable of transmitting an intracellular signal after TCR engagement. This function is accomplished by the CD3 complex of polypeptides and other signaling proteins that are associated with the TCR.

The T-cell receptor (TCR) α and β chains indicate the regions encoded by different TCR gene segments.

The positions of the complementarity-determining regions (CDRs) of both the β and the α chains are also shown. These are the most variable parts of the TCR and the most important for TCR binding to the peptide/major histocompatibility complex. CDR1 and CDR2 of both the α and the β chains are encoded within the variable region genes. CDR3 is formed by the rearrangement of V, D, and J gene segments of the β chain and V and J segments of the α chain and is encoded by the distal part of the V region segment through part of the J segment. The different parts of the TCR are not drawn to scale.

The γδ TCR is an alternative form of TCR that is similar in overall structure to the αβ receptor. Although some γδ cells express CD8, most are CD4 ⁻ and CD8 ⁻ (double negative). CD8 expression is largely confined to those γδ cells residing in the small intestine. The γδ TCR is also expressed in association with the CD3 complex. Although γδ T cells form a minor proportion of the T cells in the thymus and secondary lymphoid organs, they are abundant in various intraepithelial locations, such as in the skin, intestines, and lung.

T-Cell Receptor Structure and T-Cell Receptor Repertoire Formation

Genes encoding the TCR are organized similarly to Ig genes ( eFig. 13-3 ). In a manner similar to that described previously for B cells, rearrangement of gene segments, junctional diversity, and combinatorial joining of the two chains is responsible for the diversity of the TCR repertoire. However, in contrast to B cells, TCR genes do not undergo somatic mutation. Thus nearly the entire TCR αβ repertoire is formed during T-cell development in the thymus and before any interaction with antigen. Because the T-cell repertoire is shaped to recognize self-MHC antigens, it is believed that extrathymic somatic mutation might result in the generation of deleterious self-reactive T cells.

The components of the αβ TCR heterodimer are shown in Figure 13-4 , and the ribbon backbone structure of the Vα and Vβ portions of a human TCR are shown in Figure 13-5 . The upward-pointing loop structures are the CDRs. These regions are the most variable part of the TCR α and β chains and are most important for binding of the TCR to the MHC/peptide complex. The CDR1 and CDR2 regions of the α chain and β chain are encoded within the germline TCRAV and TCRBV gene segments, and variability in their sequences distinguishes the different V region subfamilies. The most diverse part of the α and β chains is the CDR3, which directly interacts with peptide in the binding groove of the MHC molecule.

A ribbon backbone structure of the Vα and Vβ portions of a human T-cell receptor (TCR).

The upward-pointing loop structures are the complementarity-determining regions (CDRs). These regions are the most variable part of the TCR α and β chains and are most important for TRC binding to peptide/major histocompatibility complex. The CDR1 and CDR2 regions of the α chain and β chain are encoded within the germline TCRAV and TCRBV gene segments, respectively. The highly variable CDR3 region is formed by the rearrangement of V, D, and J gene segments. Junctional nucleotide substitutions and deletions at the margins of rearrangement add to the potential diversity of the CDR3 region.

(Adapted from Kotzin BL, Kappler J: Targeting the TCR in rheumatoid arthritis. Arthritis Rheum 41:1907, 1998.)

Antigen-Presenting Cells and the Major Histocompatibility Complex

There are two major varieties of MHC molecules (and genes) involved in the presentation of antigens to T cells. MHC class I molecules include human leukocyte antigen (HLA)-A, HLA-B, and HLA-C molecules. MHC class II molecules include the HLA-DR, HLA-DQ, and HLA-DP molecules. Class I molecules are expressed on nearly all nucleated cells. In contrast, class II molecules have a limited distribution and are normally present only on cells involved in antigen presentation to T cells, including dendritic cells, macrophages, B cells, and thymic epithelial cells. The limited expression of class II antigens in different tissues may be extremely important in preventing various types of autoimmune reactions. After activation or exposure to certain cytokines, such as interferon-γ (IFN-γ), other human cell types such as activated T cells and epithelial cells can express class II molecules.

The general structures of the two classes of MHC molecules are shown in Figure 13-6 . For MHC class I antigens, the α chain (encoded within the MHC) is complexed to beta ₂ -microglobulin (encoded outside the MHC). The α chain is highly polymorphic (variable between individuals), whereas beta ₂ – microglobulin is invariant. The extracellular portion of the α chain is divided into three domains: α1, α2, and α3. The outer α1 and α2 domains represent the polymorphic components of the molecule, and the α3 domain is relatively constant. MHC class II molecules are composed of an α and a β chain, both of which are encoded within the MHC. The NH ₂ -terminal domains of each chain (α1 and β1 domains) represent the polymorphic regions of the molecule and are important in antigen presentation, whereas the α2 and β2 domains are relatively constant.

Comparison of the composition of class I and class II major histocompatibility complex (MHC) molecules.

The class I MHC α chain is variable (polymorphic) among different individuals. It is expressed with beta ₂ -microglobulin, which is encoded outside the MHC region and does not differ among different individuals. The class II MHC molecules are composed of α and β chains. For human leukocyte antigen (HLA)-DR molecules, only the β chain is variable, whereas for the HLA-DQ and HLA-DP molecules, both the α and the β chains are encoded by polymorphic alleles. In class I MHC molecules, the peptide-binding region is formed between the α1 and the α2 domains, which are polymorphic. The peptide-binding region of class II MHC is formed between the α1 and the β1 domains of the α and β chains, respectively.

The structures of MHC class I and class II molecules have provided remarkable insight into how antigenic peptides are bound and presented to T cells. In Figure 13-7A , the peptide-binding groove of a class I molecule is viewed from the top, showing the surface that is contacted by a TCR. MHC class I pockets can usually only bind peptides of 8 to 10 amino acids, which are bound in a typical extended conformation with both the NH ₂ terminus and the carboxy (COOH) terminus anchored in the peptide-binding groove. In the case of MHC class II, the peptide-binding groove is formed by the interaction of the NH ₂ -terminal domains of the α and β chains (see Fig. 13-7B ). The structure of MHC class II allows peptides of varying lengths to bind because both ends of the peptide are free, and the peptide shown in Fig. 13-7B has the typical polyproline-like extended helical conformation seen in all class II MHC-bound peptides.

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