The immunoglobulin supergene family is a group of structurally similar glycoproteins, which mediate antigen recognition and cellular interactions. They are derived from a family of genes which evolved from a common primordial gene. The products of these genes are transmembrane glycoproteins characterized by a common structural motif of functional domains. Some important members of this immunoglobulin supergene family are the immunoglobulins, MHC, TcR, secretory component, and adherence proteins such as ICAM-1.
The basic structure of all immunoglobulin molecules consists of two identical L chains and two identical H chains (Fig. 1-15). The antibody molecule consists of three major domains connected by a hinge region. As shown in Fig. 1-15, digestion of antibody molecule with a proteolytic enzyme-papain results in the separation of these three domains. Two domains are identical and are called fragment antigen binding (Fab), and the third domain is called fraction crystallizable (Fc). However, treatment with proteolytic enzyme pepsin results in a fragment that contains both antigen binding arms (Fab')2 and several pieces of the Fc fragment. Fab interact with the antigen, and Fc bind to Fc-receptors on different cells.
Figure 1-15. Prototypic structure of immunoglobulins. The complementarity regions (e.g., antigen receptor sites that make specific contact with ligands) sites of the V region are shown in the insert.
Various forms of immunoglobulins such as IgG and IgE are found as monomers, secreted IgA as dimers and IgM as pentamers (Fig. 1-16). Consequently, two distinct regions of the assembled immunoglobulin occur: the first, which binds to an antigenic determinant and the second, which has other functions, such as binding to special cells and the first component of complement. The two H chains and each H chain and L chain are linked by disulfide bonds. Each chain is divided into two regions: the C region at the carboxyl-terminus and the variable region at the amino-terminus. The C region of each L chain consists of about 107 amino acids and has an invariant structure except for isotypic features (kappa or lambda) and allotypic variants (e.g., molecular structures that are individually inherited). V and C regions of H chains are further divided into domains characterized by folding of the polypeptide chain into 110 amino acid loops. V regions of H and L chains display great variability in the sequence of amino acids. Localized areas of these hypervariable regions of H and L chains interact to form antigen binding sites (i.e., CD1, CD2 and CD3; Fig. 1-15). In contrast, C regions of H chains dictate other functions of immunoglobulins, including binding to cell surface receptors. Eight immunoglobulin isotypes, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE, are produced by B cells as a result of rearrangements of V genes for H chains (VH), D genes for H chains (DH), J genes for H chains (JH), V genes for L chains (VL), J genes for L chains (JL), and C region genes (vide infra) The special properties of each immunoglobulin class are as follows (Table 1-3).
Figure 1-16. Diagram of various forms of immunoglobins; IgG and IgE are found as monomers, secreted IgA as dimers and IgM as pentamers. Dimers and pentamers are held together by the J chain.
IgG: IgG is a monomeric, four-chain structure consisting of two gamma heavy chains and two kappa or lambda light chains. The C region of the H chain of the molecule consists of three domains. Inter-chain disulfide linkages between the Cgamma1 and Cgamma2 domains stabilize the structure and define the hinge region of the molecule. IgG is the dominant immunoglobulin in extracellular fluids and is the only immunoglobulin transported across the placenta, and directly acts as an opsonin.
There are four subclasses of IgG, each of which displays unique antigenic determinants on the C region of the H chains. The approximate proportion of each subclass in blood is IgG1, 70%; IgG2, 20%; IgG3, 8%; and IgG4, 2%. The antibody specificities are distributed in somewhat specific patterns in each subclass. Neutralizing antibodies to protein toxins are mostly found in IgG1, antibodies to polysaccharides in IgG2, and antibodies to viruses in IgG3.
IgM: IgM is a pentamer of 4-chain units that are bound to a separate peptide called the J chain. IgM molecules consist of µ H chains and kappa or lambda L chains. Monomeric IgM is the principal antigen receptor on B cells. IgM is found principally in blood, but also occurs in external secretions. It binds most efficiently the C1q subunit of the first component of complement (vide infra), and is the first immunoglobulin expressed in B cell development.
IgA: IgA consists of a heavy chains and kappa or lambda light chains. There are two principal molecular forms of IgA, monomers whose basic structure and numbers of domains are similar to IgG and dimers that bind to J chains. Monomeric IgA, the second most common immunoglobulin in adult serum, is primarily produced by plasma cells in the bone marrow, whereas dimeric IgA, the dominant immunoglobulin in external secretions, is produced by plasma cells at mucosal sites.
Dimeric IgA is complexed and transported with a secretory component to form secretory IgA (Fig. 1-17). Dimeric IgA binds to polymeric immunoglobulin receptors (secretory component) on the basolateral membranes of epithelial cells; the complex is internalized and transported across the cells in an endocytic vesicle to the apical pole of the cell where it is secreted as secretory IgA. The addition of a secretory component not only facilitates the transport of dimeric IgA, but protects the molecule from proteolysis.
Figure 1-17. Assembly and secretion of secretory IgA.
There are two subclasses of IgA, IgA1 and IgA2. IgA1 predominates in the blood; there is an equal distribution of the two subclasses in external secretions. IgA2 is more resistant than IgA1 to bacterial IgA proteases that attack the hinge region of the molecule.
IgD: IgD is a monomeric four-chain polypeptide structure that is similar to IgG but its heavy chain (delta) is unique. Although this protein is expressed along with monomeric IgM on mature B cells, only small amounts of it are found in extracellular fluids.
IgE: IgE is also a four-chain polypeptide structure that is similar to IgG, but its heavy chain (epsilon) is distinct. Only trace amounts of this immunoglobulin are found in serum. IgE binds avidly to circulating blood basophils and mast cells in the submucosal sites and the skin. Cell-bound IgE antibodies defend against tissue parasites and initiate the pathogenesis of immediate hypersensitivity by triggering the release of low-molecular weight vasoactive compounds, including histamine, leukotrienes, and platelet-activating factor and certain proinflammatory cytokines such as TNF-alpha and IL-5, once they are cross-linked by antigens.
Initial exposure to an antigen results in the production of low affinity antibodies, but continued exposure to antigen leads to the production of high affinity antibodies. In the primary antibody response (the first immunization), B cells are activated to produce IgM antibody. By 3-5 days, specific antibodies, mainly of the IgM isotype, appear in the serum and the concentration (titer) increases until a peak is reached in 10-14 days (Fig. 1-18). Antibody titers then fall to preimmunization levels after some weeks. Upon reimmunization, there is a more rapid and extensive development of antibody-producing cells in regional lymph nodes, and many of them undergo an isotype switch to produce IgG or other immunoglobulin classes of specific antibodies. As a result, in most cases following re-immunization, serum antibodies are primarily IgG and have a greater affinity for antigens; also, the antibody titers are higher and persist for much longer periods.
Figure 1-18. Isotypes of serum antibodies in primary and secondary immunization.
There are a number of important consequences of antibody binding to antigens, depending upon the nature of the antigen. These include the neutralization of adherence sites or toxins from bacteria, the formation of opsonins, and the activation of the classical pathway of the complement system for that purpose or to create other bioactive factors that enhance inflammatory reactions. 1) In the case of IgM, antigen-antibody complexes are created that most efficiently activate the classical pathway of the complement system and thereby lead to the formation of functional complement fragments including opsonins that facilitate the removal of the complexes by the RES. 2) IgG antibodies, which are the dominant immunoglobulins in extracellular fluids, neutralize toxins and viruses, opsonize particles for ingestion by phagocytes, or when complexed to antigens, activate the classical pathway of complement. 3) Secretory IgA antibodies defend mucosal sites by binding toxins and preventing adhesion of microbial pathogens. 4) IgE antibodies on the surface of mast cells and basophils play an important role in defense against parasites and development of immediate hypersensitivity as previously noted.
Specific antibodies are generated as a consequence of immunoglobulin gene rearrangement, i.e., recombination of V, D, J, and C genes (Fig. 1-14). The immune system generates millions of different antibody molecules from the pool of V genes. Separate sets of V genes encode the variable domains of immunoglobulin H and L chains. The two chains are produced separately, but the mechanisms by which their diversity is achieved are similar in principle.
Light Chain Formation: Most antibody molecules use the kappa light chain. The kappa gene cluster consists of several hundred (~300) VL genes; a few J genes (~4) and one C gene. These germline genes are tandemly arranged on the chromosome and are transcriptionally inactive. As the B cell matures, genes are arranged (recombined) so that one V gene is joined to a J gene, and the rearranged VJ segment together with the C gene is transcribed. The portion of DNA between the joined segments is deleted, and the transcripts are processed by splicing to produce the messenger RNA for the L chain. kappa chains are encoded by a separate cluster of V, J and C genes, but the rearrangement and transcription are similar to that of the lambda chain. Any given B cell uses only one type of L chain to produce the immunoglobulin molecule. The L chain combines with the H chain during their transport from polyribosomes to the membrane.
Heavy Chain Formation: The H chain gene system has a design that is similar to that of light chain, but is slightly more complex (Fig. 1-14). In addition to ~ 1,000 VH genes, there are > 10 D genes and ~ 4 J genes. Furthermore, this genetic cluster has nine C genes that encode different immunoglobulin isotypes. The mature B cell (Fig. 1-14) rearranges its immunoglobulin genes, joins them together, and deletes the DNA between the joined segments. The rearranged VDJ gene segment is transcribed together with a Cµ or Cdelta gene, and this long transcript is spliced into VDJCµ or VDJCdelta messages resulting in the expression of IgM and IgD, respectively, on the B cell surface. Both immunoglobulin molecules use the same VDJ segment and, therefore, possess the same immunological specificity. The B cell is now ready to bind to a specific antigen and become further differentiated.
Antibody diversity is generated by the following mechanisms.
Immunoglobulin Gene Rearrangements: 1) The joining of various V, D and J genes is entirely random that results in ~ 50,000 different possible combinations for VDJ(H) and ~ 1,000 for VJ(L). Subsequent random pairing of H and L chains brings the total number of antibody specificities to ~107 possibilities. 2) Diversity is further increased by the imprecise joining of different genetic segments. 3) Rearrangements occur on both DNA strands, but only one strand is transcribed (allelic exclusion). 4) Only one rearrangement occurs in the life of a B cell because of irreversible deletions in DNA. Consequently, each mature B cell maintains one immunologic specificity and is maintained in the progeny or clone. This constitutes the molecular basis of the clonal selection; i.e., each antigenic determinant triggers the response of the pre-existing clone of B lymphocytes bearing the specific receptor molecule. It also follows that deletion of the B cell clone results in immunologic unresponsiveness to the antigen.
Somatic Mutations: This mechanism leads to a fine-tuning of the antibody specificity after immunization. Rearranged VDJH, and VJL genes in the B cells are uniquely susceptible to point mutagenesis by enzymes that become activated following stimulation of the cell by antigen. The clonal progeny of an antigen-driven B cell thus produce antibodies that may differ in one or more amino acid positions in the regions of the protein that are responsible for antigen binding. Cells producing the mutant antibody with highest affinity for the antigen are preferentially stimulated and thus eventually dominate the response. Therefore, antibodies produced after repeated immunization commonly display numerous point mutations (derived by somatic mutations in B cells found in peripheral lymphoid organs) and have higher affinities for antigens (affinity maturation), as compared to antibodies produced in the primary immune response.
Antibody Function: Antibody molecules perform a number of important functions that are necessary for mounting an effective immune response against microbial pathogens. CH region genes encode the biological functions of immunoglobulins (Table 1-3). For example, IgM and IgG bind to the C1q subunit of C1, IgG crosses the placental barrier to the fetal circulation, and polymeric immunoglobulins, particularly dimeric IgA, are transported across epithelial cells into mucosal secretions.
To accomplish these functions, B cells switch their immunoglobulin isotype. The VDJ genes which are associated with Cµ or Cdelta, which are the original constant genes expressed in mature B cells, become associated with another C gene (Fig. 1-11). This has been termed the isotype switch, because the C gene determines the antibody isotype. The switch is accomplished by genetic recombination, whereby the VDJ gene segment is transferred from the Cµ/Cdelta junction onto another C region gene downstream (Fig. 1-11). Because the Cµ/Cdelta and other interposed genes are deleted, the switch is irreversible. The new antibody maintains the same L chain and the same VH region (encoded by VDJ), but has new properties determined by the acquired C gene. The isotype switch mechanism is promoted by physical interactions between T and B cells (for example, the binding of CD40 on B cells to its ligand on T cells) and by specific cytokines from T cells (for example, IL-5 and IL-10 promote IgA production; IL-4 promotes IgE production).
Furthermore, each antibody molecule may exist in either a membrane-bound or secreted form. Every C gene contains a 3' sequence encoding the hydrophobic cytoplasmic tail of the H chain, so that the immunoglobulin molecule produced by the B cell is inserted in the surface membrane to function as the receptor for antigen. When the B cell differentiates into a plasma cell, an enzyme is activated that modifies the RNA transcript. Consequently, the translated protein ends with a hydrophilic peptide and is secreted from the cell.
The specific receptor for antigen on T lymphocytes, the TcR (Fig. 1-8), is a heterodimeric protein with motifs that are similar to immunoglobulin molecules, but whose structure is encoded by a different set of V, J, D, and C genes. Moreover, T cells consist of two subsets carrying different receptors, that have been designated alpha/beta and gamma/delta.
A minority of T cells express a TcR consisting of gamma and delta chains and those cells are primarily CD4+. These chains are encoded by very few genes; the gamma/delta repertoire is accordingly very limited. The gamma gene cluster consists of seven V genes, two J genes, and four C genes. The delta genes are interspersed within a gene locus that appears to include 10 V genes, two D genes, two J genes, and one C gene. Mature gamma/delta T cells seem to migrate primarily to mucosal and cutaneous tissues. The functions of gamma/delta T cells are not yet understood. Moreover, the recognition of antigen by gamma/delta T cells are not MHC-restricted.
Most T cells express the alpha/beta type of TcR. The genomic organization of the alpha and beta genes is more complicated than that of the immunoglobulin genes. Indeed, although a locus genes are interspersed with genes for the delta TcR (vide infra), alpha and beta genes are rearranged and expressed at different times and on different T lymphocytes.
The smaller alpha chain is encoded in a gene cluster consisting of ~100 V genes, ~50 J genes (a high number compared to immunoglobulin J genes) and one C gene. The alpha chains of various binding specificities are generated by a random genetic recombination of one V and one J gene, which are then joined with Calpha, by a mechanism analogous to that of the immunoglobulin L chain. The heavier beta chain is encoded by ~ 30 V genes, two D genes, >10 J genes and two C genes. The random joining of one of each V, D and J genes and their rearrangement to one C gene is similar to the process described for immunoglobulin genes. Rearranged VJCalpha and VDJCbeta DNA encodes the alpha and beta chain transcripts, respectively.
TcR genes are rearranged as lymphocytes mature in the thymus. Mature T cells, which are released from the thymus, are irreversibly committed to recognize one specific antigenic epitope in complex with self-MHC molecule.
Generation of TcR Diversity: The combinatorial diversity of TcR is greatly increased by junctional diversity, i.e., the variability of the junctions between different VDJ genes. New nucleotide base pairs are often added at the junction. Indeed, the junctional diversity of TcR is several orders of magnitude greater than that of an immunoglobulin gene. On the other hand, rearranged TcR genes are not subject to somatic mutations that contribute significantly to the generation of antibody diversity. The lack of somatic mutations appears to be related to the fact that the alpha/beta T cells always recognize a complex of antigenic fragment with the self MHC molecule. The receptor mutation could divert the specificity towards self molecules.
The TcR cannot bind to soluble antigens but they recognize antigenic peptides bound to MHC molecules (i.e. class I or class II). Even after TcR binds MCH-peptide complex, it cannot transmit optimal signal necessary for T cell activation. Intracellular signalling of T cells requires non-covalent association of TcR with cell surface CD3 complex (Fig. 1-8). The CD3 complex consists of four transmembrane peptides designated gamma, delta, epsilon, and zeta. The CD3 complex itself does not recognize the antigen and does not have variable domains. However, the CD3 complex transmits the biochemical signals generated by the TcR/antigen/MHC interaction on the surface that lead to lymphocyte activation.
General Features: These molecules play a very important role in the recognition of self and non-self antigens by T cells. The MHC consists of a cluster of >100 genes on chromosome 6 that encode a number of biologically important molecules (Fig. 1-9). These molecules are responsible for the rejection of tissue grafts by genetically disparate individuals, as the name histocompatibility indicates. These molecules present antigens to T lymphocytes; govern interactions between T cells, B cells and accessory cells; and control the intrathymic development of the TcR repertoire against foreign antigens (positive selection) and against self (negative selection) Human MHC protein products are called human leukocyte antigens (HLA).
Genes and Structures: The two most important HLA glycoproteins are designated as class I and class II molecules (Fig. 1-9).
MHC Class I/II Molecules: MHC class I molecules are ubiquitous on somatic cells whereas MHC class II molecules are restricted to monocytes, macrophages, dendritic cells, B cells, Langerhans cells, keratinocytes, activated T cells and certain types of epithelial cells. MHC class I molecules have three extracellular domains (alpha1, alpha2 and beta1), and a cytoplasmic tail. In contrast, MHC class II molecules have four extracellular domains (alpha1, alpha2, beta1 and beta2).
Three genes encode three independently expressed MHC class I molecules: HLA-A, -B and -C. Each gene contains three exons for the domains 1, 2 and 3. The MHC class II cluster, HLA-D, also contains three distinct genes, DP, DQ and DR, each of which has a separate set of exons for the alpha and beta chain.
MHC Alleles: An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) exists in different forms, or alleles. HLA alleles are designated by numbers and subscripts. For example, two unrelated individuals may carry class I HLA-B, genes B5, and Bw41, respectively. Allelic gene products differ in one or more amino acids in the alpha and/or beta domain(s). Large panels of specific antibodies are used to type HLA haplotypes of individuals using leukocytes that express class I and class II molecules. HLA typing is used for matching donors and recipients for organ/tissue transplantation and to predict the risk of certain diseases . In addition, the polymorphism of HLA genes has major implications for the function of class I and class II molecules (vide infra).
Role in Antigen Presentation: MHC molecules are required for antigen presentation to T cells (Fig. 1-12). Peptides associated with MHC class I and class II molecules are recognized by CD8+ and CD4+ T cells, respectively. Foreign protein antigens are taken up by various types of cells in the body, internalized and subjected to enzymatic degradation called antigen processing. Antigenic peptide fragments bind to MHC class II molecules and are then transported to the cell surface. This MHC/antigen complex is recognized by the TcR on CD4+ T cells. A CD4+ T cell activated by an appropriate class II/peptide antigen complex on an antigen-presenting cell, such as a B cell or macrophage, may become a helper cell for antibody or cell-mediated immune responses.
A different scenario is found for viral antigens in infected cells or tumor antigens. These antigens are processed to fragments, which are expressed in association with the class I molecule. The MHC class I/antigen complex is recognized via the TcR by CD8+ T lymphocytes, which become activated and differentiate into CTLs that destroy infected cells.
Variable domains of MHC class II molecules encoded by some allelic genes may be unable to bind a given antigenic peptide and thus fail to present the peptide to antigen-specific T cells. As a result, an immune response to this antigen cannot be mounted. Because of the association of high and low responses to a specific antigen with particular MHC class II alleles, MHC genes have been termed immune response genes. HLA typing reveals that individuals carrying certain alleles are at a higher risk of developing diseases such as ankylosing spondylitis, myasthenia gravis or type I diabetes mellitus. It is likely that this association reflects an underlying immunopathologic reaction involving MHC class I/II molecules or an association with other genes in the MHC.
MHC products control the selection of the immune repertoire of T lymphocytes. T cells interact with MHC class I/II molecules during their maturation in the thymus. This interaction kills immature cells whose TcR have a high affinity for self-MHC or for an MHC/self-protein complex by a mechanism called apoptosis or programmed cell death. Potentially autoreactive T cells would be eliminated in this fashion (negative selection). Furthermore, the selection process by MHC molecules determines the T cell repertoire of the individual against various foreign antigens (positive selection). This negative and positive sorting of T cells is called thymic selection.
T Cell Activation: Although, the presentation of antigen in the context of MHC molecules is essential for T cell recognition of peptide antigens, interactions between the MHC-bound peptide and TcR and the MHC class I or class II molecules, respectively, with CD8 or CD4 is not sufficient to activate T cells. Other ligands on antigen-presenting cells and their receptors on T cells are required to complete the process. These ligand-receptor interactions include the ligands ICAM-1, LFA-3, and B7-1/2 on antigen-presenting cells binding to their receptors LFA-1, CD2, and CD28/CTLA-4, respectively, on T cells (Fig. 1-19). These and other counterstructures for B7-1 and B7-2 appear to precisely control the extent of T cell activation.
Figure 1-19. Ligand-receptor interaction necessary for optimal T-cell activation. The requirements for CD8+ T cells are the same except for interactions between MHC class I molecules and CD8 molecules.
The immune system has evolved to distinguish between self and non-self antigens and to largely eliminate self-reactive lymphocytes. Because the repertoire of immune specificities is vast and largely random, it is not surprising that many nascent lymphocytes possess receptors for self-antigens. The mechanism of intrauterine tolerance is not well understood, but much has been learned about the mechanisms for excluding or inactivating self-reactive lymphocytes, particularly by using the model of experimentally induced immune tolerance to foreign antigens. When an antigen is introduced into immunologically immature newborn animals, they may, upon reaching maturity, become unresponsive to immunization with that antigen (neonatal tolerance). This immunological tolerance is characterized by the absence of both antibody and cell-mediated responses, and it is specific for the original antigen.
Subsequent experiments revealed that the induction of antigen-specific tolerance is not always restricted to immature organisms. Unresponsiveness can also be induced in adults by using relatively higher doses of soluble antigen (high dose tolerance). The induced state of unresponsiveness to the antigen is sometimes accompanied by the appearance of suppressor T cells that actively and specifically inhibit the responses of B and T cells. Recent studies also reveal that IgM+IgD- B cells and mature T lymphocytes may be directly inactivated by small doses of antigen in vitro (low dose tolerance). In that model, short exposure of lymphocytes to the antigen, either at a critical concentration or in a certain modality, leads to an inactivation rather than a stimulation of the cells.
Collectively, the experiments on tolerance induction demonstrate that the unresponsiveness to self is likely to be achieved at several levels. During normal development, the self-reactive lymphocyte clones may be inactivated or deleted by exposure to self macromolecules during the early stages of maturation in the thymus (Fig. 1-6). The autoselection is dependent upon MHC class I molecules for CD8+ T cells and class II molecules for CD4+ T cells. Those cells that are not eliminated and reach their full immunological potential may be inactivated, when self molecules are presented to these cells at high concentrations or in a form that is tolerogenic rather than immunogenic. Also, it is possible that some self-reactive lymphocytes are suppressed by other regulatory cells, such as CD8+ suppressor T cells.
The failure of any of the mechanisms involved in self recognition and elimination or down regulation of self-reactive clones may result in autoimmunity. Autoimmune disorders in genetically prone individuals may be generated by a) changes in the expression of self macromolecules or alterations in their presentation to lymphocytes, b) release of sequestered self-antigens into the circulation, or access of immunogens to normally immunologically privileged sites, and c) alterations in lymphocyte maturation and immune regulation. In addition, foreign antigens such as bacteria and viruses that cross-react with self antigens may augment or initiate any of the above mechanisms.