ACP medicine, 3rd Edition

Immunology/Allergy

Adaptive Immunity: Antigens, Antibodies, and T Cell and B Cell Receptors

Cox Terhorst Ph.D.1

John R. David M.D.2

1Professor of Medicine, Harvard Medical School, Chief, Division of Immunology, Beth Israel Deaconess Medical Center

2Richard Pearson Strong Professor, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Professor of Medicine, Harvard Medical School

The authors have no commercial relationships with manufacturers of products or providers of services discussed in this chapter.

June 2004

Antigens

An antigen is any substance capable of generating an immune response; that is, antigens react with T cells and B cells to induce the formation of antibodies and to sensitize lymphocytes, and those antibodies and sensitized cells then react with the antigens. The first antigens to be studied were microorganisms and foreign proteins, and it remains true that foreign proteins are almost universally antigenic. In addition, polysaccharides can induce antibody formation when coupled to proteins, and certain purified polysaccharides are themselves effective antigens. One such example is purified pneumococcal polysaccharide, which can be used as a vaccine against the particular strain of pneumococcus from which the polysaccharide is obtained. Although most antigens are macromolecules, some small molecules can also be antigenic. Antibodies to DNA or RNA occur in many patients with rheumatic diseases, especially systemic lupus erythematosus.

ANTIGEN RECOGNITION

Antigens are recognized not only by antibodies but also by antigen-specific B cell receptors (BCRs) [see B cell receptors, below] and T cell receptors (TCRs) [see T Cell Receptors, below], which are located on the surfaces of B cells and T cells, respectively. In general, TCRs and antibodies recognize different antigenic determinants.

Two distinct types of TCRs exist: TCR-αβ and TCR-γδ. Each has different mechanisms for recognizing antigens [see 6:IV Cell-Cell Interactions, Cytokines, and Chemokines in Immune Response Mechanisms]. For example, T cells bearing TCR-αβ recognize antigens that have been processed by antigen-presenting cells (APCs) to become peptide fragments bound to major histocompatibility complex (MHC) class I or class II molecules on the surface of the APCs. In contrast, T cells bearing TCR-γδ appear not to require antigen presentation by MHC molecules. Helper T cells recognize only peptide fragments bound to MHC class II molecules, whereas cytotoxic T cells recognize processed viral antigens presented by both MHC class I and MHC class II molecules on the surface of virus-infected cells. Pure lipids from mycobacteria can be presented as antigen to TCR-αβ by CD1 molecules rather than by MHC class I or class II molecules.

ADJUVANTS

Adjuvants are substances capable of increasing the immunogenicity of antigens and are critical in the production of vaccines. Many microbial products have been used as adjuvants,1 including substances from Mycobacterium tuberculosis, bacillus Calmette-Guérin (BCG),Corynebacterium parvum, Brucella abortus, and Bordetella pertussis, as well as toxoids from Vibrio cholerae and Clostridium tetani. Adjuvants have also been derived from vaccinia virus and other poxviruses, BCG, and Salmonella by transfecting the organism with genes for an antigen of interest. Others have come from lipopolysaccharide derivatives, such as mono phosphoryl lipid A. Freund complete adjuvant, which consists of dead mycobacteria in oil emulsified with an antigen in aqueous solution, is now infrequently used because it generally causes a strong local inflammatory reaction. Other adjuvants are extracts from the soap bark tree (Quillaja saponaria), polymers (e.g., inulin), peptide complexes, and a number of cytokines. The aluminum salt alum is approved for general use as an adjuvant in humans. The complex of antigen and complement fragments, particularly those derived from C3, probably serves as the physiologic adjuvant.

Antibodies

Antibodies are a heterogeneous group of serum proteins called immunoglobulins. Immunoglobulins are secreted by differentiated B cells called plasma cells. According to Sir Francis MacFarlane Burnett's clonal selection theory, a single plasma cell produces only one specific antibody. This theory is aptly illustrated by the disease multiple myeloma, in which malignant proliferation of plasma cells occurs in bone marrow. The multiple myeloma plasma cell produces an abnormal monoclonal immunoglobulin called a myeloma protein. Its homogeneity is clearly visible on electrophoresis as a single dense band. Certain antibodies that are produced in response to highly homogeneous antigens, such as streptococcal polysaccharide, may also be relatively homogeneous.

Immunoglobulin molecules consist of two identical heavy chains and two identical light chains [see Figure 1a]. Each light chain is attached to a heavy chain by disulfide (S—S) bonds, and the heavy chains are also attached to each other by one or more S—S bridges. Amino acid sequences in both heavy and light chains are divided into regions that are either constant or variable [see Figure 1b]; in addition, each variable region contains sequences that are hypervariable.

 

Figure 1. Structure of Immunoglobulin Molecule

(a) The immunoglobulin molecule is a Y-shaped protein made up of four polypeptide chains. Two heavy chains (blue) are joined to two light chains (green) by disulfide bonds. Blue squares represent intrachain S—S bonds; blue bars indicate interchain S—S bonds. The heavy chains extend from the stem of the Y into the arm; the two light chains are confined to the arms. Each polypeptide has regions whose amino acid sequences are constant (white and yellow) and variable (red). The variable regions also contain hypervariable regions. All antibodies of a given type have the same constant regions, but the variable regions differ from one clone of a B cell to another. The heavy- and light-chain variable regions fold to create an antigen-binding site. (b) Schematic model of the domain structure of an antibody molecule. The domains have a characteristic folding pattern, which is also seen in the T cell receptor and proteins of the major histocompatibility complex.17

CLASSIFICATION OF IMMUNOGLOBULINS

There are five classes of immunoglobulins, and each class contains a specific heavy chain: IgG contains two γ chains; IgM, two µ chains; IgA, two α chains; IgD, two δ chains; and IgE, two ε chains. There are also two types of light chains, κ and δ, which can be differentiated antigenically. One form of IgM, the secreted form, is a pentamer. The monomeric form of IgM is expressed on the extracellular surface of B cells. IgA exists as a monomer or a dimer. The polymeric forms of IgM and IgA have an additional J (joining) chain that facilitates polymerization.

IgG

IgG is the major immunoglobulin in the serum, where it exists as a monomer. IgG has a half-life in the blood of approximately 23 days. It is the main antibody raised after antigenic challenge. There are four subclasses of IgG—IgG1, IgG2, IgG3, and IgG4—each different in structure and biologic properties. For example, only IgG1 and IgG3 bind the first component of complement and adhere to monocytes. The antibodies that coat microorganisms and render them more susceptible to phagocytosis (i.e., opsonization) are of the IgG class. IgG antibodies can also neutralize viruses and toxins such as diphtheria toxin. Human antibodies to polysaccharides are mainly of the IgG class, but lesser amounts of IgM and IgA are also produced. Although the fetus does not produce this class of immunoglobulin, IgG readily crosses the placenta; therefore, IgG antibodies found in the newborn are from the mother.

Clinically, IgG has been used successfully for reconstituting the immunity of patients with primary immune deficiencies, such as agammaglobulinemia, and for preventing hemolytic disease in the newborn. Women with Rh-negative blood who bear a fetus with Rh1-positive red blood cells are sensitized at the first delivery by Rh1-positive red blood cells from the fetus. The mother then produces anti-Rh1 IgG antibodies that will cross the placenta during subsequent pregnancies; these antibodies react with fetal red blood cells, causing hemolytic disease. Erythroblastosis fetalis can be prevented by injecting IgG rich in anti-Rh-positive antibodies (RhoGAM) into an Rh1-negative mother at the time of delivery or abortion. These antibodies presumably combine with any fetal Rh1-positive red blood cells present and prevent them from immunizing the mother.

IgG can be split into three fragments by the proteolytic enzyme papain. Two of the fragments are similar and are called Fab; the third is called Fc [see Figure 1a]. The Fc portion is responsible for the biologic activity of the various immunoglobulins; among other things, the Fc portion controls the ability of immunoglobulins to bind to cells, fix complement, and traverse the placenta. Another proteolytic enzyme, pepsin, splits the IgG molecule behind the S—S bonds that bridge the heavy chains, leaving one large fragment, F(ab′)2, which is able to bind and precipitate antigen because of its bivalency and capacity to form a lattice.

IgA

IgA is the predominant immunoglobulin in secretions, where it is usually found as a dimer and is released as such by local plasma cells. Monomeric IgA constitutes 15% of the serum immunoglobulins. In the serum, it has a half-life of 5 to 6 days. There are two subclasses of IgA: IgA1 and IgA2. The IgA dimer combines with the secretory piece, which is a polypeptide chain produced by local epithelial cells. In this form, IgA is quite resistant to proteolytic digestion. Unlike serum IgA, IgA combined with the secretory piece can undergo active transport across the mucosal epithelium by endocytosis [see Figure 2].

 

Figure 2. IgA Secretion

Plasma cells secrete IgA molecules into the general circulation as either monomers or dimers. The circulating dimer can combine with a mucosal transport receptor on the surface of an epithelial cell. As the antibody-receptor complex is transported through the cell, the receptor is cleaved. The portion of the receptor that remains attached to the antibody dimer is called the secretory piece. The secretory piece is joined to the constant region of IgA by disulfide bonds. The mucosal transport receptor contains five immunoglobulin-like domains and is anchored in the membrane by a proteolytically labile segment.18

IgA is present in saliva, tears, and colostrum. It also occurs in the respiratory and gastrointestinal tracts, in the vagina, and in the prostate. The increased levels of antibodies to dietary antigens that are found in persons with IgA deficiency suggest that the IgA class of immunoglobulin normally limits the absorption of such antigens.

IgA may play an important role in local immunity by neutralizing viruses and by combining with viruses and bacteria, thereby preventing their adherence to mucosal surfaces. Although IgA does not bind to the first component of complement, as IgG and IgM do, it can lead to activation of the alternative complement pathway [see 6:II Innate Immunity]. One of the complement components generated by this pathway, C3b, aids in the opsonization of bacteria, enhancing their uptake and killing by phagocytes.

IgM

Most B cells have monomeric IgM on their surface. However, IgM exists primarily as a pentamer and is found mainly in the serum, where it makes up 10% of the immunoglobulins. In the immune response, IgM is the first immunoglobulin raised in response to antigen stimulation. Cells that produce IgM or their precursors do not become memory cells, so that a second challenge with antigen produces no more IgM antibody than the first stimulus. Because the IgM response to antigen is short-lived, the presence of specific IgM in the serum may be helpful in establishing the diagnosis of acute infection with a particular pathogen. The fetus makes IgM antibodies to certain microorganisms, which can be helpful in the diagnosis of fetal toxoplasmosis, rubella, or syphilis. Not all fetuses infected by these organisms produce such antibodies, however.

As a pentamer, IgM is highly efficient at fixing complement. Molecule for molecule, IgM is 20 times as effective as IgG in agglutinating bacteria and red blood cells and 1,000 times as active in bactericidal reactions. Isohemagglutinins, such as anti-A and anti-B, are of the IgM class. Waldenström macroglobulinemia is a disease characterized by the monoclonal production of IgM.

IgD

IgD, which is a monomer, occurs in the serum in trace amounts. It is found in relatively high concentrations in umbilical cord blood. Most of the B cells of umbilical cord blood have IgD on their surface, and most B cells in adults have both IgM and IgD on their surface. Plasma cells that produce IgD have been found in the tonsils and adenoids, although they are very rare in other lymphoid tissues. The function of IgD is unknown.

IgE

IgE is present in trace amounts in the serum, constituting only 0.004% of the immunoglobulins. Plasma cells that produce IgE are found in the tonsils and adenoids and on the mucosa of the respiratory and GI tracts.

Distinct receptors for IgE are found on the surface of mast cells, B cells, T cells, macrophages, and eosinophils. IgE binds to its receptor on these cells by its Fc portion; heating the antibody destroys its cell-binding ability. Formerly referred to as reagin, IgE plays a primary role in immediate hypersensitivity—namely, the immune reaction in hay fever, extrinsic asthma, wheal-and-flare reactions, and anaphylaxis. IgE binds tightly to mast cells and basophils. When these IgE-coated cells interact with specific antigens, termed allergens, they release potent mediators of immediate hypersensitivity, including histamine, slow-reacting substance of anaphylaxis (SRS-A), and an eosinophilic chemotactic factor [see 6:X Allergic Response]. Levels of IgE are higher than normal in persons with atopic dermatitis, as are the levels of IgE antibody specific for allergens to which the individual is susceptible. In patients with allergies, specific IgE antibodies are detected by means of a radioimmunoassay called the radioallergosorbent test (RAST).

The exact function of IgE is unknown. Certainly, the manifestations of immediate hypersensitivity, such as hay fever and extrinsic asthma, do not appear to serve any useful purpose for the person affected or for the species in general. Therefore, the observation that IgE levels are sometimes elevated in persons living in the tropics, and especially in those afflicted with helminthic parasites, was greeted by immunologists as a possible indication that IgE plays a protective role against parasites. The mediators released could affect the parasite either directly or by producing an increase in vascular permeability and the release of eosinophilic chemotactic factor, which may lead to the accumulation of other necessary antibodies (e.g., IgG) and cells to attack the parasite. In this context, it is of interest that eosinophils can mediate IgG-dependent damage to schistosomula (the larval form of the parasite Schistosoma mansoni). In addition, parasite-specific IgE immune complexes can induce a macrophage-mediated cytotoxic response to schistosomulum organisms.

Antigenic Differences of Immunoglobulins

Immunoglobulins have three types of serologic, or antigenic, determinants: isotypic, allotypic, and idiotypic.

Isotypic determinants

Isotypic determinants distinguish between the constant regions of the various classes and subclasses of heavy chains and light chains; they represent the different constant-region genes. For example, there are four IgG heavy-chain isotypes: γ1, γ2, γ3, and γ4, representing the subclasses IgG1, IgG2, IgG3, and IgG4, respectively. There is only one κ light-chain isotype and one λ light-chain isotype.

Allotypic determinants

Allotypic determinants distinguish between immunoglobulins of a particular isotype; they represent different alleles of immunoglobulin genes and therefore are genetically determined according to mendelian laws in a manner similar to the way that the ABO blood groups are determined. The γ heavy chains have more than 20 different allotypic markers, which are collectively termed Gm. In addition, κ light chains contain a set of at least three allotypic markers, collectively called Km. There are no known allotypic markers for the µ, δ, and ε heavy chains or for the λ light chain.

Idiotypic determinants

An idiotope is defined as a single antigenic determinant on the hypervariable region of an antibody. An idiotype is the antigenic character of the variable region of an antibody. Idiotypic determinants distinguish one immunoglobulin from another of the same allotype.

Genetic Source of Antibody Diversity

The carboxyl-terminal halves of all κ light chains have almost identical amino acid sequences; this portion of the molecule is therefore called the constant, or C, domain. The amino-terminal half has a variable sequence of amino acids and is known as the variable, or V, domain [seeFigure 1] The first 110 amino acids of the amino-terminal portion of the λ light chain and the heavy chain are also variable. The remaining 75% of the heavy chain is constant and contains three homologous regions.

Within the variable regions, three areas—referred to as the hypervariable, or complementarity-determining, regions—show great variation; these areas correspond to the antigen-binding site of the antibody. X-ray analysis has shown that immunoglobulin molecules are built up from compact globular units connected by short segments of more or less linear polypeptide chains [see Figure 1]. As expected, the hypervariable regions are located at the interface between immunoglobulin and antigen.

The most intriguing aspect of the genetic control of immunoglobulin synthesis is the diversity of the product: plasma cells can make antibodies that react with an indefinite number of different antigenic sites. How can DNA code for such a large number of antigens, many of which have only recently (on the evolutionary time scale) come into existence?

VDJ RECOMBINATION

In all cells, DNA for the κ light chain codes for more than 300 variable (V) regions, five joining (J) regions, and one constant (C) region. The V and J regions are separated from the C region by an intervening stretch of DNA. Thus, in the so-called germline configuration, DNA encodes the information for at least 1,500 different combinations of V and J regions; in other words, at least 1,500 different κ light chains are possible.

The emergence of individual plasma cell lines is the result of somatic recombination in the DNA and RNA splicing [see Figure 3]. As the pre-B cell differentiates into a plasma cell, rearrangements and deletions in the DNA bring one of the V genes, chosen at random, adjacent to one of the J genes. This V-J unit and the remaining J regions are separated from the C gene by a short length of DNA. In the next step, the DNA is transcribed to nuclear RNA, and the stage is set for a second event.

 

Figure 3. Variable-region Genes

Variable-region genes are constructed from gene segments. Light-chain variable genes are constructed from two segments (center panel). A variable (V) and a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain variable-gene-region gene. The constant region is encoded in a separate exon and is joined to the variable-region gene by RNA splicing of the light-chain message to remove the L to V and the J to C introns. Immunoglobulin chains are extracellular proteins, and the V gene segment is preceded by an exon encoding a leader peptide (L), which directs the protein into the cell's secretory pathways and is then cleaved. Heavy-chain variable regions are constructed from three gene segments (right panel). First the diversity (D) and J gene segments join, then the V gene segment joins to the combined DJ sequence, all at the genomic DNA level. The heavy-chain constant-region sequences are encoded in several exons: note the separate exon encoding the hinge domain (purple). The constant-region exons together with the L sequence are spliced to the variable-domain sequence during processing of the heavy-chain gene RNA transcript. Posttranslational alterations remove the L sequence and attach carbohydrate moieties.19

This event begins when an enzyme cleaves the nuclear RNA to produce messenger RNA (mRNA). In a process called RNA splicing, the segment that separates the V-J unit from the C region is removed, along with any superfluous J segments. The remaining V-J-C segment is now translated into one of the 1,500 κ light-chain proteins. Actually, the number of possible proteins is higher because the joining of any V region to a J region can involve one of a variety of base pairs at the recombination site.

Variability in the heavy chain makes an important contribution to the specificity of an antibody and also results from somatic recombination and RNA splicing. The germline configuration of the DNA carries instructions for several hundred different heavy-chain V genes, six J genes, 10 to 20 diversity (D) genes, and nine C genes (these C genes code for the heavy-chain classes: IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgE). DNA deletion, transcription to nuclear RNA, and RNA splicing produce the final V-D-J-C sequence in the mRNA that is translated by ribosomes to a heavy-chain protein. This assembly process produces more than 18,000 possible varieties of heavy-chain proteins (antibody specificity does not vary with class, so the nine C genes do not enter into the calculation).

The combination of more than 1,500 light-chain varieties with the 18,000 heavy-chain varieties yields more than 27 million different kinds of antibodies with different antigen-binding sites. In addition, somatic hypermutation occurs, particularly during affinity maturation, and the rate of somatic hypermutation is relatively high (one base pair per 1,000 cell divisions). Therefore, the potential number of specific antibodies in a single person is indefinite.

Although some of the mechanisms of V-D-J rearrangement are unique to B cells (and T cells, as synthesis of TCRs occurs by a similar mechanism [see T Cell Receptors, below]), the general mechanisms of DNA repair are also engaged.2 Two genes involved in V-D-J rearrangement in B cells are the recombination-activating genes rag1 and rag2.3,4,5 Disruption of the rag1 or rag2 gene causes a block in B cell development before the transition from pro-B cell to pre-B cell, as found in patients with severe combined immunodeficiency syndrome or Omenn syndrome.6 Disruption of the surface IgM gene, the J region of the heavy chain, or the J region of the κ light chain leads to a similar block in B cell development.

ALLELIC EXCLUSION

In an individual B cell, only one of the chromosomes undergoes complete V-D-J recombination leading to expression of heavy and light chains. A mechanism exists that prevents the other chromosome from being rearranged and therefore expressed in the same cell. This is called allelic exclusion. It prevents a B cell from expressing two entirely different immunoglobulins or BCRs. A similar mechanism operates during synthesis of TCRs in T cells [see T Cell Receptors, below].

Primary and Secondary Antibody Responses

When antigen is first introduced into the body, a primary response occurs that is characterized by a lag phase that lasts several days, during which no antibody is detected. Increasing amounts of IgM antibody appear in the serum, usually reaching a peak level after 7 days. After 6 to 7 days, IgG antibody is also detected. The IgM titer begins to wane before the maximal IgG titer is reached, about 10 to 14 days after the antigen is introduced. Antibody titers then decrease, and very little antibody is detected 4 to 5 weeks after a single dose of antigen.

If the antigen is encountered a second time, a secondary response (also called an anamnestic or booster response) occurs because of the existence of memory B cells. Both IgM and IgG titers rise exponentially, without the lag phase seen in the primary response. Whereas the peak IgM level during the secondary response may be the same as, or slightly higher than, the peak IgM level during the primary response, the IgG peak level during the secondary response is much greater and lasts longer than the peak level during the primary response. This variation in response is an apt illustration of immunologic memory and is caused by a proliferation of antigen-specific B cells and helper T cells during the primary response. The characteristics of the primary and secondary responses explain the need for booster injections in immunization programs.

AFFINITY MATURATION BY HYPERMUTATION

The binding properties of antibodies change with time by a process termed affinity maturation, which involves somatic hypermutation and selection. After the first stimulation, the antibodies have progressively greater affinity for the antigen as antigen exposure progresses, and increasingly stable antigen-antibody complexes are formed. In addition, the antibody becomes less specific, and cross-reactions with related antigens increase. The lessening specificity reflects the fact that cross-reactions were previously too weak to detect; they become apparent as antibody develops greater affinity for antigen.

IMMUNOGLOBULIN CLASS SWITCH RECOMBINATION

The genes that code for the IgM and IgD heavy chains (the µ and δ genes, respectively) play a critical role in the primary immune response. Whereas IgM antibodies are unable to act in many tissues of the human body, IgG, IgA, and IgE serve functions in the peripheral immune system. Class switching means that the same variable region can be transferred from the heavy chain of IgM to one of the other antibody heavy chains. In the switch from IgM to IgG production that constitutes the booster response, constant-region genes are deleted before the DNA is transcribed to RNA.6,7 If the cell switches to production of IgG3, for example, the genes for µ and δ are deleted [see Figure 4]. After transcription, RNA splicing produces an mRNA with the sequence V-D-J-Cγ3, which is translated into protein.

 

Figure 4. Booster Response

In the booster response, a plasma cell switches from IgM production to IgG production, a process called class switching. In this hypothetical model of heavy-chain class switching to Cγ3, the heavy-chain C-region exon clusters (yellow) and the switch regions (green) are indicated (a). The switch region is a stretch of DNA that directs the deletion events. In this model, recombination of the switch regions Sµ and Sγ3 and the deletion of the intervening DNA occur first to produce a DNA sequence in which the gene for Cγ3 has been brought into close proximity to the V-D-J segment (b). Further processing and transcription of this DNA yields the messenger RNA (mRNA) encoding IgG3 (c).

MECHANISMS INVOLVED IN SOMATIC HYPERMUTATION AND CLASS SWITCH RECOMBINATION

An exciting recent breakthrough explains the mechanism underlying both somatic hypermutation (SHM) and class switch recombination (CSR). This was the discovery of activation-induced cytosine deaminase (AID), an enzyme necessary for both processes. SHM and CSR occur only in the B cells in germinal centers of lymph nodes that have been stimulated by antigen. Further, AID is expressed only in antigen-stimulated B cells.

AID converts a cytosine to uracil in the variable region of the antibody gene. The cell regards this as an error, because uracil does not belong in DNA, and the correction process can introduce a variety of mutations. If a glycosylase removes the uracil, then during the subsequent replication or repair process, low-fidelity or error-prone DNA polymerase may fill in the gap with a different base or may fill it in and extend it by strand displacement. If a glycosylase does not remove the uracil, then in the subsequent replication process, a high-fidelity DNA polymerase may recognize the uracil as thymine and pair it with an adenine [see Figure 5].

 

Figure 5. Antibody Diversity

Antibody diversity is promoted by the enzyme activation-induced cytosine deaminase (AID), which generates mutations by converting cytosine (C) to uracil (U) in the variable region of the antibody gene. During replication, high-fidelity DNA polymerase will recognize the uracil as a thymine (T) and pair it with an adenine (A). Alternatively, the uracil may be removed by a glycosylase. Subsequently, mutations may occur during replication, as a low-fidelity DNA polymerase fills the gap more or less at random, or during repair, as an error-prone DNA polymerase preferentially fills the gap with thymine or as a low-fidelity DNA polymerase fills in the gap and extends it by synthesizing bases by strand displacement, which mostly occurs opposite adenine and thymine.20

Enhancers in the intron DNA also play a role in determining the location of the hypermutation. The AID enzyme plays a similar role in CSR, determining which heavy chain the VDJ will be switched to from the µ chain. The processes are not exactly parallel, however. For instance, SHM occurs in the G2 phase of the cell cycle and involves homologous recombination and a µ intronic enhancer element, whereas CSR occurs in the G1 phase and involves nonhomologous end joining and a 3′ immunoglobulin heavy (IgH) gene enhancer element. Thus, the same unique mechanism is employed in the development of two quite different properties of antibodies, with their high specificity and their function depending on SHM and CSR, respectively.

Immunoglobulin Receptors

B CELL RECEPTORS

In addition to being secreted, immunoglobulins are also expressed on the surface of B cells, where they act as antigen receptors.8 These cell surface membrane immunoglobulins (SmIgs) differ from secreted immunoglobulins in that they have a transmembrane domain and are monomeric. The first SmIg that a B cell expresses is IgM; at a later stage of B cell development, IgD is coexpressed. SmIgs do not travel to the cell surface by themselves; the process requires formation of a complex consisting of the immunoglobulin and two polypeptide chains called Igα and Igβ, which takes place in the endoplasmic reticulum [see Figure 6a]. Binding of the resultant BCR with antigen drives the B cell to maturation. Stimulation by the helper T cell activates the B cell, causing it to differentiate into a plasma or memory cell that produces secretory antibody specific for the antigen encountered. Igα and Igβ are not expressed after terminal differentiation. The mature plasma cell ceases to express SmIgs, although it may retain the SmIg mRNA.

 

Figure 6. Cell Surface Membrane Immunoglobulins

(a) Cell surface membrane immunoglobulins form a complex with the proteins Igα and Igβ. Igα and Igβ are linked by disulfide bonds, but the exact stoichiometry is unknown. The exact ratio of these two proteins to each immunoglobulin molecule is also unknown. (b) The TCR-CD3 complex is shown. A T cell receptor for antigens is composed of six distinct polypeptide chains. Two of the chains, α and β, are the disulfide-bonded chains of the heterodimer TCR that binds to antigen. The four other chains—γ, δ, and two ε chains—are collectively called CD3. CD3 associates with TCR and transports it to the T cell surface. When antigen binds to the TCR, CD3, along with a homodimer of ζ chains, sends a signal to the nucleus, via intracellular signaling pathways. Specific genes are then transcribed, and cytokines, chemokines, and other immunodulatory molecules are produced that mediate the antigen-specific immune response.

BCRs play important roles in regulation of the immune response. B cell responses to antigen can become anergic, thus providing a control mechanism for B cell responses and antibody production. A precursor of the BCR expressed on the surface of pre-B cells is thought to control allelic exclusion. In addition, BCRs interplay with Fc receptors.

FC RECEPTORS

Fc receptors bind the Fc portion of an immunoglobulin; they are expressed on a multitude of cells, including mast cells, macrophages, eosinophils, and tumor cells. Fc receptors are composed of a family of molecules. The Fc receptor for IgE (FcεRI) is the model for all Fc receptors and consists of three polypeptide chains, designated α, β, and γ. FcεRI mediates signal transduction in the mast cell when IgE binds to the receptor. FcεRIα is the binding site for the Fc portion of IgE. FcεRIβ is a transmembrane molecule that connects FcεRIα with FcεRIγ, the chain responsible for recruiting signal transduction molecules.9,10 FcRIIβ1, another Fc receptor that is expressed on B cells, provides a negative feedback signal to the BCR, which leads to the termination of humoral immune responses.11

Engineered Antibodies

LYMPHOCYTE HYBRIDOMA

The development of the lymphocyte hybridoma, a product of cell fusion, has had a revolutionary impact on immunology and clinical medicine. B lymphocyte hybridomas are the means by which extraordinarily high titers of very specific, pure antibodies can be produced for experimental and clinical purposes. The B lymphocyte hybridoma, as developed by Köhler and Milstein,12 is the product of the fusion of a mouse myeloma cell and a lymphocyte from the spleen of a mouse immunized with a specific antigen. The hybrids can be cloned and selected for specific antibody production.

HUMAN MONOCLONAL ANTIBODIES

Several methods of generating human monoclonal antibodies exist. One method entails taking the complementary DNA coding for a mouse monoclonal antibody and systematically replacing the mouse sequences with human sequences. Another method of humanizing mouse monoclonal antibodies entails making a transgenic mouse containing large segments of human DNA, including several variable regions and all the human constant regions.13 Because this mouse still has its own immunoglobulin regions, it is bred with a mouse in which there has been a targeted disruption of the mouse J region of the immunoglobulin heavy-chain and κ light-chain genes. The progeny of this breeding can then be injected with any antigen, and they will produce humanized monoclonal antibodies to it. Because the transgenic mouse contains a limited number of human variable regions, the potency of these antibodies relies on the natural somatic mutation and affinity maturation that occurs in the mouse. A third method of generating human monoclonal antibodies entails constructing expression libraries of human variable regions either in bacteria or in bacteriophages. In theory, all the variable regions can be cloned. The antibody can be expressed on the surface of the bacterium or bacteriophage and selected by affinity to the antigen of interest.

Monoclonal antibodies are being used in many therapies. For instance, humanized monoclonal antibodies to tumor necrosis factor-α (TNF-α) have been used successfully in the treatment of Crohn disease and rheumatoid arthritis. In other applications, monoclonal antibodies are used to remove T cells and tumor cells before bone marrow transplantation and during acute transplant rejection. Other potential uses of monoclonal antibodies are the production of anti-IgE antibodies and anti-hormone receptors to prevent allergy and modulate endocrine abnormalities, respectively. A monoclonal anti-IgE, omalizumab, was recently approved by the FDA for treatment of allergic asthma.

T Cell Receptors

Unlike the immunoglobulin receptors on B cells, which recognize free antigens, TCRs recognize antigens only in conjunction with autologous MHC antigens, which are expressed on the surface of professional APCs (e.g., dendritic cells, macrophages, and B cells). The CD4+ helper T cells (as well as the few CD4+ cytotoxic T cells) require MHC class II molecules, and the CD8+ cytotoxic T cells require MHC class I molecules [see 6:V Adaptive Immunity: Histocompatibility Antigens and Immune Response Genes]. This phenomenon is referred to as MHC restriction. The ability of T cells to recognize these self-MHC molecules is determined during development in the thymus before the lymphocytes are exposed to antigen [see 6:I Organs and Cells of the Immune System].

The molecules that make up the TCR have been identified, and the genes that encode these molecules have been isolated and cloned. The TCR is composed of six distinct polypeptides, known as the TCR-CD3 complex.14,15,16 From 85% to 95% of normal peripheral blood lymphocytes carry TCR-αβ; only 5% to 15% carry TCR-γδ. The antigen-recognizing portion of the TCR-αβ complex consists of two glycosylated polypeptide chains, termed TCR-α and TCR-β, that are linked by disulfide bonds to form a heterodimer. The corresponding polypeptide chains in the TCR-γδ complex consist of TCR-γ and TCR-δ. The TCR-α, TCR-β, TCR-γ, and TCR-δ chains each contain variable and constant portions that are analogous to those of immunoglobulin molecules.

TCR-αβ and TCR-γδ heterodimers are closely associated with the CD3 proteins CD3γ, CD3δ, CD3ε, and CD3ζ [see Figure 6b]. The CD3 proteins are present on all peripheral blood T cells and on 90% of thymocytes. Expressions of the CD3 and the TCR complexes on the cell surface are mutually dependent: neither complex is observed on the surface of the T cell without the other. Structural and functional data suggest that the activities of the TCR are distributed among the subunits of the TCR-CD3 complex: the TCR polypeptides (α, β, γ, and δ) bind to antigen and MHC gene products, and the CD3 proteins transduce the binding signal to the cytoplasm of the T cell, which results in activation of T cell functions.

By the use of mice containing spontaneous and engineered mutations of various genes of TCRs, the development pathway of the T cell has been confirmed. The organization of the genes that encode the human TCR-α, -β, -γ, and -δ chains is analogous to that of the immunoglobulin heavy-chain genes: there are V, D, and J segments, which are flanked by recognition sequences that mediate site-specific recombination [see Antibodies, above]. Thus, the diversity of TCRs is generated by many of the same mechanisms that are used by B cells for the production of immunoglobulins. T cells and B cells may in fact use the same recombination enzyme, or recombinase.

The genomic sequences that encode the TCR-β chain contain two very similar constant-region genes, Cβ1 and Cβ2, each of which is associated with a cluster of six or seven J genes and a single D gene. There are at least 70 Vβ genes that are associated with the two Cβ genes. These variable regions are distinct from the immunoglobulin variable regions. Rearrangement of the β-chain gene segments can lead to the production of approximately 3,600 different β chains.

The TCR-α genes are arranged differently. A single Cα gene is preceded by a very large stretch of DNA containing at least 50 distinct J genes. A Dα gene segment has not been demonstrated directly. Some Vα genes are organized as families of related genes. Rearrangement of the α-chain gene segments can account for approximately 2,500 different polypeptides. No somatic hypermutation has been detected in TCRs. Thus, 107 TCR-αβs can be formed. The genes for CD3γ, CD3δ, CD3ε, and CD3ζ are transcribed in all T cells; however, these genes do not undergo rearrangement.

Acknowledgments

Figures 1 through 6 Seward Hung.

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Editors: Dale, David C.; Federman, Daniel D.