Adaptive Immunity: Antigens, Antibodies, and T Cell and B Cell Receptors Part 2

Affinity Maturation by HYPER Mutation

The binding properties of antibodies change with time by a process termed affinity maturation, which involves somatic hy-permutation 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 ^ and 6 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 ^ and 6 are deleted [see Figure 4]. After transcription, RNA splicing produces an mRNA with the sequence V-D-J-C y3, which is translated into protein.


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.

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 Cy3, 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 S^ and Sy3 and the deletion of the intervening DNA occur first to produce a DNA sequence in which the gene for Cy3 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).

Figure 4 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 Cy3, 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 S^ and Sy3 and the deletion of the intervening DNA occur first to produce a DNA sequence in which the gene for Cy3 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).

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 gly-cosylase 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].

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 m 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 k 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 recep-tors.8 These cell surface membrane immunoglobulins (SmIgs) differ from secreted immunoglobulins in that they have a trans-membrane 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 Iga and IgP, 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. Iga and Ig| are not expressed after terminal differentiation. The mature plasma cell ceases to express SmIgs, although it may retain the SmIg mRNA.

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 (FceRI) is the model for all Fc receptors and consists of three polypeptide chains, designated a, and y. FceRI mediates signal transduction in the mast cell when IgE binds to the receptor. FcERIa is the binding site for the Fc portion of IgE. FceRI| is a transmembrane molecule that connects FcERIa with FceRIy, the chain responsible for recruiting signal transduction molecules.9,10 FcRII|1, 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 Kohler and MUstein,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 immunoglob-ulin 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 k 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-a (TNF-a) 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.14-16 From 85% to 95% of normal peripheral blood lymphocytes carry TCR-a|; only 5% to 15% carry TCR-y6. The antigen-recognizing portion of the TCR-a| complex consists of two glycosylated polypeptide chains, termed TCR-a and TCR-| , that are linked by disulfide bonds to form a het-erodimer. The corresponding polypeptide chains in the TCR-y6 complex consist of TCR-y and TCR-6. The TCR-a, TCR-|, TCR-y, and TCR-6 chains each contain variable and constant portions that are analogous to those of immunoglobulin molecules.

TCR-a and TCR-y6 heterodimers are closely associated with the CD3 proteins CD3y, CD36, CD3e, and CD3Z [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 (a, y, and 6) 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.

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.

Figure 5 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.

(a) Cell surface membrane immunoglobulins form a complex with the proteins Iga and Igp. Iga and Igp 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, a and p, are the disulfide-bonded chains of the heterodimer TCR that binds to antigen. The four other chains—y, 8, and two e 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 Z 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.

Figure 6 (a) Cell surface membrane immunoglobulins form a complex with the proteins Iga and Igp. Iga and Igp 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, a and p, are the disulfide-bonded chains of the heterodimer TCR that binds to antigen. The four other chains—y, 8, and two e 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 Z 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.

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-a, -y, and -8 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-| chain contain two very similar constant-region genes, C|1 and C|2, each of which is associated with a cluster of six or seven J genes and a single D gene. There are at least 70 V| genes that are associated with the two C| genes. These variable regions are distinct from the immunoglobulin variable regions. Rearrangement of the chain gene segments can lead to the production of approximately 3,600 different | chains.

The TCR-a genes are arranged differently. A single Ca gene is preceded by a very large stretch of DNA containing at least 50 distinct J genes. A Da gene segment has not been demonstrated directly. Some Va genes are organized as families of related genes. Rearrangement of the a-chain gene segments can account for approximately 2,500 different polypeptides. No somatic hypermuta-tion has been detected in TCRs. Thus, 107 TCR-a|s can be formed. The genes for CD3y, CD38, CD3e, and CD3Z are transcribed in all T cells; however, these genes do not undergo rearrangement.

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