Immunoglobulin & TCR Genetics IMMUNOGLOBULIN GENETICS Lee S.F. Soderberg, Ph.D. I.

IMMUNOGLOBULIN (Ig)

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The designation of the chemical fraction comprising antibodies, Ig is found primarily in the gammaglobulin () serum electrophoresis fraction. A. Ig Classes There are 5 classes of Ig which differ slightly in their structures and functions. These are designated IgG, IgM, IgA, IgD, and IgE. . Class IgG H chain (isotype) Characteristics Monomer (150 kD). Major Ig class in serum. Only Ig class that can cross the placenta. Pentamer (900 kD, incl. J chain) in serum Monomer on B cells Monomer is a minor serum component. Dimer (with J chain and SC) is the major Ig class in secretions. Monomer on B cells Monomer adsorbs to mast cells and basophils Function Acts in tissues to inactivate toxins and viruses, promote phagocytosis, and activate complement (IgG1, IgG2, IgG3) to try to kill microorganisms. Similar to IgG, but appears earlier after antigen stimulation. Antigen receptor on B cells Immunity in the gut on other mucous membranes Antigen receptor Mediates immediate hypersensitivity (allergy)

IgM IgA IgD IgE

1. 2.

J Chain - 15-kD peptide included in IgM and IgA polymers to lend stability. Secretory component (SC) - as dimeric IgA crosses the epithelium at mucous membranes, it picks up the 70-kD SC glycoprotein to give it resistance to mucous membrane enzymes.

II. ANTIBODY STRUCTURE A. Primary structure Each of the 5 Ig classes shares a common basic unit structure. 1. 4 polypeptide chains a. 2 large chains (50 kD) called "heavy (H) chains". b. 2 small chains (25 kD) called "light (L) chains". B. Symmetry Antibodies are always symmetrical with 2 identical H chains bound together by disulfide bonds and 2 identical L chains bound by disulfide bonds, usually to the H chains.

C. Constant/Variable Regions The H and L chains are divided into constant (C) and variable (V) regions. The amino acid sequences of the C region of H and L chains are conserved with minor variations. The V regions of the H and L chains are highly variable, except when antibodies have the same antigen-binding specificity.

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Antigen-binding Site. The variable region of the H chain (V H) aligns with the variable region of the L chain (VL). Together the two V regions comprise the antigen-combining site. There are two antigen-binding sites per antibody molecule (monomer). IMPORTANT CONCEPT 1: Amino acid sequence determines antibody specificity

2.

Complementarity-determining Regions (CDR). Within each V region are 3-4 small regions of very high amino acid variability. The CDR, also called hypervariable regions, are the points of most intimate contact with antigens.

D. Hinge Region - allows flexibility between the two antigen combining sites. E. Enzyme-defined Fragments 1. Papain - cleaves antibody molecules at the hinge region into 2 fragments (Fab), which retain antigen-binding activity, and 1 fragment (Fc), which contributes the following functions to certain Ig classes: a. Binding to Fc receptors on certain cell types (macrophages, B cells, NK cells). b. Imbedded in B cell membrane (when Ig is antigen receptor). c. Site of complement (see SDL Unit #13) activation (IgM, IgG1, IgG2, IgG3 only). d. Interaction site for crossing the placenta (IgG only). 2. Pepsin - digests Fc, leaving a single fragment with 2 antigen-binding sites, (Fab') 2. (Fab')2 fragments are useful in clinical diagnostic tests, where cell binding through Fc might give false positive results. Secondary and Tertiary Structure Inter-chain disulfide bonds hold H and L chains together. Intra-chain disulfide bonds create domains; 2 in L chains (V L and CL) and 4 or 5 in H chains (V H, CH1, CH2, CH3, and, in IgM and IgE, CH4)

F.

III. ANTIGENIC DETERMINANTS ON IMMUNOGLOBULINS IMPORTANT CONCEPT 2: Unique sequences in H and L chains are useful in classifying antibodies

A. Isotype 1. Defines Ig classes. Determinants in the C H region are common to all antibodies within an Ig class (e.g. IgM) and unique to that Ig class (IgM determinants are not present on IgG, IgA, IgD or IgE). Heavy chains with these determinants are designated by the corresponding Greek letter ( for IgM). Antibodies specific for the isotypes are used clinically to measure patient serum levels of each Ig class. 2. Other H chain constant region determinants define subclasses of Ig (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) 3. L chain isotype determinants do not correspond to Ig class. There are only 2 types of L chains, and . Each antibody unit has 2 identical or 2 identical chains (never 1 of each). B. Allotype Like isotype determinants, allotype determinants are present on the constant region of H or L chains. However, allotype determinants vary from
2

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person to person in the same fashion as blood type or tissue type. Allotype determinants have no known functional significance, but are useful for studying the inheritance of immune disorders and Ig-producing tumors. C. Idiotype Idiotype determinants are formed by the unique molecular configuration of antigen binding sites. Antibodies with a particular antigenic specificity have V region configurations peculiar to that antigen. Large amounts of antibody with identical V regions will stimulate the production of anti-idiotype antibodies. Such antibodies may specifically block or enhance immune responses (see Lecture #17). IV. IMMUNOGLOBULIN GENES IMPORTANT CONCEPT 3: Relatively few genes code for many antibodies To code for the very large number of antibody structures (~10 8-109 different specificities) and conserve DNA, a complex process called gene rearrangement, briefly described below, has evolved. A. Variable Region Genes 1. VH Region Each VH region is encoded by 3 genes; a V (variable) gene (from a total of about 200 V genes), a D (diversity) gene (from about 12 genes), and a J (joining) gene (from about 4 genes). During cell differentiation, intervening DNA segments are eliminated to bring 1 V gene, 1 D gene, and 1 J gene together. Once formed, the gene grouping, VDJ, is conserved during further lymphocyte differentiation. 2. VL Region Unlinked, but similar to, VH region, except that there are no D genes; only V (about 350) and J (about 4). 3. Since VH and VL genes are independently selected and both contribute to specificity. Further variability is generated through junctional diversity (imprecise joining, particularly of the J genes), N-region additions (codons at the junction may be added or a frame-shift corrected), and, after activation by antigen, by somatic mutation (single base change, particularly in the CDR or hypervariable regions). B. Constant Region Genes 1. There are at least 2 CL gene groups, corresponding to and . 2. There are at least 9 CH genes, corresponding to the 5 major Ig classes plus subclasses of IgG and IgA (1, 2, 3, 4, , 1, 2, , ). Each CH gene is made up of a number of exons corresponding to the various domains. 3. The rearranged DNA, including the variable region gene cluster (VDJ or VJ) and the nearest constant region genes (either in the H chain or

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4.

or in the light chain) is transcribed. The RNA transcript is then spliced to remove intervening segments. Translation produces first chain and then, with L chain, IgM, which then appears on the B cell membrane. As B cells mature, RNA transcripts are rearranged, such that VDJ is associated with either the genes or the genes. Thus, both IgM and IgD are produced and expressed on the cell membrane at the same time. Following specific immune activation, isotype switching occurs. The VDJ gene cluster in the DNA rearranges to associate with a different C gene, such as 1 to produce IgG1. Because this Ig class switching occurs at the DNA level, not RNA transcripts, it is irreversible. Since the VDJ gene cluster is conserved, antigenic specificity is not changed, although it can be modified by somatic mutation.

C. Allelic Exclusion Even though each B cell has two sets of chromosomes and, hence, two sets of immunoglobulin genes, only one H chain VDJ is expressed and only one L chain VJ is expressed. This is called allelic exclusion. The net effect of allelic exclusion is that each B cell expresses one and only one antigenic specificity. V. IMMUNOGLOBULIN PROPERTIES The concentration of IgG immunoglobulin far exceeds other classes of Ig in the serum of normal individuals. Immunodeficiency diseases may decrease some or all Ig classes. Cancer involving B cells (myeloma) may increase certain Ig classes and specificities, decreasing others. IgE may be increased in allergic patients. Maternal IgG crosses the placenta, providing protection to babies. Infants can get IgA and IgG through nursing, but such antibodies largely stay in the gut. VI. T CELL RECEPTORS (TCR) T cells express specific antigen receptors (TCR) on their membranes; a single specificity per cell. The TCR are of two types, and (referring to their chain designations), that are present on distinct T cell populations. Approximately 90% of T cells express the type TCR. Cells of the type TCR tend to be concentrated in mucosal areas of the body. Like Ig, the TCR chains have variable and constant regions that are encoded by V, D, and J genes and by C genes, respectively. During T cell maturation in the thymus, the V, D, and J genes rearrange to form the chain V region and V and J genes rearrange to form the chain V region. Allelic exclusion guarantees that only one and one chain will be synthesized per cell and, hence, each cell will have only a single antigenic specificity. Unlike Ig, TCR gene rearrangements sometimes omit the D gene and, with two sets of D and J genes, the genes can undergo two successive rearrangements. Also unlike Ig, TCR do not undergo class switching, even though there are two alternative chain C genes.

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A major difference between Ig and TCR is in their specificities. While antibodies can bind a wide array of antigenic compounds and free antigens, TCR are much more restricted in their specificities. IMPORTANT CONCEPT 4: MHC molecules TCR only recognize protein antigens bound to

The specificity of TCR is restricted to foreign peptide antigens bound to MHC gene products. T cells with TCR specificities that do not recognize self-MHC or that recognize self-peptides associated with self-MHC are eliminated in the thymus during maturation. Thus, the TCR binding site includes epitopes that bind to the foreign antigen and epitopes that bind to the MHC heterodimer . The expression of the antigen-MHC complex on the cell surface, where it can be recognized by TCR, is called antigen presentation. In their native form, most antigens are too large to fit into the groove of the MHC molecules and must be cleaved to an appropriate size, a process called antigen processing.

READING: The Immune System, 3rd Ed., Ed: P. Parham, Chapter 4 and 5-1 5-5.