Exposure of the immune system to certain antigens results in the production of antibody reactive with antigen. Later in this chapter the interaction of T and B cells in the regulation of antibody production is discussed. After the administration of an antigen, there is a relatively abundant production of IgM antibody, which continues and then falls off, while the production of IgG increases beginning shortly after that of IgM and does not fall as rapidly after peaking (Figure 19.4). Administration of a second dose of the antigen some 4 weeks later results in a relatively rapid increase in the level of IgG with some IgA and IgE but with much less increase in IgM. This is continued even after a third injection. The IgG that is produced following the second and third injections usually has a much greater affinity for the antigen, while that of IgM increases only slightly (Janeway and Travers, 1996).
The production of antibody is also regulated in a feedback manner. Administration of spe- cific antibody to an animal immunized against the antigen specific to the antibody results in an inhibition of the formation of the specific antibody. This result has been clinically useful in the prevention of Rh incompatibility in pregnant women after the initial sensitizing pregnancy
Figure 19.4 Synthesis of antibody types during the primary and secondary and later responses to the administration of an antigen at the periods shown by the numerical boxes and arrows on the X axis. (Adapted from Janeway and Travers, 1996, with permission of the authors and publisher.)
(Clarke, 1973). While the suppression by IgG molecules is antigen-specific, such antibodies binding to one epitope of a complex antigen may suppress the antibody response to all epitopes of this antigen. An epitope is the simplest antigenic determinant specifically reactive with an antibody molecule, as depicted in Figure 19.5. Although the exact mechanism of this regulation is not understood, it appears that intact Fc regions of the molecules are required for the effect (cf.Heyman, 1990).
Another as yet largely theoretical but potentially very important mechanism of the regula- tion of antibody formation is the proposed network theory of the immune system in which idio- typic determinants are primarily involved in the recognition and regulation of other immune response products and mechanisms (Jerne, 1974). An idiotype, as noted in Figure 19.5, consists of epitopes found in or near the antigen-combining sites of immunoglobulins and, as discussed below, in the T cell receptor. Since the variable regions possess an extremely wide variety of idiotopes (see below), the opportunity for the immune system to produce antibodies to such id- iotopes is always present. While as yet there is not substantial evidence that anti-idiotype anti- bodies play a major role in the regulation of antibody formation or the immune system in general, there is evidence that anti-idiotypic antibodies are formed within the organism and pos- sibly play a significant role in regulating the formation of autoantibodies, i.e., antibodies to the proteins of the organism or “self” antigens (Dwyer, 1992; Rodey, 1992).
A variety of other factors also regulate antibody response—including drugs, hormones, and dietary factors—but a detailed discussion of these topics is beyond the scope of this text.
B Lymphocytes and the Antibody Response
For the production of circulating antibodies, B cells must mature into secretory plasma cells. However, the immune response is highly specific, and thus only certain B cells will respond to certain antigens. For many years it had been argued that such a response was the result of a
Figure 19.5 Terminology of the immunoglobulin-antigen interaction. A paratope is the antigen-combin- ing site formed by the combined variable region segments of the heavy and light chains of immunoglobu- lins or the appropriate chains of the T-cell receptor. Idiotopes are epitope conformations in or around the antigen combining site, the paratope. The region labeled as paratope may also serve as an idiotope. (Repro- duced from Rodey, 1992, with permission of the author and publisher.)
direct specific interaction of the antigen with an immunoglobulin-like molecule on the surface of the B cell, such an antibody being specific for the epitope of the antigen. During the last decade, the general structure of such a receptor on B cells has been described, as shown in Figure 19.6. In this figure, the antigen receptor complex is composed of two heavy chains that are transmem- brane and two light chains that are extracellular. Associated in the membrane with this immuno- globulin are two other components termed Ig-α and Ig-β. These molecules are known to bind several different protein-tyrosine kinases which in turn are capable of activating signal transduc- tion pathways, which ultimately result in an increase in replication and differentiation of the B cell interacting with the specific antigen (DeFranco, 1992; Justement, 1994).
In the uncommitted B cell that has developed from the stem cells of the bone marrow or other sites of lymphopoiesis, the receptor immunoglobulin is the monomeric transmembrane form of IgM or IgD. The further differentiation of the uncommitted B cell and the production of IgG, IgA, and IgE involve complex genetic rearrangements, as discussed below.
B-Cell Differentiation and the Genetics of Antibody Production
As noted above, millions of B lymphocytes mature, having on their surface millions of different IgM and IgD receptor molecules complementary to what many feel are billions of antigen spe- cies. An understanding of the mechanism of this highly complicated biological phenomenon has evolved rapidly over the last several decades, since the determination of the structure of immu- noglobulin molecules. Contributory to such an understanding was the “clonal selection theory” proposed initially by Burnet (1970) and refined by many others. Major support for this theory came from the finding that myelomas in different patients each secreted a single molecular spe- cies of immunoglobulin and thus were clonal for that species. The receptor concept developed above was in part based on such findings. The remaining dilemma, however, was the mechanism for the production of the extreme diversity of antibodies to respond to an even greater diversity of antigens. Two general theories to explain this were entertained. The first, the germline theory, argued that immunoglobulin genes are expressed in exactly the same way as those for any other protein. This model required an enormous number of genes simply coding for the millions of immunoglobulin molecules. A second theory argued that there were only a limited number of immunoglobulin genes, which somehow diversified as the antibody-forming B lymphocytes de- veloped from their stem cells. However, at the time, the theory demanded a completely original
Figure 19.6 Model for the major portion of the antigen-receptor complex of B lymphocytes. mIg is composed of two heavy chains of the types depicted in Figure 19.2 that are transmembrane and two light chains (κ or λ) as shown. (Reproduced from DeFranco, 1992, with permission of the author and publisher.)
mechanism for such diversification. An answer to this dilemma came from the studies of Tone- gawa and associates (cf. Brack et al., 1978), who demonstrated that DNA of the embryo con- tained a number of λ gene sequences, only one of which could be found in plasma cell DNA. Tonegawa and associates argued that the most likely explanation of these data was that somatic recombination had occurred during the development of plasma cell DNA coding for a single molecular species of the λ chain, suggesting that embryonic DNA contained a number of possi- ble λ chains. Subsequent studies by numerous investigators (Goldman and Goldman, 1984; Leder, 1983; Early et al., 1980) confirmed and extended these findings. In the genome, immuno- globulin genes occur in various places throughout the karyotype (cf. Matsuda and Honjo, 1996). Within each immunoglobulin genomic region there are multiple exon segments coding for dif- ferent portions of the heavy or light chain. As a B cell matures and becomes committed to the production of a specific antibody, rearrangements, recombinations, and deletions of the vast ma- jority of genes coded for the portions of the peptide chains occur. The result is that the mature B cell possesses a single pair of genes coding for a functional specific antibody. A diagram of the generation of a specific κ chain resulting from gene rearrangement and RNA processing may be seen in Figure 19.7. For the light chain, some species may have as many as 200 variable region genes in the embryo as well as in most non–antibody-producing cells in the adult. Each of the V
Figure 19.7 A diagram of κ gene expression. Note that the pathway from germline κ genes to proteins that are secreted involves alterations in the structure of DNA, RNA, and protein. The critical and unique component is the extensive DNA recombination which joins one of many germline Vκ regions to one of several J gene segments, resulting in a rearranged gene which is then transcribed, processed, secreted by cleavage of the signal peptide, finally resulting in a κ chain to be associated with the heavy chain. (After Max, 1993, with permission of the author and publisher.)
genes, whether of the κ or λ type, retains certain structural features, including a “leader” se- quence coding for a hydrophobic sequence of amino acids involved in the secretion of the anti- body molecule through the cell membrane. As noted in Figure 19.7, there are also a series of small sequences termed the J or joining sequences for the human κ chain. Five J sequences are present, each capable of joining with variations of the V gene (Figure 19.7). In the human λ chain system there are six C genes, each one linked to its own J sequence, while in the mouse there are four such combinations. During lymphocyte development, one V gene with its leader sequence is recombined with one of the J sequences along with a single C gene to form an active light chain. During RNA processing, intervening sequences and extra J genes in the transcript are spliced out to yield a coherent messenger RNA, which is then translated into a protein. If the protein is secreted, the leader sequence is first cleaved off.
The formation of the heavy-chain variable region is similar to that of the light chain except that the V genes are situated at a much greater distance from the C genes in the DNA of the embryo, and a second region, the D or diversity gene, is also present between the V and J genes. The D genes are segments from 12 to 15 base pairs in length, which in the human comprise about 30 different sequences. In Table 19.1 may be seen an estimate of the number of the light and heavy chain combinations. The table is taken from data in Janeway and Travers (1996) and is of the human. Similar numbers in the mouse vary somewhat from those of the human but are generally similar overall. Such values for other species have not been established as yet. In addi- tion, as other factors enter into the diversity so that the total number of potential antibody struc- tures is many orders of magnitude more than the final number in Table 19.1 (see below).
The formation of the heavy-chain constant region is considerably more complex than that of the light chain, since there are at least five classes of heavy chains, as shown diagrammatically in Figure 19.8. The final formation of the C genes in the plasma cell DNA results from “isotype switching,” in which various segments of the DNA in the C gene region are deleted with sub- sequent recombination. By a similar technique, the V-D-J combination may be moved to other immunoglobulin genes. In the figure, 5′ of the C genes are long stretches of repeated noncoding sequences. In noncommitted B cells expressing IgM or IgD, the gene structure retains two C genes, those for the µ and the δ constant region. In this case one of the sequences is excised during RNA processing so that only a single heavy chain is produced (cf. Janeway and Travers, 1996).
It should be noted that in any single B cell, only one of the pair of genes coding for an immunoglobulin molecule is expressed. This phenomenon, termed allelic exclusion, indicates that if, for a given immunoglobulin gene in a single B lymphocyte, the alleles, one from each parent, are different, the lymphocyte will express only one of the alleles. The mechanism for this phenomenon is not known, although recent studies suggest a role for DNA methylation in allelic exclusion (Mostoslavsky et al., 1999; Bergman, 1999). A further factor in antibody diversity is the fact that antibody genes exhibit somatic hypermutation, especially in domains around the rearranged V region of the molecule (Neuberger and Milstein, 1995). The mutation rates in these regions have been estimated to be as high as one point mutation per 1000 bp per cell generation,
Figure 19.8 Diagrammatic representation of the heavy-chain genes and formation of the mRNA and protein products. The coding segments of the variable regions, diversity (D) and junctional (J), are comparable to those in Figure 19.17. The multiple constant region genes occurring in the germline DNA are removed by isotype switching that involves recombination between specific DNA sequences 5′ to each of the constant region genes. After establishment of a specific constant region gene together with individual variable, D and J segments, somatic recombinations occur, leading to a single V(D)J-joined rearranged DNA from which is transcribed the nuclear RNA that matures to the mRNA from which the heavy chain gene is translated and subsequently associated with light chains as shown in the figure. The figure as shown is the general case with the constant region (C) following isotype switching, representing any of the various constant regions shown in genomic DNA with individual exons separated by introns. (Adapted and modified from Janeway and Travers, 1996.)
which is many orders of magnitude higher than the spontaneous mutation rate (cf. Storb, 1996). This hypermutation occurs during a narrow period of B-cell differentiation in germinal centers of lymphoid tissues, involves primarily transitions and transversions, especially the former (Neuberger and Milstein, 1995); and is not a result of defective DNA repair mechanisms (cf. Jacobs et al., 1998). A recent study (Muramatsu et al., 2000) suggested that RNA editing may be involved in this process, which increases the diversity potential and also generates antibodies of a higher affinity, with subsequent selection for the expression of those antibodies producing a more effective immune response (French et al., 1989).