IMMUNOLOGY - Roit I. - Mir 2000

Chapter 8. Sources of Diversity in Antigen-Recognizing Structures

■ Thanks to the immense diversity of Antibodies synthesized by B Cells and antigen-recognizing receptors expressed by T cells, The Immune System is capable of recognizing and responding to a vast array of different Antigens.

■ An immunoglobulin molecule is composed of heavy and light chains; light chains can belong to either the k or λ type. The total number of possible antigen-combining site variants is calculated as the product of the numbers of different heavy and light chains.

■ Immunoglobulin light chains are encoded by V and J Gene segments; heavy chain encoding also involves V and J segments, with D segments contributing additional diversity.

■ Recombinations of a limited number of V, D, and J gene segments generate an infinite number of variable domains with diverse specificities.

■ Following antigenic stimulation, somatic hypermutations occur within the genes for immunoglobulin light and heavy chains. In contrast, T-Cell receptor genes do not undergo these changes.

■ TCR encoding involves four gene groups: α and β genes are expressed by the majority of peripheral T cells, whereas γ and δ genes are expressed by a thymic T-cell subpopulation and a small fraction of peripheral T cells.

■ TCR diversity, much like antibody diversity, is generated through recombinations between V, D, and J gene segments occurring within each of the α-, β-, γ-, or δ-chain loci with minor mechanistic differences.

■ The recombination of V, D, and J gene segments encoding IMMUNOGLOBULINS and T-cell receptors is regulated, at least in part, by two recombination-activating genes (RAG-1 and RAG-2).

■ Beyond simple rearrangements of V, D, and J gene segments, antibody and TCR diversity is further shaped by the insertion of extra NUCLEOTIDES ("N-region" Variability), shifts in the joining positions of gene segments, and alterations in the reading frames of D segments.

■ Immunoglobulin isotype switching is driven by the recombination of VDJ genes with different C genes, coupled with differential RNA splicing.

The immune system's ability to recognize antigens relies entirely on antibodies synthesized by B cells and antigen-binding receptors expressed by T cells. While both cell populations can recognize a multitude of diverse antigens, they do so through different pathways. Although antibodies differ from T-cell receptors (TCRs), the antigenic Specificity of both is generated by highly similar mechanisms, which will be explored in this chapter.

Thanks to their striking diversity in antigen-binding site specificity, antibodies ensure the recognition of millions of different environmental antigens. Furthermore, each Class of antibodies features a characteristic effector region; for instance, IgE can bind to mast cell Fc receptors, whereas IgG is capable of attaching to phagocytes. It has been estimated that the body produces far more structural antibody variants than all other Proteins combined. The number of antibody variants synthesized by the Organism actually exceeds the total number of genes in our genome. How can such a massive SCALE OF DIVERSITY arise? Although early concepts of antibody formation have evolved significantly over the years, it remains astonishing how close Ehrlich came to modern views at the turn of the century with his side-chain theory (Fig. 8.1). His idea of antigen-mediated Selection of antibody-producing cells is nearly identical to the modern clonal selection theory, with the sole exception that he envisioned multiple receptors of different specificities residing on the same cell.

Fig. 8.1. Ehrlich's proposed side-chain theory. Ehrlich postulated that the binding of an antigen to a pre-existing receptor On the surface of a B cell (now known to be membrane-bound immunoglobulin) triggers The Cell to synthesize and secrete elevated amounts of such receptors. Although, as illustrated, Ehrlich believed a single cell could produce antibodies capable of binding more than one type of antigen, he nevertheless anticipated both the clonal selection theory of Immunity and the fundamental concept that antigen receptors exist prior to the immune system's contact with the antigen.

THEORIES OF ANTIBODY FORMATION

In the post-Ehrlich era, concepts of antibody formation lost their initial simplicity. The need to revise these theories arose when chemists learned to synthesize novel Organic compounds absent in nature, and, as Landsteiner demonstrated, the immune system proved capable of responding to each of them by producing specific antibodies. The notion that natural selection could generate the genes required for antibodies against all these novel synthetic substances within immune cells seemed implausible. This led to the instructive hypothesis of antibody formation, which proposed that an antigen acts upon a flexible immunoglobulin molecule ("instructing" it) to shape a complementary binding site. The rapid progress of molecular biology in the 1950s and 1960s rendered the instructive hypothesis untenable, as it became clear that no mechanism of "instructive" antigen action actually exists. With this new turn in scientific thought, selectionist ideas regained favor. Almost simultaneously, Jerne and Burnet put forward the clonal selection theory, asserting that each lymphocyte produces immunoglobulins of only a single specificity and that an antigen selectively activates cells carrying antibodies specific to it alone.

However, the question regarding the sources of antibody diversity remained unanswered. The theoretical assumption that a distinct gene exists for antibodies of each of the myriad specificities immediately opened up another problem. Half of the Amino Acid Sequence of any immunoglobulin light chain, and a quarter of any heavy chain, is always variable, while the remainder is constant. Given a vast repertoire of putative antibody genes, how is it possible to preserve an invariant sequence within the constant regions of immunoglobulin chains? This question was answered by Dreyer and Bennett, who proposed that Variable and constant regions are encoded by separate genes, with many genes existing for variable (V) regions and one or a very limited number of genes for constant (C) regions. It now remained to explain the source of variable region diversity! The foundation for this was the idea of somatic mutagenesis, according to which a vast array of modified, i.e., mutated, genes arises from a relatively small number of germline genes over an individual's lifetime. Additionally, it was suggested that a complete V gene might be generated through the recombination of several gene segments. During the cutting and joining of DNA fragments, extra nucleotides can be inserted between them, generating additional variability termed N-region diversity, since the resulting nucleotide sequence differs from the germline. Alternatively, Gene Conversion involving a pool of pseudogenes was proposed as a source of variable region diversity. Ultimately, five potential sources of antigen-recognizing Structure diversity were identified:

✵ multiplicity of germline V-region genes:

✵ somatic mutagenesis;

✵ somatic recombination between segments forming a complete V gene;

✵ gene conversions;

✵ insertion of extra nucleotides.

Today it is known that mammals can utilize all five of these mechanisms to generate antibody diversity (Fig. 8.2). Notably, sharks possess a substantial number of antibody-encoding genes and "have no need" for somatic recombination, whereas chickens have a limited number of germline antibody genes and rely heavily on gene conversion (see Chapter 15).

Fig. 8.2. Five possible sources of structural diversity in the V regions of immunoglobulin H and L chains.

1. Germline gene multiplicity. There is A large number of distinct unrearranged genes (V1-Vn), each encoding a V domain of a specific affinity.

2. Somatic mutagenesis. During B-cell ontogeny, Mutations occurring within the germline V gene in different B-cell clones give rise to diverse V genes.

3. Somatic recombination. During B-cell ontogeny, a series of gene segments (J1-Jn) recombine and join the main body of the V gene. As a result, a protein is synthesized whose individual elements are encoded by different gene segments.

4. Gene conversion. DNA segments belonging to a series of pseudo-V genes can be copied into a functional V gene, altering its original nucleotide sequence.

5. Insertion of extra nucleotides. During recombination, prior to the joining of excised V and J DNA segments, additional nucleotides encoding extra amino acid residues in the V regions may be inserted between them.

All five of these mechanisms serve as sources of antibody diversity in mammals.