Fundamentals of Immunology - Lecture Course by M. V. Skok - Kyiv 2002

Section I. Immunochemistry

Lecture 4. Immunoglobulin Genes. Antibody Biosynthesis

For a long time, a major challenge in immunology was explaining the vast diversity of Antibodies. According to Macfarlane Burnet's clonal Selection theory, antibodies and immune Cells of various specificities pre-exist in the body, and the antigen acts merely as a selection factor that promotes the expansion of a Cell clone with a specific reactivity. Given the Central dogma of molecular biology established by Beadle and Tatum in 1943—one Gene, one polypeptide chain—the number of genes required to encode antibodies of all possible specificities would exceed the size of the vertebrate genome. Following the Discovery of the polyfunctional nature of antibodies, this estimated number decreased significantly (down to 10⁴ - 10⁶ variants), yet remained exceptionally high. A revolutionary breakthrough came with the realization that in immunoglobulin genes, a single polypeptide chain is encoded by multiple gene segments, and the combinatorial joining of these segments achieves the immense diversity of antibodies.

In 1965, Dreyer and Bennett first proposed that the variable (V) and constant (C) domains of IMMUNOGLOBULINS are encoded by separate genes. These genes were subsequently identified, and it was revealed that they change their chromosomal positions during organismal development. In 1976, DNA was extracted from mouse embryonic cells and from myeloma cells producing immunoglobulin light chains. The DNA was cleaved with restriction Enzymes, fractionated by agarose gel Electrophoresis, and hybridized with radioactive V- and C-gene probes. It was found that in embryonic cells, the V and C genes reside in different Regions of the DNA, whereas in mature myeloma DNA, they are localized together. Thus, it was proven that during ontogeny, immunoglobulin genes undergo rearrangement—a reorganization process that brings V and C genes into close proximity and joins them.

Susumu Tonegawa was awarded the 1987 Nobel Prize for his research on The Structure of immunoglobulin genes.

Structure of immunoglobulin genes.

There are three immunoglobulin gene clusters: H, k, and λ. They are located on different Chromosomes. These clusters comprise V and C genes for heavy and light chains, as well as J genes (from joining) and D genes (from diversity).

1. The λ-chain gene cluster consists of 29 V genes and four pairs of J and C_λ genes. The V genes feature leader sequences. Between the V and J genes lie hundreds of thousands of Base Pairs of an intron, while a small intron separates the J and C genes.

2. The k-chain gene cluster contains 40 V genes, 5 J genes, and one C_k gene.

3. The H-chain gene cluster has the most complex Organization. It contains over 1000 V genes (of which 51 are functional), 27 D genes, 6 J genes, and C genes encoding the heavy chains of all immunoglobulin classes.

As B lymphocytes mature, these genes undergo sequential reorganization:

1) site-specific VJ recombination (for light chain genes);

2) site-specific DJ recombination (1) and V+DJ joining (2) for heavy chain genes;

3) METABOLISM/31.html">Transcription of the entire V(D)J-intron-C block;

4) RNA splicing to yield the functional V(D)JC transcript;

5) Translation;

6) post-translational Cleavage of the leader sequence.

The transcription promoter is activated only after recombination has occurred; therefore, transcription of unrearranged DNA is impossible.

Mechanism of recombination (using VJ as an example).

V and J genes possess Two Types of recognition signals: a heptamer (7 nucleotide base pairs) and a nonamer (9 base pairs). In the V gene, these signals are separated by 11–12 base pairs, whereas in the J gene, they are separated by 21–23 base pairs. Consequently, the recombination mechanism is said to follow the 12/23 rule. The crossover sites are brought together by the recombinase enzyme, which recognizes the heptamer and nonamer signals on both genes undergoing recombination. Recombinase is encoded by two genes, RAG1 and RAG2. The onset of expression of these genes serves as the trigger for immunoglobulin gene rearrangement to begin within The Cell. Recombination initiates the transcription of the rearranged chain, which in turn halts the recombination of other variants of that gene, thereby determining the clonal nature of lymphocytes: each cell produces antibodies of a single Specificity.

It is important to note that the hypervariable regions of V domains (CDRs) are encoded by:

- on the light chain: 1 and 2 within the V gene, 3 at the VJ junction;

- on the heavy chain: 1 and 2 within the V gene, 3 within the D segment.

Thus, the STRUCTURE OF THE antibody Active Site is formed by the products of V, D, and J genes.

Mechanisms of immunoglobulin diversification.

1. Large number of functional V genes: λ – 29; k – 40; H – 51. They are divided into families based on Sequence Homology, indicating that they are products of the duplication of a single ancestral gene.

2. Combinatorial diversification:

1) L chain: VJ: 27V x 4J λ = 116; 40V x 5J k = 200; 116 + 200 = 316 L-chain variants;

2) H chain: DJ: 27 x 6 = 162; VDJ: 51 x 162 = 8,262; the D gene has three potential reading frames: 8,262 x 3 = 24,786

3) Combination H x L: 316 x 24,786 = 7.8 x 10⁶

3. Mutations at recombination sites add further diversity, exceeding 10⁸.

4. An additional diversification factor is TdT (terminal deoxynucleotidyl transferase), an enzyme that adds NUCLEOTIDES during the rearrangement of V, D, and J segments.

Thus, through combinations of a limited number of V, D, and J genes, a vast array of potential antibody variants (and correspondingly, B-lymphocyte receptors) is generated. And that is not all. These mechanisms constitute the preimmune repertoire. Upon encountering an antigen, mature B cells located in Secondary Lymphoid Organs undergo somatic mutations. Special morphological structures known as germinal centers form in the Spleen and Lymph Nodes, where a hypermutation mechanism is switched on within the immunoglobulin genes (the exact mechanism remains unknown), and the already rearranged V genes begin to change rapidly. The mutation accumulation rate reaches 10^-3 per base pair per cell generation. This means that one mutation occurs per 1,000 base pairs in every cell generation—10⁶ times more frequently than mutations in other genes. As a result of somatic mutations, numerous antibody variants are produced that differ only slightly from one another. The present antigen serves as a selection factor, promoting the proliferation (Replication) of those cells whose receptors fit it best. This fine-tunes the B-cell repertoire, with some cells partially differentiating into memory cells. Upon re-exposure to the antigen during a secondary response, these specific B cells are activated, rendering secondary-response antibodies more specific than primary ones. This process, known as the maturation of the Immune Response, increases its affinity and avidity for the antigen. The phenomenon of hypermutation is unique to immunoglobulin genes.

It should be noted that the described diversification mechanism does not exist in all animal species. In sheep, for instance, antibody diversity is generated through point mutations in immunoglobulin genes occurring in Peyer's patches. In birds, so-called Gene Conversion takes place, involving the incorporation of pseudo-V genes in the bursa of Fabricius. Thus, evolutionary trials tested various mechanisms for achieving antibody diversity, and different animal species selected different strategies.

Allelic exclusion.

Experiments have shown that each B cell produces antibodies of a single specificity. This is readily understood based on the immunoglobulin gene rearrangement mechanism discussed above. However, every heterozygous Organism possesses two sets of genes, derived from the maternal and paternal genomes. Immunoglobulin genes are subject to allelic exclusion, the precise mechanism of which is not yet fully understood. It is believed that the functional rearrangement of one allele acts as a negative signal for the other. Using transgenic mouse models, it has been demonstrated that introducing pre-rearranged immunoglobulin genes into mouse embryos suppresses the rearrangement of their endogenous genes.

A B-cell precursor possesses two sets of gene clusters for the H, k, and λ chains. First, one H-gene allele begins to recombine, either paternal or maternal (the selection is entirely random). Following several cycles of proliferation, one of the L-gene alleles is activated (also at random). DJ recombination occurs in both chromosomes (maternal and paternal), whereas VDJ recombination occurs in only one. If the rearrangement is successful, The production of a full-length μ chain halts further reorganization in the H genes and signals the initiation of rearrangements in the L genes. Initially, the VJ GENES OF THE k chain recombine; if this attempt is unsuccessful, other VJ k variants are tested; if this fails again, the λ-chain genes undergo recombination. The first successful combination yields a functional IgM and halts any further reorganization (shutting off RAG genes). The presence of two L-chain gene pools, k and λ, increases the reliability of forming a complete immunoglobulin molecule. The high probability of this success is evidenced by the fact that the majority of immunoglobulins bear k rather than λ chains.

Switching from membrane-bound to secreted forms of IgM.

Immunoglobulins M, G, A, and E can exist in both membrane-bound (B-lymphocyte receptors) and soluble (antibodies) forms. They differ in The Nature of their C-terminal peptide: in the membrane-bound form, it is hydrophobic and serves as the transmembrane domain of the receptor, whereas in the secreted form, it is hydrophilic. Let us examine the switching mechanism using IgM as an example. Both membrane and secreted IgM are encoded by the same genes. Within the Cμ gene, There is a DNA fragment encoding the hydrophilic peptide and another fragment encoding the hydrophobic peptide. Their transcription is halted by two respective stop codons. Initially, when the cell is not yet secreting immunoglobulins, the stop codon Functions.

2. A transcript is produced that carries information for both the hydrophobic and hydrophilic Peptides, and the fragment corresponding to the hydrophilic peptide is excised as an intron via mRNA splicing. When the cell receives appropriate signals and begins to secrete immunoglobulins, stop codon 1 comes into play, and a shorter gene lacking the hydrophobic peptide is transcribed.

Class switching of immunoglobulins.

All B cells initiate immunoglobulin gene reorganization with the rearrangement of the μ-chain gene and produce IgM. Switching to IgD occurs prior to antigen encounter, allowing the cell to produce both classes—IgM and IgD—with the same specificity. Following antigenic stimulation, a switch to the secreted form of IgM occurs, followed by a switch to one of the other immunoglobulin classes. Since a single rearranged V gene (VDJ) can combine with different C genes, antibodies of the same specificity can belong to different classes, possess different Fc fragments, and consequently perform distinct biological functions in various Tissues of the organism.

Class switching occurs via two distinct pathways.

1) Switching from IgM to IgD occurs through alternative RNA splicing of a transcript containing both Cμ and Cγ gene sequences. This is why a cell can simultaneously express both IgM and IgD.

2) Switching from IgM to IgG, IgE, and IgA occurs through recombination and deletion (removal) of all intervening DNA and is irreversible: a cell that has begun secreting IgG can no longer produce IgM (IgG- or IgA-secreting myelomas lack the DNA for other immunoglobulin classes). Switching to a specific class or subclass is regulated by cytokines produced by various T-lymphocyte subpopulations.

Antibody Biosynthesis.

Following transcription and translation, the gene products of the heavy and light chains enter The Endoplasmic reticulum (endoplasmic reticulum, ER). There, the assembly of the immunoglobulin molecule takes place. In accordance with the General Principles of oligomeric protein assembly, no chain can exit the ER until it has been incorporated into the oligomer. The chains are bound to specialized chaperone Proteins that retain them in the ER until oligomer assembly is complete. For example, the heavy chain remains bound to the BiP chaperone until a light chain attaches to it. Once assembled, the immunoglobulin molecule leaves the ER and moves to the Golgi apparatus, where glycosylation occurs. Fully formed antibodies accumulate in vesicles and are secreted via exocytosis. The B-cell receptor, which incorporates the membrane-bound form of immunoglobulin, also contains additional components necessary for signal Transduction from the receptor into the cell interior. During B-cell receptor synthesis, the entire complex exits the ER together.

Following antigenic stimulation, the B cell receives differentiation signals and gradually transforms into a plasma cell. Unlike resting lymphocytes, which feature a large Nucleus and scant Cytoplasm, plasma cells possess a well-developed ER and function essentially as antibody-producing factories. They cease expressing many markers typical of B cells, rendering them unresponsive to numerous external stimuli. Cytoplasmic staining reveals that their cytoplasm is packed with antibodies. Having fulfilled its function, a plasma cell does not revert to its previous state and eventually dies (the lifespan of a standard B cell is 3–4 weeks). Long-lived memory B cells branch off into a separate Lineage at Cytology/cytology/16.html">Early stages of B-Cell Differentiation.

Hybridomas and Monoclonal Antibodies.

Serum antibodies represent a heterogeneous mixture of immunoglobulins spanning various classes and specificities. Obtaining homogeneous antibodies has long been the ultimate dream of immunologists, as it would allow them to study their properties, crystallize them for X-Ray Diffraction Analysis, and apply them in practical fields. This role was partially fulfilled by myeloma antibodies—natural monoclonal antibodies derived from the progeny of a single malignant B cell. Notably, malignancies of The Immune System are almost exclusively B-cell disorders. This is widely believed to be a consequence of somatic hypermutation, which frequently generates transformed cells. Thus, lymphoproliferative disorders are the evolutionary price paid for the high Specificity of the immune response. Although myeloma antibodies were successfully employed in numerous experiments, the dream of generating monoclonal antibodies of a desired specificity remained unfulfilled. Initial attempts to isolate them from serum mixtures by fractionating populations based on charge, Hydrophobicity, and other physicochemical properties yielded limited success.

By the early 1970s, Methods for culturing immune cells in vitro were well established. Biochemists used these cultures to study, among other things, the enzymes involved in purine and pyrimidine biosynthesis. It was discovered that normal cells utilize two purine biosynthetic pathways: de novo synthesis and the salvage pathway, which recycles breakdown products. De novo synthesis is mediated by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Certain tumor cell lines were found to be deficient in this enzyme, rendering them unable to synthesize nucleotides de novo. If such cells are cultured in the presence of aminopterin—which blocks the nucleotide salvage pathway—they perish. Normal cells survive under these conditions because they retain The ability to perform de novo nucleotide synthesis. Concurrently, cell Hybridization techniques—involving the fusion of cell membranes via electrical pulses or chemical Treatment with polyethylene glycol—were undergoing intensive study. Building upon these experiments, G. Köhler conceived the idea of hybridizing a normal, antibody-producing B lymphocyte with an HGPRT-deficient myeloma cell line. When cultured with aminopterin, the parental tumor cells die because they cannot synthesize Purines de novo, while normal lymphocytes perish because they cannot proliferate in culture without additional growth factors. Consequently, only the hybrids survive, having inherited immortality from the myeloma cells and the de novo nucleotide synthesis pathway from the normal lymphocytes. By screening these survivors for cells producing antibodies of the desired specificity, one can establish a clone and, ultimately, obtain homogeneous monoclonal antibodies. C. Milstein, in whose laboratory Köhler worked, initially opposed the experiments, believing the probability of finding antigen-specific clones in such a mixture to be exceedingly low. Nevertheless, Köhler pursued the experiment at his own risk and successfully isolated numerous positive clones. We now know that the probability of finding them is actually quite high: during a strong immune response, as many as 1 in 20 generated clones may be positive. Köhler's experiment was truly revolutionary—it fulfilled the dream of generations of immunologists and rightfully earned a Nobel Prize. Today, this is a fairly routine Procedure. Cells produced via hybridization are termed hybridomas, and the process itself is known as hybridoma technology. It is worth noting that The Development of this technology relied entirely on advances in biochemistry, in vitro cell culture techniques, and immunological assays capable of analyzing antibody specificity across a vast number of resulting clones.

Summary.

Immunoglobulin genes are unique in that a functional gene encoding a single polypeptide chain is assembled from several distinct gene segments that draw together and join during lymphocyte development. Through a multitude of V-gene variants, VJ and VDJ combinations, heavy and light chain pairings, and several auxiliary mechanisms, a limited genetic repertoire gives rise to a virtually limitless array of antibody specificities, which is further diversified by somatic mutations. Immunoglobulins can exist in both membrane-bound and soluble forms, a versatility achieved by utilizing alternative stop codons during gene transcription. Isotype class switching occurs either via alternative RNA splicing or through irreversible deletion of intervening DNA segments. During antibody biosynthesis, exit from the endoplasmic reticulum occurs exclusively after the complete and final assembly of all polypeptide chains.