Immunogenetics

Immunogenetics is the study of the mechanisms of autoimmune diseases, tolerance in organ transplantation, and immunity to infectious diseases—with a special emphasis on the role of the genetic make-up of an organism in these processes. Historically, the launch of immunogenetics could be traced back to the demonstration of Mendelian inheritance of the human ABO blood groups in 1910. The most important influence on the development of immunogenetics is the studies of the MCH gene family.

1. The genetic basis of antibody structure

Introduction How can the immune system recognize an almost unlimited number of foreign antigens? Remarkably, each mature lymphocyte is genetically programmed to attack one and only one specific antigen: each mature B cell produces antibodies against a single antigen, and each T cell is capable of attaching to only one type of foreign antigen. Question: Every person has the ability to generate 1015 to 1018 different Ig or TCR, since genomes of many species have only 30000-40000 gene, how do so few genes produce so many different antigen receptor molecules?

The investigators over the last 30yrs, found that V and C regions of Ig are coded for by different genes—many different V region genes that can be linked up to a single C region gene. Susumu Tonegawa found that antibody genes can move and rearrange themselves within the genome of a differentiating cells: a variable (V) region gene can be located in one position in the DNA of an inherited chromosome but then move to another position on the chromosome during lymphocyte differentiation.

Review Genome: linear arrays of genes in the DNA strands of the various xmes. Every diploid cell in the human body contains the same set of genes as every cell—except the lymphocytes which differ from other cells and each other in the content of genes coding for their antigen-specific receptor. The expression of a specific pattern of genes determines the cell’s function. E.g.: only pancreatic  cells express Insulin, only B cells express antibodies. Most of genes coding for a protein have exons and introns (spliced out during RNA processing). Genes coding for cell surface proteins have a leader sequence (L exon) at the 5’end, coding for signal peptide.

General gene structure and gene expression

Genetic events in the synthesis of Ig chains VL is coded for by two separate gene segments: a variable (V) segment, which codes for the amino-terminal 95 residues. And a small joining (J) segment, codes for about 13 (96-108) residues at the carboxy-terminal end of the variable region. One V gene and one J gene are brought together in the genome to create a gene unit that, together with the C region gene, codes for an entire Ig L chain. This unique gene rearrangement mechanism-refered to as V(D)J recombination, is used only by genes coding for IgL and H chains and genes coding for TCRs. V J C

The genetic events leading to the synthesis of a kappa light chain (On chromosome 2)

An enzyme complex, known as V(D)J recombinase, mediates the rearrangement of receptor genes in B and T cells. RAG-1 and RAG-2 (recombination-activating genes) are required in the first stages of cutting Ig- and TCR-DNA. There are approximately 40 different V genes, and 5 J genes.

 Chain synthesis The  genes are found on chromosome 22 in the human. The synthesis of  chains is similar in principle to the synthesis of  chains in that it involves rearrangement of DNA, which joins a V  gene with a J  segment. There are about 40 V , 4 J  and 4 C .

Organization and rearrangement Heavy chains In contrast to the variable region of a light chain that is constructed from 2 gene segments, the variable region of a heavy chain is constructed from 3 gene segments (VH, DH (diversity segment) & JH). The D and J segments code for aa sequences in the 3rd hypervariable, or complementarity determining region (CDR3) of the heavy chain. The second feature of the H chain genes is the presence in the germline of multiple genes coding for the C region of the Ig.

V D J C Five different classes: , , , , and . Immunoglobulin heavy chain isotypes Isotype Heavy Chain IgM IgD IgG IgA IgE      IgM IgD IgG IgA IgE V D J C

The heavy chain synthesis uses the same mechanisms of rearrangement as that of L chain—the use of the V(D)J recombinase to mediate the joining of different gene segments. In the early stages of the life of a particular B cell, two rearrangements of germ line DNA must occur. The 1st brings one D segment alongside one J segment. The 2nd brings one V segment to the DJ unit (fig.) The rearranged DNA is then transcribed along with the closest C region genes  and .

Organization and rearrangement of heavy chain genes IgM IgD IgG IgA IgE (On chromosome 14) IgM IgD

Regulation of Ig Gene Expression  

This mechanism of allelic exlusion ensures that every B cell and the antibodies it synthesizes are monospecific. The arrangements occur in the absence of antigen in the early stages of B cell differentiation.

Class or isotype switching A single B cell can switch to make a different class of antibody, such as IgG, IgE, or IgA. This phenomenon is known as class or isotype switching. Class switching changes the effector function of the B cell but does not change the cell’s antigenic specificity. Class switching occurs in antigen-stimulated B cells synthesizing IgM and IgD, and involves further DNA rearrangement juxtaposing the rearranged VDJ genes with different heavy chain. In addition to antigen, class switching is dependent on the presence of cytokines released by T cells.

The cytokine that affect class switching induce further rearrangement of B cell DNA and produce switching to other Ig classes in a downstream progression. The S region, at the end of every H chain C region, permits any of the CH genes (other than C) to associate with the VDJ unit (Fig.). The cytokines present when antigen activates B cells play a key role in selection during isotope switching. Each cytokine is thought to loosen the DNA double helix structure at only certain points along the Ig locus, allowing an enzyme “ switch recombinase” to recognize DNA coding for specific C region.

Class or isotype switching (switch region) IgG1

Class switching allows an antibody with a single antigenic specificity to associate with a variety of different constant region chain thus have different effector function. For example. An antibody with a VDJ units specific for a bacterial antigen may be linked to C to produce an IgG molecules; IgG interact with cells such as macrophages that express receptors for Fc. The same VDJ unit may be linked to C to produce an IgE molecule; IgE interact with cell such as mast cells that express receptors for Fc.

Generation of antibody diversity 1. VJ and VDJ combinatorial association The association of any V gene segment with any J segment can occur to form L chain. The same, any V can associate with any D or J segments to make H chain L chains: 40 V X 5 J = 200  chains 40 V X 4 J = 160  chains H Chains: 50 V X 20 D X 6 J = 6000 H chains 2. Random assortment of H and L chains Any H chain may associate with any L chain, thus 1.2 x 106 different -containing Ig molecules (200 x6000) and 0.96 x 106 different -containing Ig molecules (160 x6000)

3. Junctional and Insertional Diversity The precise position at which the genes for the V , (D), and J segments are fused together are not constant, and inprecise DNA recombination can lead to changes in the amino acids at these junction sites (junctional diversity) that affect the antigen-binding site. In addition, small sets of nucleotides may be inserted (insertional diversity, N region diversity) at the V-D and D-J junctions (mediated by the enzyme terminal deoxynucleotidytransferase ( TdT)

4. Somatic hypermutaion Mutation that occur in V genes of heavy and/ or light chains during the lifetime of a B cell also increase the variety of antibodies produced. Generally an antibody of low affinity is produced in the primary response to antigen, DNA and amino acid sequences of this antibody closely match the sequences encoded by germ line DNA. As the response matures, the affinity for antigen of the antibodies synthesized increases, and the amino acid sequences of these antibodies diverge form those coded for in the germ line DNA. This divergence results predominantly from point mutaions in the VDJ recombined unit of antibody V genes, which result in changes in individual amino acids. This phenomenon is referred to as somatic hypermutaion because it occurs at a rate at least 10000 fold higher that normal rate. This results in the observed increased affinity of antibodies for antigen in the secondary response.

5. Somatic gene conversion Many species other than humans and mice (e.g. birds and rabbits) rely on somatic gene conversion (involving non-reciprocal exchange of sequences among genes ) and somatic hypermutaion to generate diversity within the primary Ig repertoire.

6. Receptor editing Under some circumstances, a cell in the B cell lineage can undergo a second rearrangement of its L chain variable gene segments, after it has formed a recombined VJ unit, this process is known as receptor editing. Unrearranged segments are used in the second rearrangement. Second rearrangement First rearrangement

Brief: VJ and VDJ combinatorial association Random assortment of H and L chains Junctional and insertional diversity Somatic hypermutaion Somatic gene conversion Receptor editing All these mechanisms contribute to the formation of a huge library or repertoire of B lymphocytes that contain all the specificities required to deal with the universe of diverse epitopes. Estimates of the number of total Ig specificities that can be generated in an individual are on the order of 1015-1018

2. Blood groups, and cell-surface molecules

First transfusions unpredictable –some recovered and some died. It was appreciated early in the 20th century that it was not possible to transfuse blood at random between individuals. There are over 20 antigens (cell surface molecules) on erythrocytes, and they vary between individuals. The most important and well known are those that comprise the ABO blood group system. Karl Landsteiner described the human ABO blood groups in 1901. He identified 3 types of blood (A, B, & O). In 1910, a rare 4thtype was found (AB) More than 26 blood types have been identified.

ABO Blood Group Differences in glycosylation of red cell glycoproteins give rise to three variants of the glycoprotein called A, B or O. The groups are co-expressed so that individuals can be O, A, B or AB. The O (or H) antigen is generated in all individuals, and consists of a particular carbohydrate group that is added to proteins. The ABO locus codes for a galactosyltransferase enzyme that adds a further sugar group to the O antigen. The specificity of this enzyme determines the blood group. A Allele—adds an extra N-acetylgalactosamine to O antigen to form A antigen.

B Allele—adds a galactose to O antigen to form B antigen. People with both transferases produce both A and B antigen (AB blood type); those who lack these transferases produce O antigen only (O blood type).

An Example of Blood Type Incompatibility person with type A blood who is transfused with type B blood will have antibodies that recognize and clump the red blood cells carrying type B. Red blood cells burst, releasing hemoglobin.

Rh Incompatibility The designation of blood type usually also includes whether the person has or does not have the Rh factor on the red blood cell. Rh individuals normally do not have antibodies to the Rh factor, but they make them when exposed to the Rh factor. Rh incompatibility occurs when occurs when an Rh- (no Rh antigen)mother has an Rh+(has Rh antigen) child.

If an Rh– mother has an Rh+ baby the woman will be immunised against the Rh+ antigen during childbirth and produce IgG antibodies to Rh. First Rh incompatible pregnancy—Fetal cells recognized as foreign, Mother’s immune system attacks fetal cells, Produces a mild reaction, few antibodies present. Second Rh incompatible pregnancy—Large response, plentiful antibodies, Destruction of fetal blood cells, much damage.

Other Blood Groups MNS system which also code for red-blood-cell antigens. It is important in binding specific glycoproteinson RBC Lewis encodes for enzyme (FUT3) that adds antigen to fructose and the product of the H gene necessary for ABO expression. Secretor gene causes A, B, H antigens in body fluids.