CHAPTER 13 Immunogenetics
Innate Immunity
Cell-Mediated Innate Immunity
Phagocytosis
Two major cell types go on the offensive when a foreign microorganism invades—macrophages and neutrophils. Macrophages are the mature form of circulating monocytes that migrate into tissues and occur primarily around the basement membrane of blood vessels in connective tissue, lung, liver, and the lining of the sinusoids of the spleen and the medullary sinuses of the lymph nodes. They are believed to play a key role in the orchestration of both the innate and adaptive responses, and can recognise invading microorganisms through surface receptors able to distinguishing between self and pathogen. Recognition of the foreign material leads to phagocytosis by the macrophage, followed rapidly by neutrophils recruited from the circulation during the inflammatory process. The activation of the macrophage triggers the inflammatory process through the release of inflammatory mediators. The invading organism is destroyed by fusion with intracellular granules of the phagocyte and exposure to the action of hydrogen peroxide, hydroxyl radicals, and nitrous oxide (Figure 13.1).
The Toll-like Receptor Pathway
There are 10 TLRs in man, each receptor being responsible for recognition of a specific set of pathogen-associated molecular patterns. TLR2 has been well characterised and has an essential role in the detection of invading pathogens, recognizing peptidoglycans and lipoproteins associated with gram-positive bacteria, as well as a host of other microbial and endogenous ligands. TLR2’s primary function is therefore lipoprotein-mediated signaling, and activation of the pathway by recognition of its ligand results in activation of the transcription factor NF-κB, which in turn results in the increased expression of co-stimulatory molecules and inflammatory cytokines (Figure 13.3). These cytokines help mediate migration of dendritic cells from infected tissue to lymph nodes, where they may encounter and activate leukocytes involved in the adaptive immune response. The signaling pathways used by TLRs share many of the same proteins as the interleukin-1 receptor (IL-1R) pathway (Figure 13.2). Activation of TLR leads to recruitment of the MyD88 (this is sometimes known as the MyD88-dependent pathway) which mediates the interaction between IL-1R associated kinases 1 and 4 (IRAK1 and IRAK4).
The activation of the Toll pathway has several important effects in inducing innate immunity. These effects include the production of cytokines and chemokines, including IL-1, IL-6, and TNF-α (tumor necrosis factor-alpha), which have local effects in containing infection and systemic effects with the generation of fever and induction of acute phase responses, including production of C-reactive protein. One important medical condition related to the Toll pathway is septic shock, as activation of the Toll pathway by certain ligands induces systemic release of TNF-α. There are also important health-related consequences that result from TLR2 deficiency or mutation. TLR2 deficient mice are susceptible to infection by Gram-positive bacteria as well as meningitis from Streptococcus pneumoniae.
Humoral Innate Immunity
Complement
The complement system is a complex of 20 or so plasma proteins that cooperate to attack extracellular pathogens. Although the critical role of the system is to opsonize pathogens, it also recruits inflammatory cells and kills pathogens directly through membrane attack complexes. The complement system can be activated through three pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway (see Figure 13.3).
Complement nomenclature, like much else in immunology, can be confusing. Each component is designated by the letter C, followed by a number. But they were numbered in order of their discovery rather than the sequence of reactions. The reaction sequence is C1, C4, C3, C5, C6, C7, C8, and C9. The product of each cleavage reaction is designated by letters, the larger fragment being ‘b’ (b = big), and the smaller fragment ‘a’. In the lectin pathway, MBL in the blood binds another protein, a serine protease called MASP (MBL-associated serine protease). When MBL binds to its target (for example, mannose on the surface of a bacterium), the MASP protein functions like a convertase to clip C3 into C3a and C3b. C3 is abundant in the blood, so this happens very efficiently. The other two complement pathways also converge toward C3 convertase, which cleaves C3. C3a mediates inflammation while C3b binds to the pathogen surface, coating it and acting as an opsonin. The effector roles of the major complement proteins can be summarized according to function as follows (Figure 13.4):
Specific Acquired Immunity
Humoral Specific Acquired Immunity
The main mediators of humoral specific acquired immunity are immunoglobulins or antibodies. Antibodies are able to recognize and bind to surface antigens of infecting microorganisms, leading to the activation of phagocytes and the initiation of the classic pathway of complement, resulting in the generation of the MAC (see Figure 13.4) and availability of other complement effector functions. Exposure to a specific antigen results in the clonal proliferation of a small lymphocyte derived from the bone marrow (hence ‘B’ lymphocytes), resulting in mature antibody-producing cells or plasma cells.
Immunoglobulins
Immunoglobulin structure
The immunoglobulin molecule is made up of four polypeptide chains—two ‘light’ (L) and two ‘heavy’ (H)—of approximately 220 and 440 amino acids in length, respectively. They are held together in a Y-shape by disulfide bonds and non-covalent interactions. Each Fab fragment is composed of L chains linked to the amino-terminal portion of the H chains, whereas each Fc fragment is composed only of the carboxy-terminal portion of the H chains (Figure 13.5).
Immunoglobulin isotypes, subclasses, and idiotypes
There are five different types of heavy chain, designated respectively as γ, µ, α, δ, and ε, one each for the five major antibody classes—the isotypes: IgG, IgM, IgA, IgD, and IgE, respectively. The L chains are of two types—kappa (κ) or lambda (λ), and these occur in all five classes of antibody, but only one type occurs in each individual antibody. Thus, the molecular formula for IgG is λ2γ2 or κ2γ2. The characteristics of the various classes of antibody are outlined in Table 13.1. In addition, there are four IgG subclasses—IgG1, IgG2, IgG3, and IgG4—and two IgA subclasses—IgA1 and IgA2—that differ in their amino acid sequence and interchain disulfide bonds. Individual antibody molecules that recognize specific antigens are known as idiotypes.
Immunoglobulin allotypes
The five immunoglobulin classes occur in all normal individuals, but allelic variants, or what are known as antibody allotypes of these classes, have also been identified. These are the Gm system associated with the heavy chain of IgG, the Am system associated with the IgA heavy chain, the Km and Inv systems associated with the κ light chain, the Oz system for the λ light chain and the Em allotype for the IgE heavy chain. The Gm and Km systems are independent of each other and are polymorphic (p. 135), the frequencies of the different alleles varying in different ethnic groups.
Generation of Antibody Diversity
Multiple myeloma
People with multiple myeloma make a single or monoclonal antibody species in large abundance, which in a proportion of patients is detected in their urine. This is known as Bence Jones protein and consists of antibody L chains. The amino-terminal ends of this protein molecule in different patients are quite variable in sequence, whereas the carboxy-terminal ends are relatively constant. These are called the variable, or V, and constant, or C, regions, respectively. However, the V regions of different myeloma proteins show four regions that vary little from one antibody to another, known as framework regions (FR 1–4), and three markedly variable regions interspersed between these, known as hypervariable regions (HV I–III) (see Figure 13.5).
DNA studies of antibody diversity
There are estimated to be some 60 different DNA segments coding for the V region of the H-chain, 40 for the V region of the κ L-chain, and 30 for the λ L-chain V region. Six functional DNA segments code for the J region of the H-chain, five for the J region of the κ L-chain, and four for the J region of the λ L-chain. A single DNA segment codes for the C region of the κ L-chain, seven for the C region of the λ L-chain and 11 functional DNA segments code for the C region of the different classes of H-chain. There are also 27 functional DNA segments coding for the D region of the H-chain (Figure 13.6).
FIGURE 13.6 Estimated number of the various DNA segments coding for the κ, λ, and various heavy chains.
The genomic regions in question also contain a large number of unexpressed DNA sequences or pseudogenes (p. 17). Although the coding DNA segments for the various regions of the antibody molecule can be referred to as ‘genes’, use of this term in regard to antibodies has deliberately been avoided because they could be considered an exception to the general rule of ‘one gene–one enzyme (or protein)’ (p. 167).
Antibody gene rearrangement
The genes for the κ and λ L-chains and the H-chains are located on chromosomes 2, 22, and 14, respectively. Only one of each of the relevant types of DNA segment is expressed in any single antibody molecule. The DNA coding segments for the various portions of the antibody chains on these chromosomes are separated by DNA that is non-coding. Somatic recombinational events involved in antibody production involve short conserved recombination signal sequences that flank each germline DNA segment (Figure 13.7). Further diversity occurs by variable mRNA splicing at the V–J junction in RNA processing and by somatic mutation of the antibody genes. These mechanisms readily account for the antibody diversity seen in nature, even though it is still not entirely clear how particular DNA segments are selected to produce an antibody to a specific antigen.
Class Switching of Antibodies
There is a normal switch of antibody class produced by B cells on continued, or further, exposure to antigen—from IgM, the initial class of antibody produced in response to exposure to an antigen, to IgA or IgG. This class switching involves retention of the specificity of the antibody to the same antigen. Analysis of class switching in a population of cells derived from a single B cell has shown that both classes of antibody have the same antigen-binding sites, having the same V region but differing only in their C region. Class switching occurs by a somatic recombination event that involves DNA segments designated S (for switching) that lead to looping out and deletion of the intervening DNA. The result is to eliminate the DNA segment coding for the C region of the H-chain of the IgM molecule, and to bring the gene segment encoding the C region of the new class of H-chain adjacent to the segment encoding the V region (see Figure 13.7).
