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Antibody – A Specific Antigen Recognition Molecule

Antibody – A Specific Antigen Recognition Molecule
Evolutionary processes came up with what can only be described as a brilliant solution to the problem of recognizing an almost infinite diversity of antigens. This solution was to design antibody molecules in such a way that not only are they able to specifically recognize the offending pathogen but they can also recruit various components of the immune response capable of subsequently destroying the pathogen.
The antibody molecules have two main parts, one called the variable region that is devoted to binding to the individual antigen (the antigen recognition function) and one called the constant region, concerned with linking to complement, phagocytes, NK cells, and so forth (the effector function). Thus the body has to make hundreds of thousands, or even millions, of antibody molecules with different antigen‐recognition sites but that all share the property of ecruiting other elements of the immune response (Figure 2.1).
Antibody opsonizes microbes for phagocytosis both directly via Fc receptors and indirectly via complement activation. The Fab (fragment antigen‐binding) part of the antibody binds specific antigen on the microbe and varies from one antibody to another. The Fc (fragment crystallizable) part is identical for all antibodies of the same class/subclass and functionally activates complement (IgM and IgG antibodies, via the classical pathway) and phagocytic cells (IgG antibody, via binding to Fc receptors [FcR] on the surface of the phagocyte). The coating of microbes with substances that are recognized by phagocytic cells is referred to as opsonization and both IgG and complement © components such as C3b, and the products of C3b breakdown iC3b, C3dg, and C3d (all of which are recognized by complement receptors [CR] on the phagocyte) can act as opsonins. In addition, complement activation leads to chemotactic attraction of the phagocytes to the site of the infection and increased vascular permeability in order to facilitate their passage from the blood circulation to the tissues.

Antibody‐mediated activation of the classical complement pathway
Human antibodies are divided into five classes: immunoglobulin M (shortened to IgM), IgG, IgA, IgE, and IgD, which differ in the specialization of their effector “rear ends” for different biological functions. In Chapter 1 we discussed the antibody‐independent alternative pathway of complement activation that relies upon stabilization of the C3bBb C3 convertase by microbial surface polysaccharides. However, the first complement pathway to be discovered, the classical pathway, required IgM or IgG antibody for its activation. Antibody of these two classes, when bound to antigen, will link to the first molecule in the classical pathway of complement, C1q, and trigger the proteolytic activity of the C1 complex (Figure 2.2). It was subsequently discovered that a variety of other sub­ stances, including members of the pentraxin family of soluble pattern recognition receptors such as C‐reactive protein (CRP), are also able to link microbial antigens to C1q and thereby activate the classical pathway.
A C1q is a homohexamer (six identical molecules that associate together) arranged into a central stem branching into six arms, each tipped with an antibody‐binding globular head. It is associated with two further subunits, C1r and C1s, in a Ca2+‐stabilized complex (Figure 2.2). Both C1r and C1s contain sequences called complement control protein (CCP) repeats. These are a characteristic structural feature of several proteins involved in control of the complement system. The changes that occur in C1q upon binding the antigen–anti­ body complex brings about the auto activation of C1r that then cleaves C1s. The activity of C1 is regulated by a C1‐ inhibitor (C1‐Inh) that dissociates C1r and C1s from C1q and thus prevents excessive activation of the classical pathway.
The next component in the pathway, C4 (unfortunately components were numbered before the sequence was established), now binds to the CCPs in C1s and is then cleaved enzymatically by C1s. As expected in a multienzyme cascade, several molecules of C4 undergo cleavage, each releasing a small C4a fragment and revealing a nascent labile internal thiolester bond in the residual C4b (like that in C3, see Figure 1.31) that can then bind either to the antibody–C1 complex or to the surface of the microbe itself. In the presence of Mg2+, complement component C2 can complex with the C4b to become a new substrate for the C1s: the resulting product C4b2a now has the vital C3 convertase activity required to cleave C3 (Figure 2.3). The classical pathway C3 convertase has the same job as the C3bBb generated by the alternative pathway. Activation of a single C1 complex can bring about the proteolysis of literally thousands of C3 molecules. The resulting C3b is added to C4b2a to make it into a C5 convertase, which generates C5a, with chemotactic and anaphylactic functions, and C5b, which forms the first component of the membrane attack complex (Figure 1.33 and Figure 2.4). Just as the alternative pathway C3 convertase is controlled by factors H and I, so the breakdown of C4b2a is brought about by Factor I in the presence of either C4‐binding protein (C4bp) or cell surface C3b receptor (CR1) acting as cofactors.

Activation of the classical complement pathway. The first component, C1, of the classical pathway of complement activation is a complex composed of three subunits: C1q, C1r, and C1s. (a) C1q forms a hexamer arranged in a “bunch of tulips”‐like structure and is associated with the flexible rod‐like Ca2+‐dependent complex C1r2–C1s2, which interdigitates with the six arms of C1q. (b) Activation of the complement cascade by the classical pathway requires antibodies to be bound to antigen in order that the globular heads of the C1q hexamer can bind to the Fc part of at least two antibodies. Certain other molecules, such as C‐reactive protein, are also able to coat microbial surfaces and subsequently bind C1q to trigger the classical pathway.

The lectin and classical complement pathways merge to generate the same C3 convertase
It is appropriate at this stage to recall the activation of complement by innate immune mechanisms involving mannose‐binding lectin (MBL). On complexing with a microbe, MBL binds and activates the proteolytic activity of the MBL‐associated serine proteases, MASP‐1 and 2, which structurally resemble C1r and C1s respectively. In an analogous fashion to the C1qrs complex, MASP‐1 and MASP‐2 split C4 and C2 to generate the C4b2a C3 convertase (Figure 2.3).

Irrespective of whether activation occurs via the classical, lectin, or alternative pathway (indeed, all three pathways will often be activated in response to a particular infection although the classical pathway will have to wait for the arrival of antibody), several biologically active complement components are generated that have important roles in the immune response (Figure 2.5).

Comparison of the classical, lectin, and alternative complement pathways. The classical pathway is activated by antibody, whereas the alternative and lectin pathways are not. The molecules with protease activity are highlighted in light blue. The key central event for all three pathways is the cleavage of C3 by C3 convertase (namely C4b2a for the classical and lectin pathways, C3bBb for the alternative pathway). Beware confusion with nomenclature: the large C2 fragment that forms the C3 convertase is designated as C2a, but to be consistent with C4b, C3b, and C5b, it would have been more logical to call it C2b. Mannose‐binding lectin (MBL), when combined with microbial surface sugars, associates with the MBL‐associated serine proteases (MASP)‐1 and ‐2, which split C4 and C2.

Antibody can activate phagocytosis
Microorganisms are sometimes able to resist phagocytosis. If small amounts of antibody are added the phagocyte springs into action. It does so through the recognition of two or more antibody molecules bound to the microbe, using specialized Fc receptors on the cell surface of the phagocyte (Figure 2.1).
A single antibody molecule complexed to the microorganism is not enough because it cannot cause the cross‐linking of the Fc receptors on the phagocyte surface membrane that is required to activate the cell. There is a further consideration connected with what is often called the bonus effect of multivalency. For thermodynamic reasons, which will be discussed in Chapter 5, the association constant of ligands that use several rather than a single bond to react with receptors is increased geometrically rather than arithmetically. For example, three antibodies bound close together on a bacterium could be bound to a macrophage a thousand times more strongly than a single antibody molecule (Figure 2.6).

Other activities of antibody
In addition to the activation of complement and the facilitation of phagocytosis, antibodies mediate a variety of other functions, including participation in a process referred to as antibody‐dependent cellular cytotoxicity (ADCC), formation of immune complexes to enable the removal of antigen from the circulation, and, for the IgE class of antibody, triggering of mast cell and basophil degraulation (Figure 2.7). Aside from working with other components of the immune system, antibodies can directly neutralize the binding of viruses to their cell surface receptors, prevent adhesion of bacteria to body surfaces and neutralize bacterial toxins.

Antibodies are made by lymphocytes
The central role of the lymphocyte in the production of antibody was established largely by the work of James Gowans. He depleted rats of their lymphocytes by chronic drainage of lymph from the thoracic duct using an indwelling cannula, and showed that they had a grossly impaired ability to mount an antibody response to microbial challenge. The ability to form antibody could be restored by injecting thoracic duct lymphocytes obtained from another rat of the same strain.

The majority of resting lymphocytes are relatively small cells (approximately 10 μm diameter) with a darkly staining nucleus due to condensed chromatin and with relatively little cytoplasm (Figure 2.8a) containing the odd mitochondrion required for basic energy provision (Figure 2.8b). They are derived from hematopoietic stem cells in the bone marrow which can develop into the common lymphoid progenitors that give rise to both the antibody‐producing B‐cells and to the T‐cells (Figure 2.9). The B‐cells can be divided into two populations (B‐1 and B‐2), and the T‐cells can be divided into those with a γδ T‐cell receptor and those with an αβ T‐cell receptor. T‐cells, particularly those with an αβ TCR, can be further divided on the basis of their function into helper T‐cells, regulatory T‐cells, and cytotoxic T‐cells. The helper T‐cells provide assistance for B‐cells, cytotoxic T‐cells, and macrophages (Figure 2.10).