Events in B-Cell Responses
Activation of B Lymphocytes: Clonal Selection, Expansion, and Antibody Production
The immunologic activation of most B cells requires a series of events as outlined in the numbered steps that follow and as illustrated in process figure 1.
1. Clonal selection and binding of antigen. In this case, a pre committed B cell of a particular clonal specificity picks up the antigen on its Ig receptors and processes it into small peptide determinants. The antigen is then bound to the MHC-II receptors on the B cell. The MHC/Ag complex on the B cell is bound by T-cell receptors.
2. Induction by chemical mediators. The B cell receives developmental signals from macrophages and T cells (interleukin-2 and interleukin-6) and other growth factors, especially IL-4 and IL-5.
3. The combination of these stimuli on the membrane receptors causes a signal to be transmitted internally to the B-cell nucleus.
4. These events trigger B-cell activation. An activated B cell— referred to as a lymphoblast—enlarges and increases its syn thesis of DNA and protein in preparation for entering the cell cycle and mitosis.
5–6. Clonal expansion. The stimulated B cell multiplies through successive mitotic divisions and produces a large population of genetically identical daughter cells. Some cells that stop short of becoming fully differentiated are memory cells, which remain for long periods to react with that same antigen at a later time. This reaction also expands the clone size, so that subsequent exposure to that antigen provides more cells with that specificity. This expansion of the clone size is one factor in the increased memory response. The more numerous progeny are large, specialized, terminally differentiated B cells called plasma cells.
7. Antibody production and secretion. The primary action of plasma cells is to secrete copious amounts of antibodies with the same specificity as the original receptor into the sur rounding tissues (process figure 1, step 7). Although an individual plasma cell can produce around 2,000 antibodies per second, production does not continue indefinitely. The plasma cells do not survive for long, and they deteriorate after they have synthesized antibodies.
Fig1. Events in B-cell activation and antibody synthesis. These steps are tied to the numbered text. Only key receptors for these reactions are shown in the inset.
Products of B Lymphocytes: Antibody Structure and Functions Earlier we saw that a basic immunoglobulin (Ig)2 molecule contains four polypeptide chains connected by disulfide bonds. Because antibodies are a type of immunoglobulin, they all feature this same basic structure. Let us review it using an IgG molecule as a model. Two functionally distinct segments called fragments can be differentiated. The two “arms” that bind antigen are termed antigen binding fragments (Fabs), and the rest of the molecule is the crystallizable fragment (Fc), so called because it can be crystallized in pure form. The amino-terminal end of each antigen binding fragment (consisting of the variable regions of the heavy and light chains) folds into a groove that will accommodate one epitope of the antigen. The presence of a special hinge region at the site of attachment between the Fabs and the Fc allows swiveling of the Fabs. In this way, they can change their angle to accommodate nearby antigen sites that vary slightly in distance and position. The Fc portion is involved in binding to various cells and molecules of the immune system itself. Figure 2 shows a schematic view of an antibody (see figure 2 for a quick review of antibody genetics).
Fig2. Working models of antibody structure. (a) Diagrammatic view of IgG depicts the principal regions (Fabs and Fc) of the molecule. Note that the Fabs can swivel at the hinge region and provide flexibility in position. (b) Three-dimensional model of immunoglobulin shows the tertiary and quaternary structure achieved by intrachain and interchain bonds and the positioning of the light- and heavy-chain components.
Antibody-Antigen Interactions and the Function of the Fab The site on the antibody where the antigenic determinant binds is composed of hypervariable regions with an extremely variable amino acid content. The groove of this antigen binding site has a specific three-dimensional fit for the antigen (figure 3). Because the specificity of the two Fab sites is identical for each antigen, an Ig molecule can bind two epitopes on the same cell or on two separate cells, linking the cells together.
Fig3. Antigen-antibody binding. The union of antibody (Ab) and antigen (Ag) is characterized by a certain degree of fit and is supported by a multitude of weak linkages, especially hydrogen bonds and electrostatic attraction. A better fit (i.e., antigen in (a) vs antigen in (c)) provides greater lymphocyte stimulation during the activation stage.
The goal of secreted antibodies is to bind to the antigen that initiated the antibodies’ formation. From that point, multiple outcomes are possible. (figure 4). Antibodies called opsonins stimulate opsonization, a process in which microorganisms or other particles are coated with specific antibodies so that they will be more readily recognized by phagocytes, which dispose of them. Opsonization has been likened to putting handles on a slippery object to provide a better grip.
Fig4. Summary of antibody functions. Complement fixation, agglutination, and precipitation are covered further in chapter 17.
Antibodies can aggregate or agglutinate cells by cross-linking them into large clumps. Agglutination renders microbes immobile and enhances their phagocytosis. Precipitation is a similar reaction that occurs with small, free antigen molecules. Both processes provide a basis for certain immune tests. The interaction of an antibody with complement can result in the specific rupturing of cells and some viruses. This is seen in complement fixation, which refers to the classical pathway of complement activation.
In neutralization reactions, antibodies fill the surface receptors on a virus or the active site on a bacterial protein, which pre vents them from attaching to their target cells. Antitoxins are special types of antibodies that neutralize bacterial exotoxins.
Functions of the Crystallizable Fragment Although the Fabs bind antigen, the Fc has a different binding function. In most classes of immunoglobulin, the end of the Fc contains an effector that can bind to the membrane of cells, such as macrophages, neutrophils, eosinophils, mast cells, basophils, and lymphocytes. The effect of an antibody’s Fc binding to a cell depends upon that cell’s role. In the case of opsonization, the attachment of antibody to foreign cells and viruses exposes the Fcs to phagocytes.
Certain antibodies have regions on the Fc portion for fixing complement, and in some immune reactions, the binding of the Fc causes the release of cytokines. For example, the Fc end of the antibody of allergy (IgE) binds to basophils and mast cells, which causes the release of allergic mediators such as histamine.
Accessory Molecules on Immunoglobulins All antibodies contain molecules other than the basic polypeptides. Varying amounts of carbohydrates are affixed to the constant regions in most instances (see figure 2b and table 1). Two additional accessory molecules are the J chain, which helps keep the monomers of IgA and IgM together, and the secretory component, which helps move IgA across mucous membranes. These proteins occur only in certain immunoglobulin classes.
Table1. Characteristics of the Immunoglobulin (Ig) Classes
The Classes of Immunoglobulins Immunoglobulins exist as structural and functional classes called isotypes (compared and contrasted in table 1). The differences in these classes are due primarily to variations in the Fc. The classes are differentiated with shorthand names—Ig, followed by a letter: IgG, IgA, IgM, IgD, IgE.
The structure of IgG has already been presented. It is a monomer 4 produced by a plasma cell late in a primary response and by memory cells responding the second time to a given anti genic stimulus. It is by far the most prevalent antibody circulating throughout the blood, lymph, and extracellular fluids. It has numerous functions: It neutralizes toxins, opsonizes, and fixes complement, and it is the only antibody that crosses the placenta and provides protection to the fetus.
The two forms of IgA are (1) a monomer that circulates in small amounts in the blood, and (2) a dimer that is a significant component of the mucous and serous secretions of the salivary glands, intestine, nasal membrane, breast, lung, and genitourinary tract. The dimer, called secretory IgA, is formed in a plasma cell by two monomers held together by a J chain. To facilitate the transport of IgA across membranes, a secretory piece is later added by the epithelial cells of the mucosa. IgA coats the surface of these membranes and appears free in saliva, tears, colostrum, and mucus. It confers essential immunity against enteric, respiratory, and genitourinary pathogens by excluding foreign intruders on mucosal membranes. Its contribution in protecting newborns who derive it passively from nursing is the focus of Clinical Connections.
IgM is a huge molecule composed of five monomers (making it a pentamer) attached by the Fc portions to a central J chain. With its 10 binding sites, this molecule has tremendous capacity for binding antigen. IgM is the first class synthesized following the host’s first encounter with antigen. Its ability to agglutinate and fix complement make it an important antibody in many immune reactions. It circulates mainly in the blood and is far too large to cross the placental barrier.
IgD is a monomer found in minuscule amounts in the serum, and it does not fix complement, opsonize, or cross the placenta. Its main function is to serve as a receptor for antigen on B cells, usually along with IgM, and it is the triggering molecule for B-cell activation.
IgE is also a less common blood component unless one is allergic or has a parasitic worm infection. Its Fc region interacts with receptors on mast cells and basophils. Its biological significance is to stimulate an inflammatory response through the re lease of potent physiological substances by the basophils and mast cells. Because inflammation would enlist blood cells such as eosinophils and lymphocytes to the site of infection, it would certainly be one defense against parasites. Unfortunately, IgE has another, more insidious effect—that of mediating anaphylaxis, asthma, and certain other allergies.
Evidence of Antibodies in Serum
Regardless of the site where antibodies are first secreted, a large quantity eventually ends up in the blood by way of the body’s communicating networks. If one subjects a sample of antiserum (se rum containing specific antibodies) to electrophoresis, the major groups of proteins migrate in a pattern consistent with their mobility and size. The albumins show up in one band, and the globulins in four bands called alpha-1 (α1), alpha-2 (α2), beta (β), and gamma (γ) globulins. Most of the globulins represent antibodies, which explains how the term immunoglobulin was derived. Gamma globulin is composed primarily of IgG, whereas β and α2 globulins are a mixture of IgG, IgA, and IgM.
Monitoring Antibody Production over Time: Primary and Secondary Responses to Antigens
We can learn a great deal about how the immune system reacts to an antigen by studying the levels of antibodies in serum over time (figure 5). This level is expressed quantitatively as the titer, 5 or concentration, of antibodies. Upon the first exposure to an antigen or immunogen, the system undergoes a primary response. The earliest part of this response, the latent period, is marked by a lack of antibodies for that antigen, but much activity is occurring. During this time, the antigen is being concentrated in lymphoid tissue and is being processed by the correct clones of B lymphocytes. As plasma cells synthesize antibodies, the se rum titer increases to a certain plateau and then tapers off to a low level over a few weeks or months.
Fig5. A graphic view of the primary and secondary immune responses to antigenic stimuli. Monitoring the antibody titer or concentration throughout the initial (primary) response and the second (anamnestic) response produces a distinct pattern. Note that the anamnestic response yields an amount of antibody that is nearly 1,000 times that of the primary response (the scale is logarithmic). This in effect “boosts” this defense to even more effective levels.
When the class of antibodies produced during this response is tested, an important characteristic of the response is uncovered. It turns out that, early in the primary response, most of the antibodies are the IgM type, which is the first class to be secreted by plasma cells. Later, the class of the antibodies (but not their specificity) is switched to IgG or some other class (IgA or IgE).
When the immune system is exposed again to the same antigen or immunogen within weeks, months, or even years, a secondary response occurs. The rate of antibody synthesis, the peak titer, and the length of antibody persistence are greatly increased over the primary response. The rapidity and amplification seen in this response are attributable to the memory B cells that were formed during the primary response. Because of its association with recall, the secondary response is also called the anamnestic response. The recall effect shortens the latent or lag period and yields a faster, stronger, and longer-lasting antibody response. The main factors in creating the anamnestic effect are that the memory B cells do not have to go through the early steps in activation, and they do not re quire as many signals to form plasma cells.
The advantage of this response is evident: It provides a quick and potent strike against subsequent exposures to infectious agents. This memory effect is the fundamental basis for vaccine boosters.
Monoclonal Antibodies: Specificity in the Extreme
The value of antibodies as tools for locating or identifying anti gens is well established. For many years, antiserum extracted from human or animal blood was the main source of antibodies for tests and therapy, but most antiserum has a basic problem. It contains polyclonal antibodies, meaning that it is a mixture of different antibodies because it reflects dozens of immune reactions from a wide variety of B-cell clones. This characteristic is to be expected, because multiple immune reactions may be occurring simultaneously. Also, even a single species of microbe has many epitopes and will induce the expression of antibodies with different specificities. Antibodies produced from a single B-cell clone, and that therefore recognize a single antigen, are called monoclonal antibodies (MABs). These antibodies are used to target specific cells in the body that may differ only slightly from other cells; for instance, cancerous lung cells from healthy lung cells. With the advancement of genetic engineering techniques discussed in chapter 10, the creation of MABs has become far easier than it once was, and therapies relying on the use of mono clonal antibodies have grown exponentially, with more than 80 in current use (table 2).
Table2. Selected Monoclonal Antibody–Based Therapies
