Chapter 61: Adaptive Immunity: B Cells & Antibodies

B cells perform two important functions: (1) they differentiate into plasma cells that produce antibodies and (2) they differentiate into long-lasting memory cells that respond robustly and rapidly to reinfection. Antibodies are the principal defense used by the immune system to prevent infection because, by binding to the microbes’ surfaces, they can inhibit them from attaching to target cells and/or recruit innate immune killing mechanisms. Antibodies can also inhibit toxins such as those made by tetanus and diphtheria. Nearly all vaccines are designed to generate these protective, or neutralizing, antibodies.

Advances in cell biology have allowed the generation of large quantities of engineered monoclonal antibodies. The ability of these antibodies to strongly bind a specific antigen with very limited “cross-reactive” binding of other antigens is the basis for many common diagnostic tests and an increasing array of therapies for cancer and inflammatory and infectious diseases (see Monoclonal Antibodies section later in this chapter).

As described in Chapter 59, B cells come from stem cells called common lymphoid progenitors, which give rise to all lymphocytes. Unlike T cells, B-cell precursors differentiate into fully functional B cells in the bone marrow; they do not pass through the thymus. Like T cells, each mature B cell represents a clone, a group of cells that underwent the same heavy chain and light chain rearrangements to end up with the same B-cell receptor (BCR). And like T cells, B-cell clones undergo positive selection and negative selection to ensure that each mature naïve B cell that enters the circulation has a functional BCR that binds to self MHC and does not strongly bind self-antigens. This B-cell clone’s BCR has the same antigen specificity as the antibodies that will be produced by the clone’s offspring cells. Figure 61–1 depicts an overview of the phases of B-cell maturation.

B cells constitute about 30% of the recirculating pool of small lymphocytes. Within lymph nodes, they are located in follicles; within the spleen, they are found in the white pulp. They are also found in gut-associated lymphoid tissue (GALT) such as Peyer’s patches. They express the chemokine receptor CXCR5, which guides them toward chemokines produced in a region called the B-cell follicle. B cells reside in the follicles and survey the lymph and bloodstream for antigens. After binding an antigen, B cells are stimulated to proliferate and “class switch.”

Like T cells, B cells generally require two signals to become activated. Signal 1 is the binding of antigen to the BCR (Figure 61–2). Binding of multiple BCRs leads to cross-linking in which the BCRs are brought close to each other, increasing the amount of second messenger signals sent into the cell. The more BCRs that are cross-linked by antigen, the stronger the signal will be. As we will discuss below, signal 2 can come from a variety of sources. What the various types of signal 2 have in common is that they are inflammatory, meaning they only accompany foreign antigens that represent a real threat to the host.

Naïve B cells are short lived; without signal 2, they fail to achieve activation and are either deleted by apoptosis or become anergic, a state of nonresponsiveness. The requirement for inflammatory input from signal 2 at the time of activation is a safeguard so that B cells are not inadvertently activated by harmless antigens. Upon activation, B cells can either differentiate into long-lived plasma cells, which secrete more antibody, or into long-lived memory B cells, which wait in the follicles of the secondary lymphoid organs to respond to a reinfection.

T-Cell–Independent Activation

In some circumstances, B cells can be activated by a strong signal 1 and signal 2 and do not need T-cell help (see Figure 61–2A). Antigens that activate B cells without T-cell help are usually large multivalent molecules such as the chains of repeating sugars that make up bacterial capsular polysaccharide. The repeated subunits act as a multivalent antigen that cross-links many IgM antigen receptors on the B cell and sends a strong activating signal 1 into the B cell. Other macromolecules, such as lipids, DNA, and RNA, can also provide signal 1 to the B cell if these antigens are recognized by its surface BCR.

During T-cell–independent activation, the B cell’s signal 2 can come from various innate (T-cell–independent) inflammatory sources: (1) like many cells, B cells have pattern recognition receptors that can recognize pathogen-associated molecular patterns (PAMPs); (2) B cells have the complement receptor CR2, which can recognize cleavage products of C3b released during complement activation (see Chapter 63); and (3) a vaccine adjuvant can activate a B cell without requiring T-cell help (see Chapter 57). These responses typically occur outside the B-cell follicle, and the plasma cells generated by T-cell–independent activation are relatively short-lived.

The T-cell–independent response is significant because it is the main response to bacterial capsular polysaccharides; these molecules are not proteins that can be processed and presented by antigen-presenting cells to T cells. For example, the pneumococcal polysaccharide vaccine contains the surface polysaccharides of the 23 most common serotypes of Streptococcus pneumoniae along with an adjuvant but no carrier protein. Together, the polysaccharide (signal 1) and adjuvant (signal 2) strongly activate B cells. However, because the vaccine does not contain peptides, which are the only type of antigen recognized by T cells, the activation of B cells by these polysaccharides is considered to be T-cell–independent.

T-Cell–Dependent Activation

The previous example illustrates an important concept for vaccine design, but, in general, antibodies generated independently of T-cell help are short-lived and are less specific for their antigens compared with antibodies generated with T cell help. The strongest and most specific antibody response requires the participation of dendritic cells (DCs) and T cells. To describe T-cell–dependent activation of B cells, we first need the activation of naïve T cells (see Figure 61–2B, right side). As described in Chapter 60, CD4-positive T cells are activated by DCs presenting a foreign peptide complexed with class II major histocompatibility complex (MHC) proteins, along with co-stimulation.

Consider a T cell activated by a peptide that undergoes clonal proliferation. Some of the clone’s offspring will differentiate into T follicular helper (Tfh) cells (see Chapter 60). Recall that both DCs and T cells express the chemokine receptor CCR7. Upon activation, a Tfh cell turns off CCR7 and turns on CXCR5, enabling it to migrate from the T-cell zone into the B-cell follicle.

While this process is occurring, antigen fragments of the same foreign entity circulate into the B-cell follicles of the secondary lymphoid tissue and interact directly with the antigen receptors (which are membrane-bound IgM molecules) of naïve B cells. The recognized epitopes of these circulating antigens might be lipid, polysaccharide, or nucleic acid components, but some component of the antigen must also contain the same peptide. The B cell then uses its BCR to take up the antigen into endosomes, and the antigen is processed. This B cell can now function as a professional antigen-presenting cell. The antigen is processed and its peptide components are complexed with class II MHC molecules and presented on the B cell’s surface to interact with the T-cell receptors of Tfh cells at the border of the T-cell zone. Note that the Tfh cell recognizes the peptide, but the antibody from that B cell will recognize whatever lipid, polysaccharide, nucleic acid, or protein that initially bound to its BCR; the antibody is not necessarily determined by the peptide involved in the DC-T cell.

Class Switching & Affinity Maturation

If a Tfh cell recognizes the antigen peptide presented by the B cell’s class II MHC molecules, the Tfh cell provides two key signals back to the B cell: first, CD40 ligand (CD40L) molecules on the Tfh cell bind to CD40 on the B cell; and second, the Tfh cells produce the cytokine interleukin (IL)-21. Together, these signals have three important effects on the B cells: (1) they begin to proliferate rapidly; (2) they start to switch “class,” changing from using the Cμ segment to using one of the other heavy chain C_H segments (Cγ, Cε, or Cα) (see Figure 61–3); and (3) they start a process of somatic hypermutation. Genetic deficiency of the gene encoding CD40L causes an immunodeficiency called hyper-IgM syndrome. Patients with this disease have very high immunoglobulin (Ig) M levels and very little IgG, IgA, and IgE because their B cells are unable to receive T-cell help and therefore are unable to proliferate and “class switch.” Hyper-IgM syndrome is characterized by severe bacterial infections (see Chapter 68). As a B-cell clone divides, class switches, and hypermutates, the newly formed cluster of cells is called a germinal center.

Both B-cell class switching and somatic hypermutation are directed by the enzyme activation-induced cytidine deaminase (AID). For class switching, AID makes double-strand breaks in the DNA of the C_H locus of the heavy chain, removing the intervening DNA between the VDJ region and either Cγ, Cε, or Cα (see Figure 61–3). This causes irreversible switching of that IgM-positive B cell to instead express surface IgG, IgE, or IgA. The decision of whether to switch to IgG, IgE, or IgA is made based on the cytokine signals that the B cell receives:

1. IL-21 plus gamma interferon (IFN-γ) → IgG. This makes sense because IFN-γ is the cytokine associated with macrophage activation, and it is the same cytokine that generates the antibody most associated with opsonization and phagocytosis.

2. IL-21 plus IL-4 → IgE. This makes sense because IL-4 is one of the main cytokines associated with Th-2 immunity, and it is the same cytokine that generates the antibody most associated with mast cell, basophil, and eosinophil activity. Patients with allergic diseases caused by excess IgE often have excess IL-4.

3. IL-21 plus various “mucosal” cytokines → IgA. This makes sense because the cytokines in mucosal barriers induce antibodies that are secreted across mucosal surfaces. (A deficiency in the gene encoding the receptor for some these cytokines causes IgA deficiency, which can present with serious sinopulmonary and gastrointestinal infections.)

Recall that the variable region of an antibody is responsible for binding to antigen, and because the variable region is not affected by class switching, the resulting IgG, IgE, or IgA antibodies should have the same antigen specificities. However, AID does something else. It also makes nucleotide substitutions in the gene regions that encode the V_H and V_L chains. This results in the exchange of new amino acids into the antigen-binding hypervariable region, massively increasing the potential diversity of the B-cell pool.

With successive cell division and new mutations, the enlarging pool of B cells continue to compete for the circulating antigens that are present in the follicle; those B cells with higher affinity surface immunoglobulins will be more likely to bind and take up the antigens and therefore more likely to present the correct peptides to CD40L-positive Tfh cells, whereas B cells with lower affinity immunoglobulins will be outcompeted, will not receive the Tfh cell’s survival signals, and therefore will die. This process is called affinity maturation, and over multiple rounds of cell division, mutation, competition, and selection, a pool of highly specific B-cell clones evolves from the initial germinal center (Figure 61–4). Many germinal centers in many secondary lymphoid organs are engaged with each infection, ensuring a broad polyclonal antibody response.

T-cell “help,” in the form of IL-21 and CD40L, is also the main stimulus that drives B cells to differentiate into long-lived plasma cells that reside in the secondary lymphoid organs or in the bone marrow. This requirement of T-cell help might make class switching, affinity maturation, and plasma cell development seem unnecessarily cumbersome, but remember that the T cells have been carefully selected in the thymus not to see “self” peptides, and therefore, the involvement of T cells in B-cell activation is an additional safeguard against autoimmunity. Compared with T-cell–independent activation, activation of naïve B cells in the presence of Tfh cells generates much higher titers of IgG, IgA, and IgE antibodies, longer-lived plasma cells, and a much stronger response upon reinfection.

The concept of T-cell help for B cells was used in making an improved pneumococcal “conjugate” vaccine. The polysaccharides from common serotypes of S. pneumoniae were conjugated to a highly immunogenic protein. The vaccine is taken up by DCs, which process the protein component to be recognized by the T cells that become Tfh cells. In contrast, the B cells that are activated by the vaccine recognize the polysaccharide component, but once they bind the polysaccharide, they also take up and process the conjugated protein. Like the DCs, the B cells process this protein component and present the peptides to the newly activated Tfh cells. In this way, T-cell help is recruited to the follicle, generating high titers of antibody specific for the polysaccharide (see Figure 61–2).

The primary response occurs the first time that an antigen is encountered. This usually involves T-cell–dependent activation of B cells, as described earlier (see Figure 61–2). Most of the B cells activated from an initial exposure undergo class switching and affinity maturation and differentiate into plasma cells that produce large amounts of antibody specific for the epitope recognized by their immunoglobulin receptor. Plasma cells secrete thousands of antibody molecules per second for a life span that lasts from a few days to months.

The first antibodies are detectable in the serum after around 7 to 10 days in a primary response but can be longer depending on the nature and dose of the antigen and the route it takes to the secondary lymphoid organ (e.g., bloodstream or draining lymphatics). The serum antibody concentration continues to rise for several weeks and then declines and may drop to very low levels. As shown in Figure 61–5, the first antibodies to appear in the primary response are IgM, followed by IgG, IgE, or IgA, as more class-switched plasma cells are generated. IgM levels decline earlier than IgG levels because these plasma cells have shorter life spans.

Although most activated B cells become plasma cells, a small fraction of activated B cells become memory B cells, which can remain quiescent in the B-cell follicles for long periods but are capable of being activated rapidly upon reexposure to antigen. Most memory B cells have surface IgG that serves as the antigen receptor, but some have IgM.

When there is a second encounter with the same antigen or a closely related (or cross-reacting) antigen, months or years after the primary response, the secondary response is both more rapid (the lag period is typically only 3 to 5 days) and generates higher levels of antibody than did the primary response (see Figure 61–5). The memory B cells, which underwent some degree of affinity maturation during the primary response, now proliferate to form a new germinal center and repeat the process of competing for Tfh help (i.e., affinity maturation). This means that with each succeeding exposure to the antigen, the antibodies tend to bind antigen with higher affinity because the memory B cells are subjected to further rounds of affinity maturation (see Figure 61–4). During the secondary response, the amount of IgM produced is similar to that after the first contact with antigen. However, the secondary response also generates IgG plasma cells that are greater in number and longer-lived than those in the primary response, meaning secondary IgG levels are higher and tend to persist longer (see Figure 61–5).

This concept is medically important because the protection afforded by the first administration of a vaccine is less than that afforded by a booster shot. Furthermore, booster doses of vaccines improve antibody binding through new rounds of affinity maturation.

When two or more antigens are administered at the same time, the host reacts by producing antibodies to all of them. Competition of antigens for antibody-producing mechanisms occurs experimentally but appears to be of little significance in medicine. Combined immunization is widely used and shown to be just as effective as single immunization (e.g., the diphtheria, tetanus, pertussis [DTP] vaccine and the measles, mumps, rubella [MMR] vaccine).

The primary function of antibodies is to protect against infectious agents or their products (Table 61–1). Antibodies provide protection because they can (1) activate complement to cause direct lysis of cell membranes and promote inflammation (the “classical” pathway; see Chapter 63); (2) opsonize bacteria, which can occur with or without complement; (3) stimulate immune cells through their Fc receptors to lyse a target cell, also called antibody-dependent cellular cytotoxicity (ADCC); and (4) bind and neutralize toxins and viruses.

Note that this last mechanism—the highly specific, high-affinity, noncovalent binding of an antibody’s Fab to its target—is independent of the Fc region. This mechanism is the basis for many therapeutic blocking antibodies. Some examples include antibodies that inhibit the signaling of cytokines or chemokines used by pathogenic self-reactive cells to cause inflammatory or autoimmune diseases. Other antibodies block growth factors that these cells, or cancer cells, need to survive.

Opsonization is the process by which antibodies make microbes more easily ingested by phagocytic cells. This occurs by either of two reactions: (1) the Fc portion of IgG interacts with its receptors on the phagocyte surface to facilitate ingestion or (2) IgG or IgM activates complement to yield C3b, which interacts with its receptors on the surface of the phagocyte. Table 61–1 is a summary of the functions of immunoglobulins.

As described in Chapter 57, antibodies can be induced actively in the host or transferred passively and are thus immediately available for defense. In medicine, passive immunity is used in the neutralization of the toxins of diphtheria, tetanus, and botulism by antitoxins and in the inhibition of such viruses as rabies and hepatitis A and B viruses early in the incubation period. Table 61–2 is a summary of the properties of the various classes of immunoglobulins.

Because immunoglobulins are proteins, they themselves can also be antigens and can be detected with anti-immunoglobulin antibodies. This property is used to diagnose patients with an infection. In other words, rather than trying to detect a particular microbe, it is often easier to detect the serum antibody specific for that microbe using an anti-immunoglobulin antibody. This is the basis for many tests for infections, including human immunodeficiency virus (HIV) tests. It can also be used to determine the presence of self-reactive antibodies, which cause autoimmune diseases such as myasthenia gravis. Antibodies that bind to different portions of other antibodies are used to subdivide the target antibodies into groups called isotypes and allotypes.

1. Isotypes are defined by amino acid differences in their Fc regions. For example, the different antibody classes (IgM, IgD, IgG, IgA, and IgE) are all different isotypes; the constant regions of their H chains (μ, δ, γ, α, and ε) are different.

2. Allotypes are antibodies that might be of the same isotype but that have additional features that vary among individuals. They vary because the genes that code for the light and heavy chains are polymorphic, and individuals can have different alleles. For example, the γ heavy chain contains an allotype called Gm, which varies by one or two amino acids between individuals.

IgG

Each IgG molecule consists of two L chains and two H chains linked by disulfide bonds (molecular formula H2L2) (see Figure 61–6C). Because it has two identical antigen-binding sites, it is said to be divalent. There are four subclasses, IgG1 to IgG4, based on antigenic differences in the H chains and on the number and location of disulfide bonds. IgG1 makes up most (65%) of the total IgG. IgG2 antibody is directed against polysaccharide antigens and is an important host defense against encapsulated bacteria.

IgG is the predominant antibody in the secondary response and constitutes an important defense against bacteria and viruses (see Table 60–1). IgG is the only antibody to cross the placenta; only its Fc portion binds to receptors on the surface of placental cells (see Table 61–2). This receptor, called FcRn, transports maternal IgG across the placenta into the fetal blood. IgG is therefore the most abundant immunoglobulin in newborns. This is an example of passive immunity because the IgG is made by the mother, not by the fetus (see Chapter 57). Another important attribute of IgG is that it is one of the two immunoglobulins that can activate complement; IgM is the other (see Chapter 63).

IgG is the immunoglobulin that opsonizes. It can opsonize (i.e., enhance phagocytosis) because there are receptors for the γH chain, called Fcγ receptors, on the surface of phagocytes. These Fcγ receptors are also found on natural killer (NK) cells, which are responsible for ADCC. If a target cell’s membrane antigens are sensed as foreign and bound by the Fab portion of an IgG antibody (which might occur if the cell is from a transplant graft or if it is infected by a virus), then the Fc portion of these antibodies can in turn bind to and activate the surface Fcγ receptors of the NK cell. This triggers the NK cell to release its cytotoxic mediators, including perforins and proteases, killing the target cell.

IgG has various sugars attached to the heavy chains, especially in the C_H2 domain. The medical importance of these sugars is that they determine whether IgG will have a proinflammatory or anti-inflammatory effect. For example, if the IgG molecule has a terminal N-acetyl glucosamine, it is proinflammatory because it will bind to mannose-binding ligand and activate complement (see Chapter 63 and Figure 63–1). In contrast, if the IgG has a sialic acid side chain, then it will not bind and becomes anti-inflammatory. Thus, IgG proteins specific for a single antigen that are of the same clone (i.e., made by the same plasma cell) can, at various times, possess different properties depending on these sugar modifications.

IgA

IgA is the main immunoglobulin in secretions such as colostrum, saliva, tears, and respiratory, intestinal, and genital tract secretions. It prevents attachment of microorganisms (e.g., bacteria and viruses) to mucous membranes. Each secretory IgA molecule consists of two H2L2 units plus one molecule each of J (joining) chain and secretory component (Figure 61–6A and B). (The J chain is only found in IgA and IgM, which are the only immunoglobulins that exist as multimers. The J chain initiates the process that forms the disulfide bonds that bind multiple heavy chains into a multimer.) The two heavy chains in IgA are α heavy chains.

The secretory component is a polypeptide synthesized by epithelial cells that provides for IgA passage to the mucosal surface. It also protects IgA from being degraded by proteases in the intestinal tract. In serum, some IgA exists as monomeric H2L2.

IgM

IgM is the main immunoglobulin produced early in the primary response. It is present as a monomer on the surface of virtually all B cells, where it functions as an antigen-binding receptor. In serum, IgM is a pentamer composed of five H2L2 units plus one molecule of J (joining) chain (see Figure 61–6D). IgM has a μ heavy chain, and therefore, it cannot bind to Fcγ receptors to facilitate opsonization or ADCC. However, IgM does bind to complement, and the resulting C3b does opsonize because there are complement receptors on the surface of phagocytes (see Chapter 63). Because the IgM pentamer has 10 antigen-binding sites, it is the most efficient immunoglobulin in agglutination, complement activation, and other antibody reactions and is important in defense against bacteria and viruses. It can be produced by the fetus in certain infections. It has the highest avidity of the immunoglobulins; its interaction with antigen can involve all 10 of its binding sites.

IgD

This immunoglobulin has no known antibody function but may function as an antigen receptor. It is present on the surface of many B lymphocytes and is present in small amounts in serum.

IgE

IgE is medically important for two reasons: (1) it mediates immediate (anaphylactic) hypersensitivity (see Chapter 65) and (2) it participates in host defenses against certain parasites (e.g., helminths [worms]) (see Chapter 56). The Fc region of IgE binds to Fcε receptors on the surface of mast cells and basophils. Bound IgE then becomes a receptor for antigen (allergen). When the antigen-binding sites of adjacent IgEs are cross-linked by allergens, several mediators are released by the cells, and immediate (anaphylactic) hypersensitivity reactions occur (see Figure 65–1). Although IgE is present in trace amounts in normal serum (approximately 0.004%), persons with allergic reactivity have greatly increased amounts, and IgE may appear in external secretions. IgE does not bind to complement and does not cross the placenta.

IgE is the main host defense against certain important helminth (worm) infections, such as Strongyloides, Trichinella, Ascaris, and the hookworms Necator and Ancylostoma. The serum IgE level is usually increased in these infections. Because worms are too large to be ingested by phagocytes, they are killed by eosinophils that release worm-destroying enzymes. IgE specific for worm proteins binds to Fcε receptors on eosinophils, triggering the ADCC response with release of major basic protein from the eosinophil’s granules.

In general, the fetus and the newborn have an underdeveloped immune system that responds weakly to infections and vaccines. Antibodies in the fetus are primarily IgG acquired by transfer of maternal IgG across the placenta. This is why it is important to confirm the mother’s vaccine history to ensure the newborn will be protected.

Some antibodies can be made by the fetus if infection occurs, such as in congenital syphilis. After birth, it generally takes months until newborn infants can make IgG (and other isotypes, such as IgM and IgA). There are some exceptions, for example, the vaccine against hepatitis B is effective when given to newborns. But most vaccines are delayed by several weeks or months after birth to ensure the newborn’s immune system has developed enough to respond. During this time, maternal IgG gradually declines, and protection from maternal IgG is lost by 3 to 6 months. The risk of infections begins to increase over this time, which is why the first set of vaccines is usually recommended by 2 months.

The antibodies you make against a single vaccination or infection are usually made by many different clones of B cells (i.e., they heterogeneous, or polyclonal). Antibodies that arise from a single clone of cells are homogeneous, or monoclonal. Often, a plasma cell malignancy, such as multiple myeloma, can be diagnosed because a single proliferating clone might produce abnormally high levels of a monoclonal immunoglobulin, usually IgG.

The remarkable specificity of an antibody’s hypervariable region for its target molecule has made monoclonal antibodies an invaluable resource in laboratory research and clinical applications. In the 1970s, the first method of generating virtually unlimited quantities of monoclonal antibodies in the laboratory was described (Figure 61–7). Hybridoma cells, formed by the fusion of two different cells, can be created by (1) isolating spleen cells from an animal (e.g., a mouse) previously immunized with an antigen of interest and (2) mixing these cells in a culture dish with mouse myeloma cells (which grow indefinitely in culture but do not make antibodies) so that the two cell types fuse. The newly formed hybridoma cells produce of antibodies against the antigen of interest. More recent advances in molecular and cell biology have further refined this approach to create therapeutic antibodies (see box “Creating Therapeutic Antibodies”) that are now used in a variety of clinical situations, for example, to suppress immune responses in transplantation or autoimmune disease, to treat cancer, and to prevent infectious disease.

Evaluation of humoral immunity consists primarily of measuring the amount of each of the three important immunoglobulins (i.e., IgG, IgM, and IgA) in the patient’s serum. To evaluate patients suspected of having an immunodeficiency, it may be necessary to count B-cell numbers by flow cytometry, as described in Chapter 60 for T cells. It is also possible to test for B-cell function in vivo. This is done by determining a “baseline” titer of antibody against one or a few particular antigens (e.g., serotypes of S. pneumoniae), followed by immunization (e.g., with a S. pneumoniae vaccine), and repeat testing after several weeks. The absence of a normal rise in IgM and IgG after immunization indicates a defect, either an intrinsic defect in the B cells themselves or an extrinsic defect that, for example, inhibits T cells’ capacity to provide help to activate the B cells.