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.
Overview of B-cell activation. A: T-cell–independent Response. T-cell–independent antigens are large multivalent structures, often polysaccharides (purple) of a bacterial cell (gray circles), that cross-link many IgM receptors to achieve a strong activation signal 1. Signal 2 might include complement C3b derivatives (light blue) bound to the bacterial cell (shown on upper left of cell), or pathogen-associated molecular patterns (gray bars, shown binding to pattern recognition receptor on upper right of cell). Without T-cell help, these responses are short-lived and dominated by IgM plasma cells, although some IgG is also generated. Note that the IgM receptors initially recognize and bind the polysaccharide, so all resulting antibodies will be specific for that polysaccharide. B: T-cell–dependent Response. A T-cell–dependent antigen must contain at least some protein component. The B-cell receptor (BCR) binds a specific part of the antigen, it is endocytosed by the B cell, and the peptides are processed and complexed with class II major histocompatibility complex (MHC) proteins. During the germinal center reaction, B cells compete to present antigen to peptide-specific T follicular helper (Tfh) cells that had been previously activated by dendritic cells presenting the same peptide fragment of the antigen. In the germinal center, the B-cell clones that receive more CD40 ligand (CD40L) and cytokines are able to proliferate, class switch, and become long-lived memory B cells and plasma cells.
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.
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.
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 CH 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.
Class switching. T-cell help induces activation-induced cytidine deaminase (AID)-driven class switching. Activated IgM-positive B cells receive help from T follicular helper (Tfh) cells, including CD40 ligand (CD40L) and interleukin (IL)-21. This causes AID to create double-strand DNA breaks in the heavy chain locus that remove Cμ and Cδ and bring the VDJ region adjacent to one of the other C regions, either γ, ε, or α. After RNA splicing, the B cell begins to express IgG, IgE, or IgA instead of IgM.
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 CH 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:
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.
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.
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 VH and VL 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.
Germinal center reaction. B cells compete for antigen to receive T-cell help in the germinal center reaction. Naïve IgM-positive B cells survey for antigens in the B-cell follicle. Those that bind antigen (light brown) are selectively activated to engulf and process the antigen and present the peptides to T follicular helper (Tfh) cells. If a Tfh cell recognizes the peptide, it provides help (CD40L and interleukin [IL]-21), and B-cell clonal expansion initiates the germinal center reaction: (1) Repeated rounds of activation-induced cytidine deaminase (AID)-driven somatic hypermutation in the clones alter the specificity of the surface IgM for the antigen. Clones that out-compete their neighbors for antigens in the follicle (darker brown nuclei) enable them more interactions with Tfh cells, leading to progressive affinity maturation. (2) Tfh cytokines induce AID-driven class switching. (Note: In this case, gamma interferon (IFN-γ) signaled the B cells to class switch to IgG. If the Tfh cells provided IL-4, the B cells would class switch to IgE.) The successful clones either become long-lived plasma cells that leave the follicle or have the potential to become circulating memory B cells expressing IgG.
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).