Pre-B cells can be divided into large mitotically active pre-B cells and small non-dividing pre-B cells. Both large and small pre-B cells express Igμ heavy chains in the cytoplasm (cμ) and the pre-B cell–receptor complex on their surface (Fig. 9.w2). Large pre-B cells have successfully rearranged their Ig heavy chain genes. As these cells pass from the large pre-B cell group into the small pre-B cell group, they begin to rearrange their Ig light chain genes and upregulate RAG-1 and RAG-2. The final stage of B-cell development is the immature B-cell stage. Immature B cells have successfully rearranged their light chain genes and express IgM. Once again, RAG-1 and RAG-2 expression has been downregulated. As immature B cells develop further into mature B cells, they begin to express both IgM and IgD on their surface. These mature B cells are then free to exit the bone marrow and migrate into the periphery.

Fig. 9.w2 The pre-B cell receptor

The surrogate B-cell receptor complex is composed of a μ heavy chain and a V_pre-B and λ5 proteins (surrogate light chains). The receptor has a role in early differentiation and development of B cells. The unit shown here associates with the signaling molecules Igα and Igβ.

Q. What is the significance of the observation that RAG genes are expressed at two distinct times during B cell development?

A. The heavy and light chain genes undergo rearrangements at different times and the RAGS are expressed at the times when recombination is taking place in either the heavy or light chain gene loci.

Other phenotypic markers such as CD25 (IL-2Rα chain), CD79a (Igα), CD79b (Igβ), and c-Kit can help to identify particular populations of pro-B, pre-B or immature B cells (see Fig. 9.w1).

Cytokines required for B cell development

Several cytokines affect B-cell development. Common lymphoid progenitors (CLP) are responsive to IL-7 which promotes B-cell lineage development. Mice deficient in IL-7 or IL-7R exhibit an early arrest in B-cell development at the pro-B cell stage.

Blys (B-lymphocyte stimulator) signaling through its receptor BR3 is important for the survival of pre-immune B-cell stages from transition stage onwards. This is supported by the observation of a developmental block at the transitional B-cell stage in Blys deficient animals. Blys is also required for the later survival of mature B-cells.

In addition, a number of growth and differentiation factors are required to drive the B cells through early stages of development. Receptors for these factors are expressed at various stages of B-cell differentiation. IL-4, IL-3 and low-molecular-weight B cell growth factor (L-BCGF) are important in initiating the process of B-cell differentiation, whereas other factors are active in the later stages.

Transcriptional controls in B-cell development

B-lymphopoiesis is regulated by multiple transcription factors including:

• E2A and EBF, which coordinately activate early B-cell genes;

• Pax5, which ensures development to B-cell lineages by restricting transcription of lineage inappropriate genes and activating expression of B-lineage signaling molecules;

• Sox4 and LEF1, which promote the survival and proliferation of pro-B cells;

• IRF4 and IRF8, which terminate pre-BCR signaling by IRF4 and promote differentiation to small pre-B cells; and

• Bcl-6 expression, which is required for germinal B-cell differentiation and generation of memory B-cells.

In contrast expression of Blimp-1 supresses Bcl-6 expression and is required for development of Ig secreting cells and maintenance of long lived plasma cells.

Abnormalities in B-cell development

Several mutations in man and mouse have identified key molecules in the ontogeny of B-cells. The importance of pre-BCR signaling in B-cell development is highlighted by the immunodeficiencies, XLA in humans and Xid in mice, which lead to a block at the pro-B cell to large pre-B cell transition in the bone marrow. This defect is mostly caused by a mutation in the gene encoding the enzyme Bruton’s tyrosine kinase (Btk). Btk is a key enzyme involved in signal transduction downstream of the pre-BCR and BCR. XLA patients have very few circulating B-cells amd negligible serum immunoglobulin (Ig).

While XLA accounts for ~85% of the cases of agammaglobulinemia, the rest are characterized by mutations in μHC, λ5, Igα (CD79a), Igβ (CD79b), and BLNK (SLP-65), all affecting pre-BCR functions.

Common variable immunodeficiency (CVID) impacts later stages of B-cell development. CVID manifests in reduced serum Ig, memory B-cells, class switch recombination and B-cell activation. Mutations in CD40 ligand on T-cells, B-cell surface receptor CD19, activated T-cell costimulatory molecule ICOS, and TACI (another receptor for Blys) have all been identified in CVID patients.

B cell activation

T-independent antigens do not require T cell help to stimulate B cells

The immune response to most antigens depends on both T cells and B cells recognizing the antigen in a linked fashion. This type of antigen is called a T-dependent (TD) antigen.

A small number of antigens, however, can activate B cells without MHC class II-restricted T cell help and are referred to as T-independent (TI) antigens (Fig. 9.1).

Fig. 9.1 T-independent antigens

The major common properties of some of the main T-independent antigens are listed. T-independent antigens induce the production of cytokines IL-1, TNFα and IL-6 by macrophages. (Note: both poly-L amino acids and monomeric bacterial flagellin are T-dependent antigens, demonstrating the role of antigen structure in determining T-independent properties.)

Importantly, many TI antigens are particularly resistant to degradation. TI antigens can be divided into two groups (TI-1 and TI-2) based on the manner in which they activate B cells:

• TI-1 antigens are predominantly bacterial cell wall components – the prototypical TI-1 antigen is lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria;

• TI-2 antigens are predominantly large polysaccharide molecules with repeating antigenic determinants (e.g. Ficoll, dextran, polymeric bacterial flagellin, and poliomyelitis virus).

Q. What common characteristic can you see in many of the TI antigens, and how are such antigens recognized by the immune system?

A. Many TI antigens have pathogen-associated molecular patterns (PAMPs) recognized by Toll-like receptors. They therefore have an intrinsic ability to activate the immune system irrespective of their ability to bind to the specific antigen receptor on individual B cell clones.

Many TI-1 antigens possess the ability in high concentrations to activate B cell clones that are specific for other antigens – a phenomenon known as polyclonal B cell activation. However, in lower concentrations they only activate B cells specific for themselves. TI-1 antigens do not require a second signal.

TI-2 antigens, on the other hand, are thought to activate B cells by clustering and cross-linking immunoglobulin molecules on the B cell surface, leading to prolonged and persistent signaling. TI-2 antigens require residual non-cognate T cell help, such as cytokines.

Several signal transduction molecules are necessary for mediating TI antigen responses in B cells. These include CD19, HS1 protein and Lyn.

T-independent antigens induce poor memory

Primary antibody responses to TI antigens in vitro are generally slightly weaker than those to TD antigens. They peak fractionally earlier and both generate mainly IgM. However, the secondary responses to TD and TI antigens differ greatly. The secondary response to TI antigens resembles the primary response, whereas the secondary response to TD antigens is far stronger and has a large IgG component (Fig. 9.2). It seems, therefore, that TI antigens do not usually induce the maturation of a response leading to class switching or to an increase in antibody affinity, as seen with TD antigens. This is most likely due to the lack of CD40 activation (see below). Memory induction to TI antigens is also relatively poor.

Fig. 9.2 Comparison of the secondary immune response to T-dependent and T-independent antigens in vitro

The secondary response to T-dependent antigens is stronger and induces a greater number of IgG-producing cells, as measured by plaque-forming cells (see Method box 9.1).

There are potential survival advantages if the immune response to bacteria does not depend on complex cell interactions, as it could be more rapid. Many bacterial antigens bypass T-cell help because they are very effective inducers of cytokine production by macrophages – they induce IL-1, IL-6 and tumor necrosis factor-α (TNFα) from macrophages. The short-lived response and lack of IgG may also be due to lack of costimulation via CD40L and lack of IL-2, IL-4 and IL-5, which T cells produce in response to TD antigens.

It is possible to convert TI antigens into T-dependent antigens by altering their structure. For example, pneumococcal polysaccharides are TI antigens and do not induce immunological memory or antibodies in infants. However, conjugation of pneumococcal polysaccharides to a carrier protein induces polysaccharide-specific antibody in infants, and memory similar to T-dependent antigens (see Method boxes 9.1 and 2.1).

Method box 9.1 Measuring antibody production – the PFC assay and ELISPOT

Various methods have been developed for assaying antibody production. Two such methods are the plaque-forming cell (PFC) assay and the enzyme-linked immunospot assay (ELISPOT).

Antibody-forming cells (AFCs) are measured by means of a quantitative PFC assay (Fig. MB9.1.5), originally developed by Niels Jerne in the 1960s. B cells (e.g. spleen cells) are plated in agar with sheep red blood cells sensitized by binding the specific antigen to their surface. Antibody produced by any B cell will coat the red blood cells. Following the addition of complement, these coated cells may be lyzed, causing the appearance of a zone of lyzed cells (plaque) in the agar. Figure MB9.1.5 shows the appearance of such a plaque, with a B cell in the center, under the microscope. The plaques are then counted to give a quantitative measure of the number of PFCs.

Another way of detecting antibody-producing cells is by means of an enzyme-linked immunospot assay (ELISPOT). An ELISPOT assay starts out by coating a plastic well with antigen and adding a known quantity of B cells. The antigen coated onto the plastic will then capture any antibody in the vicinity of the activated B cell that is producing the antibody. After a period of time, the B cells are removed, and the specific antibody can be detected by adding an enzyme-labeled anti-immunoglobulin plus chromogen. Development of the label in this assay results in a spot surrounding the active B cell. Counting each spot and knowing the quantity of B cells originally added to the well allows one to enumerate the frequency of B cells producing the specific antibody. Method Box 2.1, Fig. MB2.1.3 shows the method of detecting antibodies and the appearance of the spots on the developed plates. In addition to analyzing specific antibody-secreting B cells, the ELISPOT assay has been adapted to measure the frequency of cytokine-secreting T cells and various other cell types (right panel). With the improvement in ELISPOT assay plate design and in ELISPOT detection equipment, antibody- or cytokine-secreting cells can now be detected at the single cell level.

Fig. MB9.1.5 Plaque-forming cell (PFC) assay.

T-independent antigens tend to activate the CD5⁺ subset of B cells

TI antigens predominantly activate the B-1 subset of B cells found mainly in the peritoneum. These B-1 cells can be identified by their expression of CD5, which is induced upon binding of TI antigens. In contrast to conventional B cells, B-1 cells have the ability to replenish themselves.

Activation of B cells by T-dependent antigens

T cells and B cells recognize different parts of antigens

In the late 1960s and early 1970s, studies by Mitchison and others, using chemically modified proteins, led to significant advances in understanding of the different functions of T cells and B cells. To induce an optimal secondary antibody response to a small chemical group or hapten (which is immunogenic only if bound to a protein carrier), it was found that the experimental animal must be immunized and then challenged using the same hapten–carrier conjugate – not just the same hapten. This was referred to as the carrier effect.

By manipulating the cell populations in these experiments, it was shown that:

• TH cells are responsible for recognizing the carrier; whereas

• the B cells recognize hapten.

These experiments were later reinforced by details of how:

• B cells use antibody to recognize epitopes; while

• T cells recognize processed antigen fragments.

One consequence of this system is that an individual B cell can receive help from T cells specific for different antigenic peptides provided that the B cell can present those determinants to each T cell.

In an immune response in vivo, it is believed that the interactions between T and B cells that drive B cell division and differentiation involve T cells that have already been stimulated by contact with the antigen on other antigen-presenting cells (APCs), for example dendritic cells.

This has led to the basic scheme for cell interactions in the antibody response set out in Figure 9.3. It is proposed that antigen entering the body is processed by cells that present the antigen in a highly immunogenic form to the TH and B cells. The T cells recognize determinants on the antigen that are distinct from those recognized by the B cells, which differentiate and divide into antibody-forming cells (AFCs). Therefore two processes are required to activate a B cell:

• antigen interacting with B cell immunoglobulin receptors – this involves ‘native’ antigen;

• stimulating signal(s) from TH cells that respond to processed antigen bound to MHC class II molecules.

Fig. 9.3 Cell cooperation in the antibody response

Antigen is presented to virgin T cells by antigen-presenting cells (APCs) such as dendritic cells. B cells also take up antigen and present it to the T cells, receiving signals from the T cells to divide and differentiate into antibody-forming cells (AFCs) and memory B (Bm) cells.

B-cell activation and T-cell activation follow similar patterns

In B cells, the signaling function of CD3 is carried out by a heterodimer of Igα and Igβ. Two molecules of the Igα/Igβ heterodimer associate with surface Ig to form the B-cell receptor (BCR). The cytoplasmic tails of Igα and Igβ carry immunoreceptor tyrosine activation motifs (ITAM).

Cross-linking of surface Ig leads to activation of the src family kinases, which in B cells are Fyn, Lyn, and Blk. Syk is analogous to ZAP-70 in T cells, and binds to the phosphorylated ITAMs of Igα and Igβ (Fig. 9.4). This leads to activation of a kinase cascade and translocation of nuclear transcription factors analogous to the process that occurs in T cells.

Fig. 9.4 Intracellular signaling in B cell activation

B-cell activation is similar to T-cell activation. If membrane Ig becomes cross-linked (e.g. by a T-independent antigen), tyrosine kinases, including Lck, Lyn, Fyn and Blk, become activated. They phosphorylate the ITAM domains in the Igα and Igβ chains of the receptor complex. These can then bind another kinase, Syk, which activates phospholipase C. This acts on membrane PIP₂ to generate IP₃ and diacyl glycerol (DAG) which activates protein kinase C. Signals from the other kinases are transduced to activate nuclear transcription factors.

B-cell activation is also markedly augmented by the ‘co-receptor complex’ comprising three proteins:

• CD21 (complement receptor-2, CR2);

• CD19; and

• CD81 (target of anti-proliferative antibody, TAPA-1) (Fig. 9.5).

Fig. 9.5 B cell–co-receptor complex

The B cell–co-receptor complex consists of CD21 (the complement receptor type-2), CD19 and CD81 (a molecule with four transmembrane segments). Antigen with covalently bound C3b or C3d can cross-link the membrane Ig to CD21 of the co-receptor complex. This greatly reduces the cell’s requirement for antigen to activate it. CD19 can associate with tyrosine kinases including Lyn, Fyn, Vav and PI-3 kinase (PI-3 K). Compare this with CD28 on the T cell. Receptor cross-linking causes phosphorylation of the Igα and Igβ chains of the antigen–receptor complex and recruitment and activation of Syk.

Follicular dendritic cells are known to retain antigen on their surface for prolonged periods of time as immune complexes (iccosomes). The antigen in such complexes can bind to:

• CD21 (via the complement molecule C3d); and

• surface Ig on the B cells.

Phosphorylation of the cytoplasmic tail of CD19 can then occur, leading to binding and activation of Lyn. It is likely that these kinases enhance the activation signal through the phospholipase C and phosphatidylinositol 3-kinase pathways, particularly when antigen concentration is low.

Q. If an animal was depleted of complement C3 during the primary immune response, what effect do you think this would have on the development of the secondary antibody response?

A. Lack of C3 means that immune complexes containing C3b and C3d do not form. Therefore they cannot bind to follicular dendritic cells (FDCs) via CR2 or engage with B cells via the B cell co-receptor complex (see Fig. 9.5); consequently B cell activation and the development of the secondary immune response is greatly impaired. (These experiments have been done by depleting mice of C3 using cobra venom factor.)

Direct interaction of B cells and T cells involves costimulatory molecules

Antigen-specific T-cell populations can be obtained by growing and cloning T cells with antigens, APCs and IL-2. It is thus possible to visualize directly B-cell and T-cell clusters interacting in vitro:

• the T cells become polarized, with the T-cell receptors concentrated on the B-cell side;

• the B cells also become polarized and express most of their MHC class II molecules and ICAM-1 in proximity to the T cells.

The interactions in these clusters strongly suggest an intense exchange of information, which leads to two important events in the B-cell life cycle:

• induction of proliferation; and

• differentiation into APCS.

The initial interaction between a naive B cell and a cognate antigen via the BCR in the presence of cytokines or other growth stimuli induces activation and proliferation of the B cell. This then leads to processing of the T-dependent antigen and presentation to T cells. The interaction between B cells and T cells is a two-way process in which B cells present antigen to T cells and receive signals from the T cells for division and differentiation (Fig. 9.6).

Fig. 9.6 Cell surface molecules involved in the interaction between B and TH cells

Membrane immunoglobulin (mIg) takes up antigen (Ag) into an intracellular compartment where it is degraded and peptides can combine with MHC class II molecules. Other arrows show the discrete signal transduction events that have been established. A and B are the antigen–receptor signal transduction events involving tyrosine phosphorylation and phosphoinositide breakdown. The antigen receptors also regulate LFA-1 affinity for ICAM-1 and ICAM-3, possibly through the signal transduction events. In the T cell, CD28 also sends a unique signal to the T cell (C). In the later phases of the response CTLA-4 can supplant CD28 to cause downregulation. In the B cell, stimulation via CD40 is the most potent activating signal (D). In addition, class II MHC molecules appear to induce distinct signaling events (E). Not shown is the exchange of soluble interleukins and binding to the corresponding receptors on the other cell.

(Adapted with permission from DeFranco A, Nature 1991; 351: 603–5.)

The central, antigen-specific interaction is that between the MHC class II–antigen complex and the TCR. This interaction is augmented by interactions between LFA-3 and CD2 and between ICAM-1 or ICAM-3 and LFA-1.

Q. Which costimulatory molecules, present on B cells and other APCs, promote T cell proliferation? What mechanisms are involved?

A. B7–1 (CD80) and B7–2 (CD86) act on antigen-activated T cells by ligating CD28, leading to expression of the high-affinity IL-2 receptor (see Fig. 8.1).

The interaction between B and T cells is a two-way event as follows:

• CD40, a member of the TNF receptor family, delivers a strong activating signal to B cells, more potent even than signals transmitted via surface immunoglobulin;

• upon activation, T cells transiently express a ligand, termed CD40L (a member of the TNF family), which interacts with CD40;

• CD40–CD40L interaction helps to drive B cells into cell cycle;

• transduction of signals through CD40 induces upregulation of CD80/CD86 and therefore helps to provide further costimulatory signals to the responding T cells.

Signaling through CD40 is also essential for germinal center development and antibody responses to TD antigens.

Q. Some individuals have a mutation that produces a nonfunctional variant of CD40L. In its homozygous form, what effect would you expect this mutation to have on serum antibody levels?

A. The mutation produces hyper-IgM syndrome, with high serum IgM and low IgG, IgA, and IgE levels, due to lack of germinal centers and failure of the B cells to switch isotype.

Cytokine secretion is important in B-cell proliferation and differentiation

Recent work has shown that CD4⁺ T cells in both mouse and man can be divided into different subsets, depending on their cytokine profile (see Fig. 11.4). During B-cell–T-cell interaction, T cells can secrete a number of cytokines that have a powerful effect on B cells (Fig. 9.7). IL-2, for example, is an inducer of proliferation for B cells as well as T cells.

Fig. 9.7 Cytokines and B cell development

B cell development is influenced by cytokines from T cells and APCs, and by direct interactions with TH2 cells. IL-4 is most important in promoting cell division, and a variety of other cytokines including IL-4, IL-5, IL-6, IL-10 and IFNγ, influence development into antibody-forming cells (AFCs) and affect the isotype of antibody that will be produced.

In particular, cytokines produced by TH2 cells strongly promote B cell activation and the production of IgG1 and IgE. These cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13:

• IL-4 acts on B cells to induce activation and differentiation. It also acts on T cells as a growth factor and promotes differentiation of TH2 cells, thus reinforcing the antibody response; excess IL-4 plays a part in allergic disease, causing production of IgE;

• IL-5 in humans is chiefly a growth and activation factor for eosinophils and is responsible for the eosinophilia of parasitic disease. In the mouse it also acts on B cells to induce growth and differentiation;

• IL-6 is produced by many cells including T cells, macrophages, B cells, fibroblasts and endothelial cells and acts on many cell types, but is particularly important in inducing B cells to differentiate into AFCs. IL-6 is considered to be an important growth factor for multiple myeloma, a malignancy of plasma cells;

• IL-10 acts as a growth and differentiation factor for B cells in addition to modulating cytokine production by TH1 cells;

• IL-13, which shares a receptor component and signaling pathways with IL-4, acts on B cells to produce IgE.

Additional TH subsets have been identified having distinct developmental programs and cytokine profiles. TH17 cells secrete IL-17 and are associated with immunity against extracellular bacteria and fungi, chronic inflammatory disease, and autoimmunity. Another T cell subset that helps B cells is the T follicular helper (TFH) cell, characterized by the expression of the chemokine receptor CXCR5 and localized to developing germinal centers. TFH cells help provide instructive signals that lead to Ig class switching and somatic mutation. TFH cells also produce high levels of IL-21, a cytokine that is critical for germinal center formation.

Q. Give three different ways in which IL-4 can reinforce the TH2-type response

A. It promotes differentiation of TH2 cells. It acts on B cells to promote their division and differentiation. It acts on endothelium and tissue cells to promote the synthesis of chemokines that selectively attract TH2 cells.

Cytokines can also influence antibody affinity. Antibody affinity to most TD antigens increases during an immune response, and a similar effect can be produced by certain immunization protocols. For example, high-affinity antibody subpopulations are potentiated after immunization with antigen and IFNγ (Fig. 9.8). A number of adjuvants are capable of enhancing levels of antibody, but few also potentiate affinity. As affinity markedly influences the biological effectiveness of antibodies, IFNγ may be an important adjuvant for use in vaccines.

Fig. 9.8 Cytokine influence on antibody affinity

Mice were immunized either with antigen alone (Ag) or with antigen plus 30 000 units of IFNγ (Ag + IFNγ). The affinity of the antibodies was measured either early or late after immunization. Mice receiving IFNγ show more high-affinity antibody (darker bars) in both the early and late bleeds than those mice that received antigen alone.

(Adapted from Holland, Holland & Steward. Clin Exp Immunol 1990;82:221–226.)

In addition to the effects of cytokines on B-cell proliferation and differentiation, cytokines are capable of influencing the class switch from IgM to other immunoglobulin classes.

Cytokine receptors guide B-cell growth and differentiation

Receptors for the many growth and differentiation factors required to drive the B cells through early stages of development are expressed at various stages of B-cell differentiation. Receptors for IL-7, IL-3 and low-molecular-weight B-cell growth factor are important in the initial stages of B-cell differentiation, whereas other receptors are more important in the later stages (Fig. 9.9).

Fig. 9.9 Cytokine receptor expression during B cell development

The whole life history of B cells from stem cell to mature plasma cell is regulated by cytokines present in their environment. Receptors for these cytokines are selectively expressed by B cells at different stages of development. Some of these receptors now have CD designation.

A recently identified cytokine, B-lymphocyte stimulator (BlyS), secreted by a variety of cells including monocytes and dendritic cells, is important in both the early development of B cells and their subsequent development and survival in germinal centers. BlyS acts through the receptor BR3 which appears to be important in T-dependent responses, whereas an alternative receptor TAC1 is more important in T-independent responses. BlyS is one of a pair of related cytokines belonging to the TNF family, which signal through these receptors to promote B cell differentiation.

B-cell–T-cell interaction may either activate or inactivate (anergize)

The description of B-cell–T-cell interaction suggests that the only possible outcome is activation of the B cell. However, this is not the case. It has already been seen that APC–T-cell interaction may yield two diametrically opposing results, namely activation or inactivation (clonal anergy). In the same way, B cells frequently become anergic. This is an important process because affinity maturation of B cells during the immune response, as a result of rapid mutation in the genes encoding the antibody variable regions, could easily result in high-affinity autoantibodies.

Clonal anergy and other forms of tolerance in the periphery are important for silencing these potentially damaging clones. However, the molecular details of this process are still unclear. Moreover, the respective roles of IgM and IgD, the two cell-surface receptors for antigen on B cells, are also not understood in terms of activation or inactivation.

B cell differentiation and the antibody response

Following activation, antigen-specific B cells can follow either of two separate developmental pathways:

• the first pathway involves proliferation and differentiation into antibody forming cells (AFCs) in the lymph nodes or in the periarteriolar lymphoid sheath of the spleen. These AFCs function to rapidly clear antigen. However, the great majority of these cells die via apoptosis within 2 weeks. Therefore, it is unlikely that these AFCs are responsible for long-term antibody production;

• in the second pathway some members of the expanded B-cell population migrate into adjacent follicles to form germinal centers before differentiating into memory B cells.

The mechanism that determines which path a B cell takes is unknown. However, it is likely that the decision can be influenced by the nature of the naive B cells initially recruited, the affinity and specificity of the BCR, the type of antigen driving the response, and the levels of T-cell help.

Q. Which type of APC is specifically located in the germinal center?

A. Follicular dendritic cells, which are very important in driving B cell development.

B cell affinity maturation occurs in germinal centers

The germinal center is important in that it provides a microenvironment where B cells can undergo developmental events that ultimately result in an affinity-matured, long-lived memory B-cell compartment (Fig. 9.10). These developmental events come about due to complex interactions between B cells, CD4⁺ TH cells, and follicular dendritic cells. These events include:

• clonal proliferation;

• antibody variable region somatic hypermutation;

• receptor editing;

• isotype switch recombination;

• affinity maturation;

• positive selection.

Fig. 9.10 B cell development in germinal centers

A B cell enters a germinal center and undergoes rapid proliferation and hypermutation of its immunoglobulin genes. Antigen is presented by the follicular dendritic cell (FDC), but only B cells with high-affinity receptors will compete effectively for this antigen. B cells, which do have a higher-affinity immunoglobulin, express Bcl-2 and are rescued from apoptosis by interaction with T cells (i.e. the B-cell-presenting antigen to the T cell). Interaction with T cells promotes class switching. The class switch that takes place depends on the T cells present, which partly relates to the particular secondary lymphoid tissue and the type of immune response current (TH1 versus TH2). B cells leave the germinal center to become either plasma cells or B memory cells (Bm).

The germinal center initially contains only dividing centroblasts. Shortly thereafter, it polarizes into a dark zone containing centroblasts and a light zone containing non-dividing (resting) centrocytes (Figs 9.11 & 9.12). Centroblasts proliferate rapidly in the dark zone and downregulate the expression of their surface immunoglobulin. Somatic hypermutation then occurs to diversify the rearranged variable region genes (see Chapter 3, Fig. 3.28). Somatic hypermutation allows a single B cell to give rise to variants with different affinities for the antigen.

Fig. 9.11 Schematic organization of the germinal center

The functions of the germinal center are clonal proliferation, somatic hypermutation of Ig receptors, receptor editing, isotype class switching, affinity maturation and selection by antigen. In this model, the germinal center is composed of three major zones: a dark zone, a basal light zone and an apical light zone. These zones are predominantly occupied by centroblasts, centrocytes, and secondary blasts, respectively. Primary B-cell blasts carrying surface immunoglobulin receptors (sIg⁺) enter the follicle and leave as memory B cells or AFCs. Antigen-presenting follicular dendritic cells (FDCs) are mainly found in the two deeper zones, and cell death by apoptosis occurs primarily in the basal light zone where tingible body macrophages are also located. Blue squares are iccosomes on FDC.

(Adapted from Roitt IM. Essential Immunology, 7th edn. Oxford: Blackwell Scientific Press, 1991.)

Fig. 9.12 Zoning of the germinal center of a lymph node

The Giemsa-stained section (×40) shows the light zone (left) and the more actively proliferating dark zone (right). There is a well-developed mantle of small resting lymphocytes which have less cytoplasm and therefore appear more densely packed.

(Courtesy of Dr K McLennan.)

Isotype switch recombination occurs following somatic hypermutation and requires cell cycling. Receptor editing of immunoglobulin light chain genes also occurs in centroblasts.

Following these developmental changes, the centroblasts migrate to the follicular dendritic cell- (FDC-) light zone of the germinal center and give rise to centrocytes which then re-express surface immunoglobulin BCR. In the light zone, centrocytes encounter antigen bound to the FDCs and antigen-specific TH2 cells. FDCs and T cells interact with centrocytes through:

• surface molecules such as the BCR, CD40, CD80 (B7-1), CD86 (B7-2), LFA-1, VLA-4, CD54 (ICAM-1); and

• cytokines such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-13 and lymphotoxin-α.

After the centrocytes have stopped dividing, they are selected according to their ability to bind antigen. Those with high-affinity receptors for foreign antigen are positively selected, while those without adequate affinity are induced to undergo apoptosis by ligation of the surface molecule Fas.

Self-reactive B cells generated by somatic mutation are deleted

Centrocytes that respond to soluble antigen or do not receive T-cell help are negatively selected and undergo Fas-independent apoptosis. In this way, selection provides a mechanism for elimination of self-reactive antibodies that may be generated during somatic hypermutation.

Positively selected centrocytes can re-enter the dark zone for successive rounds of expansion, diversification and selection. Somatic hypermutation and selection improve the average affinity of the germinal center B-cell population for presented antigen.

Following these B-cell developmental stages, the centrocytes exit the germinal center and lose their susceptibility to apoptosis by downregulating Fas and increasing the expression of Bcl-2. Three possible outcomes are associated with exit from the germinal center:

• antibody-secreting bone marrow homing effector B cells;

• marginal zone memory B cells;

• recirculating memory B cells.

The factors that regulate the decision to exit the germinal center are poorly understood.

In-vivo antibody responses show isotype switching, affinity maturation and memory

The earliest studies on antibody responses in vivo identified different phases in the response. Following primary antigenic challenge, there is an initial lag phase when no antibody can be detected. This is followed by phases in which the antibody titer increases logarithmically to a plateau and then declines. The decline occurs because the antibodies are either naturally catabolized or bind to the antigen and are cleared from the circulation (Fig. 9.13).

Fig. 9.13 The four phases of a primary antibody response

After antigen challenge, the antibody response proceeds in four phases: a lag phase when no antibody is detected; a log phase when the antibody titre increases logarithmically; a plateau phase during which the antibody titre stabilizes and a decline phase during which the antibody is cleared or catabolized.

It is now possible to understand the features of the antibody response in vivo in terms of the underlying cellular events, although the events can best be understood by viewing the B-cell population as a whole, rather than as a collection of individual cells. Features of the antibody response in vivo include:

• the enhanced secondary response;

• isotype switching;

• affinity maturation;

• the development of memory.

The responses following primary and secondary antigenic challenge are shown in Figure 9.14 and they differ in four major respects.

Fig. 9.14 Primary and secondary antibody responses

In comparison with the antibody response after primary antigenic challenge, the antibody level after secondary antigenic challenge in a typical immune response appears more quickly, persists for a longer period of time, attains a higher titre and consists predominantly of IgG.

The antibodies produced in a primary response to a TD antigen generally have a low average affinity. However, during the course of the response, the average affinity of the antibodies increases or ‘matures.’ As antigen becomes limiting, the clones with the higher affinity will have a selective advantage. This process is called affinity maturation.

The degree of affinity maturation is inversely related to the dose of antigen administered. High antigen doses produce poor maturation compared with low antigen doses (Fig. 9.15). It is thought that:

• in the presence of low antigen concentrations, only B cells with high-affinity receptors bind sufficient antigen and are triggered to divide and differentiate;

• in the presence of high antigen concentrations, there is sufficient antigen to bind and trigger both high- and low-affinity B cells.

Fig. 9.15 Affinity maturation

The average affinity of the IgM and IgG antibody responses after primary and secondary challenge with a T-dependent antigen is shown. The affinity of the IgM response is constant throughout. The affinity maturation of the IgG response depends on the dose of the secondary antigen. Low antigen doses produce higher-affinity immunoglobulin than do high antigen doses, because the high-affinity clones compete effectively for the limiting amount of antigen.

Although individual B cells do not usually change their overall specificity, the affinity of the antibody produced by a clone may be altered. Affinity maturation is achieved through two processes:

• somatic hypermutation of the region including the recombined VDJ gene segment which encodes the variable domain of the heavy chain;

• antigen-driven selection and expansion of mutant clones expressing higher affinity antibodies.

The mechanism by which affinity maturation occurs is thought to involve B-cell progeny binding to antigen on follicular dendritic cells (FDCs) in order to proliferate and differentiate further. Unprocessed antigen in immune complexes is captured by the FDCs via their FC and complement receptors and held there. As B cells encounter the antigen, there is competition for space on the surface of the FDC, leading to selection. When a cell with higher affinity arises, it will stay there longer and be given a stronger signal. B cells with higher affinity will thus have a selective advantage.

An alternative theory is that B cells with higher affinity receptors will compete more effectively to bind and internalize antigen and therefore they have a greater potential of presenting antigen to T cells, and receiving T-cell help.

B cell isotype switching and somatic hypermutation

Somatic hypermutation is a common event in antibody-forming cells during T-dependent responses and is important in the generation of high-affinity antibodies. Somatic hypermutation introduces point mutations at a very high rate into the variable regions of the rearranged heavy and light chain genes (see Fig. 3.28). This results in mutated immunoglobulin molecules on the surface of the B cell. Mutants that bind antigen with higher affinity than the original surface immunoglobulin provide the raw material for the selection processes mentioned above.

Receptor editing is another mechanism by which diversity can be introduced into B cells during affinity maturation. Secondary V(D)J recombination can occur in immature B cells whose antigen receptors bind self antigen. The resulting immunoglobulin rearrangement converts these cells into non-self-reactive cells. In this way, specificity for foreign antigens can be improved and self-reactivity avoided.

Somatic hypermutation occurs at the same time as isotype switching and both processes involve an enzyme, activation-induced cytidine deaminase (AID), which is highly expressed in germinal centers and is induced by IL-4 and ligation of CD40. Animals lacking this enzyme have deficient somatic hypermutation and class switch recombination. The mechanisms involved in isotype switching and somatic hypermutation are described below.

B cells recombine their heavy chain genes to switch immunoglobulin isotype

B cells produce antibodies of five major classes – IgM, IgD, IgG, IgA, and IgE. In humans, there are also four subclasses of IgG and two of IgA (see Chapter 3). Each terminally differentiated plasma cell is derived from a specific B cell and produces antibodies of just one class or subclass (isotype).

The first B cells to appear during development carry surface IgM as their antigen receptor. Upon activation, other classes of immunoglobulin are seen, each associated with different effector functions. When a mature AFC switches antibody class, all that changes is the constant region of the heavy chain. The expressed V(D)J region and light chain do not change. Antigen specificity is therefore retained.

The arrangement of the constant genes in mouse and humans is shown in Figures 9.16 and 9.17. Upstream of the μ genes is a switch sequence (S), which is repeated upstream to each of the other constant region genes except δ. These sequences are important in the recombination events that occur during class switching, as explained below.