Genes for immunoglobulin H and L chains are recombined to generate the surface receptor

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.

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:

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.

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.

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:

These experiments were later reinforced by details of how:

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:

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.