B Cells

The effector functions of the immune system comprise antibodies and complement-dependent mechanisms within body fluids and the mucosa, as well as tissue-bound effector mechanisms executed by T cells and mono­cytes/macrophages. B cells are characterized by antigen specificity. Following antigen stimulation, specific B cells proliferate and differentiate into plasma cells that secrete antibodies into the surroundings. The type of B-cell re­sponse induced is determined by the amount and type of bound antigen recognized. Induction of an IgM response in response to antigens which are lipopolysaccharides—or which exhibit an highly organized, crystal-like structure containing identical and repetitively arranged determinants—is a highly efficient and T cell-independent process which involves direct cross-linking of the B-cell receptor. In contrast to this process, antibody re­sponses against monomeric or oligomeric antigens are less efficient and strictly require T cell help, for both non-self and self antigens.

Some forms of T-cell responses involve the release of soluble mediators (cytokines), which effectively expands the field of T cell function beyond in­dividual cell-to-cell contacts to an ability to regulate the function of large numbers of surrounding cells. Other T-cell effector mechanisms are mediated in a more precise manner through cell-to-cell contacts. Examples of this in­clude perforin-dependent cytolysis and induction of the signaling pathways involved in B-cell differentiation or Ig class switching.

B-Cell Epitopes and B-Cell Proliferation

Burnet's clonal selection theory, formulated in 1957, states that every B-cell clone is characterized by an unique antigen specificity, i.e., it bears a specific antigen receptor. Accordingly, once rearrangement of the Ig genes has taken place, the corresponding protein will be expressed as a surface receptor. At the same time further rearrangement is stopped. Thus, only one ABS, or one specificity (one V_H plus V_L [either k or λ]), derived from a single allele can be expressed on a single cell. This phenomenon is called allelic exclusion. The body faces a large number of different antigens in its lifetime, necessitating that a correspondingly large number of different receptor specificities, and therefore different B cells, must continuously be produced. When a given antigen enters an organism, it binds to the B cell which exhibits the correct receptor specificity for that antigen. One way to describe this process is to say that the antigen selects the corresponding B-cell type to which it most effi­ciently binds. However, as long as the responding B cells do not proliferate, the specificity of the response is restricted to a very small number of cells. For an effective response, clonal proliferation of the responsive B cells must be induced. After several cell divisions B cells differentiate into plasma cells which release the specific receptors into the surroundings in the form of soluble antibodies. B-cell stimulation proceeds with, or without, T cell help depending on the structure and amount of bound antigen.

Antigens. Antigens can be divided into two categories; those which stimulate B cells to secrete antibodies without any T-cell help, and those which require additional T-cell signals for this purpose.

-Type 1 T-independent antigens (TI1). These include paracrystalline, identical epitopes arranged at approximately 5-10 nm intervals in a repetitive two-dimensional pattern (e.g., proteins found on the surface of viruses, bac­teria, and parasites); and antigens associated with lipopolysaccharides (LPS). Thus TI1 antigens represent structures with a repetitive arrangement, which allows the engagement of several antigen receptors at one time and results in optimal Ig receptor cross-linking; or structures which result in sub-optimal cross-linking, but which are complemented by an LPS-mediated activation signal. Either type of antigen can induce B cell activation in the absence of T cell help.

• Type 2 T-independent antigens (TI2). These antigens are less stringently arranged, and are usually flexible or mobile on cell surfaces. They can cross­link Ig receptors, but to a lesser extent than TI1 antigens. TI2 antigens require a small amount of indirectly associated T help in order to elicit a B-cell re­sponse (e.g., hapten-Ficoll antigens or viral glycoproteins on infected cell sur­faces).
• T help-dependent antigens. These are monomeric or oligomeric (usually soluble) antigens that do not cause Ig cross-linking, and are unable to induce B-cell proliferation on their own. In this case an additional signal, provided by contact with T cells, is required for B-cell activation.

Receptors on the surface of B cells and soluble serum antibodies usually recognize epitopes present on the surface of native antigens. For protein antigens, the segments of polypeptide chains involved are usually spaced far apart when the protein is in a denatured, unfolded, state. A conformational or structural epitope is not formed unless the antigen is present in its native configuration. So-called sequential or linear epitopes—formed by contiguous segments of a polypeptide chain and hidden inside the antigen—are largely inaccessible to B cell receptors or antibodies, as long as the antigen molecule or infectious agent retains its native configuration. These epitopes therefore contribute little to biological protection. The specific role of linear epitopes is addressed below in the context of T cell-mediated immunity. B cells are also frequently found to be capable of specific recognition of sugar molecules on the surface of infectious agents, whilst T cells appear to be incapable of recognizing such sugar molecules.

Proliferation of B cells. As mentioned above, contact between one, or a few, B-cell receptors and the correlating antigenic epitope does not in itself suffice for the induction of B-cell proliferation. Instead proliferation requires either a high degree of B cell receptor cross-linking by antigen, or additional T cell- mediated signals.

Proliferation and the rearrangement of genetic material—a continuous process which can increase cellular numbers by a million-fold—occasionally result in errors, or even the activation of oncogenes. The results of this process may therefore include the generation of B-cell lymphomas and leukemia’s. Since the original error occurs in a single cell, such tumors are monoclonal. Uncontrolled proliferation of differentiated B cells (plasma cells) results in the generation of monoclonal plasma cell tumors known as multiple mye­lomas or plasmocytomas. Occasionally, myelomas produce excessive amounts of the light chains of the monoclonal immunoglobulin, and these proteins can then be detected in the urine as Bence-Jones proteins. Such proteins represented some of the first immunoglobulin components acces­sible for chemical analysis and they revealed important early details regard­ing immunoglobulin structure.

Monoclonal Antibodies

A normal immune response usually involves the response and proliferation of numerous B cell clones, bearing ABS with varying degrees of specificity for the different epitopes contained within the antigen. Thus the immune re­sponse is normally polyclonal. It is possible to isolate a single cell from such a polyclonal immune response in an experimental setting. Fusing this cell with an “immortal” proliferating myeloma cell results in generation of a hybridoma, which then produces chemically uniform immunoglobulins of the original specificity, and in whatever amounts are required. This method was developed by Koeler and Milstein in 1975, and is used to produce mono­clonal antibodies (Fig. 1), which represent important tools for experimen­tal immunology, diagnostics, and therapeutics. Many monoclonal antibodies are still produced in mouse and rat cells, making them xenogeneic for hu­mans. Attempts to avoid the resulting rejection problems have involved the production of antibodies by human cells (which remains difficult), or the “humanization” of murine antibodies by recombinant insertion of the variable domains of a murine antibody adjacent to the constant domains of a human antibody. The generation of a transgenic mice, in which the Ig genes have been replaced by human genes, has made the production of hybridoma's producing completely human antibodies possible.

Fig.1  Monoclonal antibodies are produced with the help of cell lines obtained from the fusion of a B lymphocyte to an immortal myeloma cell. In the first in­stance, mice are immunized against an antigen. They then receive a second, in­travenous, dose of antigen two to four days before cell fusion. Then spleen cells are removed and fused to the myeloma cell line using polyethylene glycol (PEG). Those spleen cells that fail to fuse to a myeloma cell die within one day of culture. Next, the fused cells are subjected to selection using HAT medium (hypoxanthine, ami- nopterin, thymidine). Aminopterin blocks specific metabolic processes, but with the help of the intermediary metabolites (hypoxanthine and thymidine) spleen cells are able to complete these processes using auxiliary pathways. The myeloma cells, on the other hand, have a metabolic defect which prevents them from utiliz­ing such alternative pathways and resulting in the death of those cells cultured in HAT medium. However, once a spleen cell has fused with a myeloma cell, the fused spleen-myeloma product (hybridoma) is HAT-resistant. In this way only the suc­cessfully fused cells will be able to survive several days of culture on HAT medium. After this time, the cell culture is diluted such that there is, ideally, only one hy­bridoma within each well. Individual wells are then tested for the presence of the desired antibody. If the result is positive, the hybridoma cells are subcloned several times to ensure clonality; with the specificity of the produced antibody being checked following each round to subcloning. Production of purely human mono­clonal antibodies is carried out using mice whose Ig genes have been completely replaced by human Ig genes.

T-Independent B Cell Responses

B cells recognize antigens via the Ig receptor. However, if the antigen is in a monomeric, or oligomeric, soluble form the B cell can only mount a response if it undergoes the process of T-B collaboration. Many infectious pathogens carry surface antigens with polyclonal activation properties (e.g., lipopoly- saccharide [LPS]) and/or crystal-like identical determinants, which are often repeated in a regular pattern (linear e.g., flagella, or two-dimensional e.g., viruses) with intervals of 5-10 nm. These paracrystalline-patterned antigens are capable of inducing B-cell responses without contact-dependent T cell help. This probably occurs by means of maximum Ig receptor cross-linking. Such B-cell responses are usually of the IgM type, since switching to different isotype classes is either impossible or very inefficient in the absence of T cell help. The IgM response is of a relatively brief duration (exhibiting a half-life of about 24 h), but can nonetheless be highly efficient. Examples of this effi­ciency include IgM responses induced by many viral envelope antigens which bear neutralizing (“protective”) determinants accessible to the corresponding antibodies, and responses to bacterial surface antigens (e.g., flagellae, lipopolysaccharides) or parasites.

References

Zinkernagel, R. M. (2005). Medical Microbiology.