B cell proliferation

Two major functions of B cells are to secrete immunoglobulins and to bind antigens, process them, and present them to T cells. Each B cell produces immunoglobulins with a single specific antigen-binding site. By virtue of immunoglobulin on its surface (slg), B cells bind specific antigens which then deliver a signal to these cells to proliferate and clon-ally expand into memory cells or to differentiate into Ig-secreting cells. Since only one in 107-108 normal B cells has specificity for a particular antigen, the

Table 2 Key concepts and terms in cell cycle

Concepts Timing

Checkpoints

Conservation

The eukaryotic cell cycle is a set of ordered events characterized by DNA replication and cell division. Prokaryotic organisms have overlapping periods of replication and DNA segregation, i.e. simultaneous S and M phases of replication

Cell cycle arrest is induced if a particular condition at a checkpoint is not met. Mammals and other metazoan cells may also activate apoptotic pathways if a checkpoint is not met, while apoptosis is lacking in yeast and other lower eukaryotic cells

Basic aspects of the cell cycle are highly conserved in eukaryotic cells. The major differences are increasing complexity in higher eukaryotic cells. For example, there is one CDK gene in yeast while there are seven in mammalian cells

Terms

Restriction point The point in G, phase when commitment to DNA replication occurs. After a lag time, S phase begins. In yeast, the restriction point is called 'Start' Cyclin Regulatory subunits for CDKs. They encode cyclin destruction boxes that target cyclins for periodic or cyclic ubiquitin-mediated proteolysis, hence their name. Cyclins are classified into mitotic cyclins (cyclin B), S-phase cyclins (cyclin A) and G, cyclins (cyclins C, D, E and F in vertebrates)

CDK Cyclin-Dependent Kinases are heterodimeric serine/theronine protein kinases that require cyclin subunits for activity as well as positive and negative regulatory phosphorylations CDKI Cyclin-Dependent Kinase Inhibitors. A

family of proteins that bind CDKs and block kinase activity of cyclin:CDK complexes. There are two families of CDKI: Cip/Kip (p21, p27, p57) and Ink4 (p16, p15, p18, p19)

number of potential antigen-binding B cells are too few in number to provide an appropriate model to study the mechanisms of cell activation.

In order to stimulate larger numbers of cells via slg cross-linking without resorting to purification of specific antigen-binding cells, investigators have used antibodies which react with slg on the B cell (anti-Ig). By themselves, anti-Ig antibodies stimulate a number of measurable events reflecting B cell activation. Within 3 h, B cells enlarge in size, show enhanced expression of a number of surface determinants including major histocompatibility complex

(MHC) class II molecules, and show a substantial increase in RNA synthesis. The enhanced expression of MHC class II by antigen is an immunologically relevant event since these molecules interact with receptors on T cells, and their enhanced expression leads to enhanced cognate interaction between B and T helper cells. This interaction enhances the ability of T cells to activate B cells. To drive B cells beyond G| into S phase more efficiently, anti-lg must either be used at high concentrations or must be added in the presence of T cells. As noted below, physiologic B cell activation in vivo probably involves antigen binding to slg with coligation of CD 19 and CD21 by the action of complement breakdown products. Activation of B cells to proliferate does nor lead to Ig secretion per se. For this differcntiative event to occur, additional lymphokines including II.-4, IL-2 and IL-5 need to be added together with anti-Ig. These lymphokines support the stimulation of a polyclonal antibody response but not, in the case of T-independent antigens, an antigen-specific response, for which antigen-specific T cells are required.

Recently, the importance of CD40-mediated signaling in B cell proliferation and differentiation has been described. CD40 is constitutively expressed on B cells and engagement by CD40 ligand (gp39), which is primarily expressed on activated CD4 T cells, induces proliferation in B cells. The level of proliferation can be enhanced by the addition of 1L-4, IL-10, and in some systems, IL-2. CD40 stimulation can also rcscue B cells from anti-lg-induced apoptosis.

Discovery of the mechanisms of signal transduction by these cell surface molecules has led to the use of 'postmembrane' chemical stimuli to study B cell proliferation and activation. When surface Ig is cross-linked by anti-Ig, or by antigen, phospholipase C is activated to hydrolyze phosphatidvlinositol biphosphate (PIP2) into inositol triphosphate (IP5) and diacylglycerol (DAG). In support of the hypothesis that protein kinase C (PKC) activation and fCa21"], elevation may lead to B cell proliferation, is the finding that the combination of ionophores and phorbol esters leads to B cell proliferation (Table 3).

However, LPS (lipopolysaccharide) does not induce the hydrolysis of PIP2 or increase [Ca~ |„ but is nevertheless one of the most potent inducers of murine B cell proliferation. Moreover, unlike anti-Ig plus IL-4, LPS can further drive B cells to differentiate and secrete Ig in the absence of added lymphokines. It has been shown that when the intensity of cross-linking surface immunoglobulin is increased by coupling anti-Ig to a large molecular weight carrier molecule such as dextran, B cells can be activated at picomolar concentrations compared to micromolar

Table 3 Reagents and experimental systems used for lymphocyte proliferation in vitro

Antigen-specific T cell proliferation e.g. mixed lymphocyte culture, soluble recall antigens (tetanus toxoid, mumps, purified protein derivative, etc.), superantigens (staphylococcal enterotoxins, etc.) Antigen-nonspecific T cell proliferation

Plant lectins, e.g. phytohemagglutinin, concanavalin A, pokeweed mitogen

Antibody-induced proliferation, e.g. anti-T cell receptor antibodies, antibodies to CD2, CD28, Thy-1, and Ly-6, etc.

Viral transformation, e.g. herpesvirus saimiri, HTLV-I Oxidative mitogenesis, e.g. sodium metaperiodate, neuraminidase and galactose oxidase B cell proliferation

Plant lectins, e.g. pokeweed mitogen Receptor-induced proliferation, e.g. anti-IgM, anti-lgD, anti-CD40, SAC (Staphylococcus aureus Cowan I), LPS (lipopolysaccharide, endotoxin) Viral transformation, e.g. EBV (Epstein-Barr virus) B and T cell proliferation

Pharmacologic agents, e.g. calcium ionophores (A23187, ionomycin), pervanadate to inhibit protein tyrosine phosphatases

Agents to activate protein kinase C, e.g. phorbol esters, bryostatins, teleocidins Cytokines and chemokines concentrations that are required for unconjugated anti-Ig. Recently, an explanation for enhanced B cell activation at low antigen receptor occupancy levels was provided by studies of mice immunized with a recombinant model antigen, hen egg lysozyme (HEL), fused to the murine complement component, C3d. HEL bearing two and three copies of C3d was 1000- and 10 000-fold more immunogenic, respectively, than HEL alone. Thus, C3d is a molecular adjuvant of innate immunity that profoundly influences B cell proliferation. Understanding the mechanisms which lead to the various stages of B cell activation may ultimately provide the means to manipulate the immune response.

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