Junichi Ohara

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the switch in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.

Regulation of B‐Lymphocyte Activation, Proliferation, and Differentiation

Understanding the regulation of antibody synthesis is central to any effort to control the disordered immune responses which underlie many autoimmune disorders such as myasthenia gravis. I will discuss here the mechanisms involved in the stimulation of B lymphocytes, the cells which are the precursors of antibody-secreting cells. Emphasis will be placed on the activation of B cells from their resting state, the regulation of their proliferation, and some features of their differentiation into antibodysecreting cells, including a brief consideration of the problem of class switching. B lymphocytes are derived from precursors in hematopoietic tissue. Much has recently been learned about the biochemical and genetic events that control the assemblage of immunoglobulin ( Ig) genes in B-cell precursors. From this information, a picture is emerging of the mechanisms through which antibody diversity is achieved. Much less is known about the regulation of pre-B-cell growth and development. An important recent contribution to this field has been the introduction of a system through which pre-B cells may be cultured on monolayers of bone marrow stromal cells. In such cultures, pre-B cells proliferate and undergo some differentiation into mature B cells. The stromal cell monolayer appears to be critical to these processes, presumably reflecting the production of growth-regulatory substances by stromal cells. Efforts to characterize the stromal cells and to identify and purify the growth factors are now in progress. Work of this type promises to yield information of major significance to our ability to regulate the overall level of B-cell production.

Ia Antigen Expression and Autoimmunity in MRL‐lpr/lpr Mice

Major histocompatibility complex (MHC) class I1 glycoprotein antigens expressed on antigen-presenting cells play a critical role in L3T4+ T-cell proliferation and T-B interactions. As such, they are important regulatory molecules. In functional terms, Ia antigens constitute the target antigens for the large frequency of alloreactive T cells or they serve as restriction elements for the induction of syngeneic helper cells that recognize these determinants in association with nominal antigen (reviewed in reference 1). The particular class I1 determinants recognized and, consequently, the specificity repetoire of peripheral T cells are known to be determined by the M H C phenotype of the thymic APC.. Although autoreactive T cell clones specific for syngeneic Ia antigens have been frequently demonstrated in vitro4 and recognition of self-la antigens is known to form the basis of the in vitro autologous mixed lymphocyte reaction (AMLR),5 it is generally thought that such cells are not commonly activated in viva. Thus, as a result of negative selection of high-affinity self-Ia reactive T cells in the thymus, or changes in the antigen receptor or activation requirements of postthymic T cells, triggering of T cells in the periphery of normal mice optimally occurs following corecognition of class I1 determinants with antigen. It is clear, however, that because of the frequent demonstration of autoreactive cells in vitro. any changes in the conditions that might lead to enhanced activity of these cells [for example, increased Ia expression by antigen-presenting cells (APC) or aberrant expression by Iatissues] could be potentially harmful to the animal. If unregulated, this could be an important factor in the development of autoimmune disease. MRL-lpr/lpr mice spontaneously develop an autoimmune disease manifested clinically by arthritis, vasculitis, and immune complex glomerulonephritis. As such, this represents a murine model for systemic lupus erythematosus (SLE).6s7 Because strains homozygous for the Ipr gene exhibit a profound T-cell lymphadenopathy associated with polyclonal B-cell activation, autoantibody production, and specific B-cell ~nresponsiveness ,~~~ such mice also serve as useful models for studying the mechanisms underlying this B-cell hyperactivity, which, in addition to Ipr-linked disease, is observed following infections with parasites (e.g., malaria) and viruses (e.g., acquired immune deficiency syndrome). Although the marked lymphoprolifer-

In vitro translation of B-cell stimulatory factor-1 in Xenopus laevis oocytes

B-cell stimulatory factor-1 (BSF-1) can be translated in vitro in Xenopus laevis oocytes. This activity is blocked by an antibody to BSF-1. The RNA species coding for BSF-1 activity sediments of approximately 8-9S and is separable from RNA coding for interleukin-2 activity which sediments at approximately 11.5S. Finally, the fact that BSF-1 can be translated in vitro confirms that functions attributed to BSF-1 do not depend on contamination with other biologically active molecules such as phorbol myristate acetate.

Regulation of Macrophage Effector Function by B Cell Stimulatory Factor-1 (BSF-1)

Macrophages that accumulate during immune responses in vivo acquire a broad range of changes not present in the steady-state tissue macrophage or in cells from sterile, irritant-Induced inflammatory reactions. These Immunologically activated macrophages display increased levels of certain cell membrane components (la antigen, Fc receptors) and become cytotoxic to tumor and microbial target cells. Characterization of the lymphokines (LK) that regulate the activated macrophage, macrophage activation factors or MAF, remains one of the more controversial areas in immunology. Twenty years or even 10 years ago, there was a single, albeit uncharacterized MAF: migration inhibitory factor or MIF. This apparently simple situation rapidly evolved into a labyrinth of multiple LK factors, each with its own acronym: MIF, MAF, SMAF, MCF, MCIF, MCIF2 and others (1). Hope for resolution of this puzzle was provided by recent reports that recombinant gamma interferon (IFN) alone, induces many of the changes in macrophages that occur during immunologic activation. Indeed, several groups of investigators now propose IFN as the only MAF (2,3). But macrophage activation is not that simple. MAF physicochemically and biologically distinct from IFN have been described in both murine and human assay systems (4–8). Of special interest is the recent report that recombinant human GM-CSF activates peripheral blood monocytes to kill tumor target cells (9).

B lymphocyte activation. The roles of receptor cross-linkage and BSF-1.

Although responses to antigens introduced into an individual for the first time depend upon the small fraction of B lymphocytes which bear receptors capable of binding to epitopes on that antigen. Since antibody must be produced promptly and in sufficient amounts to deal with an infectious agent or a newly emerging malignant cell, B cell proliferation and differentiation are key to the efficient function of the immune system. Immunologists have adopted a working model for control of B lymphocyte growth and antibody responses which is predicated on the recognition that antigens may be divided into two broad classes, those with repetitive epitopes and those which bear but a single representation of any individually epitope. The importance of this distinction follows from the observation that receptor-mediated signalling requires cross-linking of membrane receptors. Since individual B lymphocytes possess receptors all of which have a common binding specificity, those antigens which bear repetitive epitopes will be capable of cross-linking receptors while those which bear but a single copy of an epitope will not. Current work indicates that responses to antigens of the latter type depends on T cell-B cell interactions in which the T cell recognizes antigenic peptides and class II major histocompatibility complex (MHC) molecules on the surface of the B cell. This type of interaction is usually referred to as cognate T cell-B cell interaction. Antibody responses to antigens with repetitive epitopes could, in principle, also use this mechanism. However, a second pathway of B cell responses may be utilized for such antigens, which is designated receptor cross-linkage-dependent B cell activation.

Regulation of B Lymphocyte Activation: The Roles of Receptor Cross-Linkage and B Cell Stimulatory Factor-1 (BSF-1)

Cross-linkage of membrane receptors of B cells leads to cellular activation and progress through the G1 phase of the cell cycle. Entry into S phase is generally determined by the action of costimulatory factors. We describe here the cellular biochemical events which result in B cell activation as a result of cross-linkage of membrane receptors and present evidence for a feedback regulatory mechanism which leads to cellular desensitization. We further describe the purification, characterization, and receptor-binding properties of B cell stimulatory factor-1 (BSF-1), a T cell-derived lymphokine whose action is important in B cell entry into S phase. We provide evidence that BSF-1 acts as a cocompetence factor in the growth regulation of B and T lymphocytes and suggest that it acts in a similar manner on all cells of hematopoietic lineage.