B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.     

Development and distribution of a human B cell subpopulation identified by the HB-4 monoclonal antibody

Monoclonal antibodies have been successfully used to identify B cell differentiation antigens, few of which mark discrete B cell subpopulations. We have produced a monoclonal antibody, HB-4, against a cell surface antigen on the human B cell line, BJAB, which has an unusual distribution on normal lymphoid cells. HB-4, an IgM antibody, was found to react with an antigen that is expressed by a subpopulation of B cells, approximately 50% of natural killer cells, and not by other types of cells in bone marrow, blood, and lymphoid tissues. In two-color immunofluorescence assays, the HB-4-reactive antigen was found on less than 5% of immature IgM+ B cells in fetal liver and bone marrow and on 25% of B cells in fetal spleen. The HB-4 antibody reacted with 40% of IgM+ cells in newborn blood and 60% of B cells in adult blood. In contrast, only 2 to 26% of IgM+ B cells in the peripheral lymphoid tissues of adults were HB-4+. HB-4+ B cells could be induced to proliferate by cross-linkage of their surface immunoglobulins but not by T cell-derived growth factors. The subpopulation of activated B cells that is responsive to T cell-derived differentiation factors was HB-4-, as were plasma cells. The HB-4 antibody was reactive with some but not all B cell malignancies and cell lines, and not with malignancies or cell lines of other lineages. The HB-4 antigen may therefore serve as a useful nonimmunoglobulin marker for the identification of a subpopulation of mature resting B cells that are present in the highest frequency in the circulation.     

A human lymphoid cell line secreting immunoglobulin G and retaining immunoglobulin M in the plasma membrane

A selected clone, LA 85.2, of a human lymphoid cell line produces μ, γ, and light chains. The cells secrete IgG but not IgM. Assembly of μ chains and light chains produces 8S IgM which is retained in the plasma membrane. IgM is produced at a slow rate and in lesser amounts than IgG. LA 85.2 cells produce a plasma membrane protein which can bind to antibody-antigen precipitates. It is suggested that this protein plays a role in holding the surface IgM in the plasma membrane.     

Induction of secretion of IgM from cells of the B cell line 38c-13 by somatic cell hybridization.

Cells of the murine B cell line 38C-13 possess immunoglobulins of the IgM class on their surface but do not secrete them. Upon hybridization of 38C-13 cells with murine myeloma cells, hybridoma clones were obtained that secreted both pentameric IgM of 38C-13 origin and the myeloma protein. All hybridoma clones synthesized and secreted large amounts of homogeneous IgM with a half disappearance time of about 2 hr, typical of mature plasma cells. Concomitantly with the induction of IgM secretion, the hybridoma cells lost their surface IgM. The possibility of separate pathways for the synthesis of membrane and secreted IgM is discussed.     

The intracellular ratio of the membrane to the secreted form of immunoglobulin M heavy chain is a measure of the developmental stage of B cell lymphomas

Tumors of B lymphocyte origin have been used as models for normal B cells “frozen” at particular stages of their development. Surface properties, amount, and intracellular location of immunoglobulin and the synthesis of J chain have all been used as indicators of developmental stages. Each requires special techniques or yields data that are difficult to compare from one experiment to the next. For these reasons, we have developed a metric for B cell development that is simple to perform and allows quick quantitative comparisons of cell lines. It has recently been established that the membrane (μ_m) and secreted (μ_s) forms of the IgM heavy chain differ at their extreme carboxy termini. The two proteins differ slightly in size and are easily distinguished when they are compared without their carbohydrate on sodium dodecyl sulfate (SDS) polyacrylamide gels. We have examined four mouse tumors derived from the B lymphocyte lineage whose phenotypes resemble late pre-B cells (internal μ only; uninduced 70Z/3), small B lymphocytes (high levels of surface IgM; LPS-induced 70Z/3, WEHI 231), lymphoblasts (both membrane and secreted IgM; WEHI 279.1), and plasma cells (copious IgM secretion; MOPC 104E). Despite the fact the 70Z/3 and WEHI 231 secrete no detectable IgM, all of the tumors synthesize at least intracellular forms of both μ_m and μ_s. The proportion of μ_m is stable and is characteristic of each tumor. The 70Z/3 cells and WEHI 231 cells synthesize about 75% of their total μ as μ_m; WEHI 279.1 cells synthesize about 30% and MOPC 104E cells about 5% of their total μ as μ_m. The population of LPS-stimulated B lymphocytes shows a similar progression during its differentiation. The proportion of μ_m correlates with other developmentally regulated parameters (Fc receptor, Ia and plasma cell antigen levels, and J chain) and can be used as a simple metric for comparison with developing B lymphocytes and determination of the developmental stage of a B cell tumor.     

Two independent pathways of helper activity provided by a single T cell clone

Data presented in this paper demonstrate the existence of two separate pathways by which a single T cell clone can induce B cell differentiation. With the use of high doses of antigen, a T cell clone can induce a primary antibody response in unprimed B cells. With the use of low doses of antigen, the same T cell clone can induce an immunoglobulin (Ig)G response in primed B cells. The primary response is accompanied by T cell proliferation and lymphokine production (interleukin 2, B cell growth factor, B cell differentiation factor for immunoglobulin M, and B cell differentiation factor for immunoglobulin G). The secondary response does not require proliferation and occurs independently of detectable lymphokine production. Variants of the wild type T cell helper clone have been isolated. One variant can provide help to unprimed B cells when high doses of antigen are used. This variant cannot provide help to primed B cells when low doses of antigen are used, nor can it provide help to CBA/N "xid" B cells at any antigen concentration tested. Additional variants have been isolated that proliferate on antigen-pulsed-presenting cells, but fail to secrete detectable lymphokines and do not induce B cell differentiation. These results suggest that a single T cell helper clone has multiple functional activities that can be independently expressed.     

Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine

B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a mutant IgG containing a mus tailpiece became largely retained within the cell; secretion was restored upon mutation of the tailpiece cysteine. Removal of Cys-575 also allowed hypersecretion of monomeric IgM by plasmacytoma cells. Following further removal of Cmu1, heavy chains were secreted in the absence of light chains. Thus, in B and plasma cells, Cys-575 is involved both in the polymerization of IgM and in intracellular retention of unpolymerized intermediates.     

T-cell regulation of B-lymphoblastoid cell line function

Seventeen B-lymphoblastoid cell lines (B-LCLs) have been studied for their ability to intereact with normal T cells and produce IgG and IgM in culture. All B-LCLs were HLA homozygous, having been derived from consanguineous donors by in vitro transformation with Epstein-Barr virus, 1 × 10⁴ B-LCLs were cultured with 0, 5, 10, or 20 times as many normal peripheral blood T cells in a 0.2-ml culture. Culture supernatants were removed after 3 and 6 days and assayed for IgG and IgM by a radioimmunoassay. Thirteen of the cell lines were able to secrete immunoglobulin (50–6000 ng/ml), primarily IgG, when cultured without T cells. Addition of T cells (sheep erythrocyte rosette-forming cells) modulated immunoglobulin production, causing either marked enhancement or suppression depending upon the B-cell line. T cells cultured without the B-LCLs did not secrete immunoglobulin above the background level of the immunoassays (6.25 ng/ml). Cell lines which did not secrete IgM when cultured alone could frequently be induced to do so when T cells from select donors were added. Under these conditions, IgM was generally found only in the supernatant fluid removed after 6 days. Taken together, these results suggest that B-LCLs contain cells of at least two stages, those that secrete IgG and resting cells capable of secreting IgM. Furthermore, cells at both stages can be regulated by normal T cells.     

T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.     

Cell cycle-related expression of the receptor for a B cell differentiation factor

Cloned, neoplastic B cells (BCL1) have been used to evaluate the expression of the receptor for the B cell differentiation factor, BCDF mu. These cells do not secrete IgM before stimulation with BCDF mu-containing T cell supernatants (SN). By inducing cell cycle synchrony in this homogeneous population, the expression of the BCDF mu receptor could be evaluated as a function of the cell cycle. Responsiveness to BCDF mu-containing SN is maximal when the cells are in S and G2 phases of the cell cycle, and a 2-hr exposure of cells to BCDF mu-containing SN during S/G2 results in optimal IgM secretion 5 days later. Cells in S/G2 are also maximally effective in absorbing BCDF mu activity from SN. These data support the hypothesis that B cells do not respond to differentiative signals until after they are committed to at least one round of cell division.     

Effects of tobacco glycoprotein (TGP) on the immune system. I. TGP is a T-independent B cell mitogen for murine lymphoid cells

It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.     

Collaboration of Th1 and Th2 T cell clones in specific antibody responses: regulation of the IgM response to phosphorylcholine

Carrier (KLH)-specific type 1 T cell clones (Th1), which are defined by secretion of IL-2 and IFN-gamma but not IL-4, and type 2 (Th2) clones, which secrete IL-4, but not IL-2 or IFN-gamma, have been isolated and analyzed for their ability to collaborate in providing help for B cells to secrete phosphorylcholine-specific IgM antibodies. The resulting antibody responses exhibited a characteristic pattern suggesting two distinct regulatory interactions among the Th1, Th2, and B cells. At low doses of antigen, Th1 cells enhanced the helper function of the Th2 cells, an effect due primarily to IL-2. At high doses of antigen, Th1 cells or IFN-gamma inhibited Th2-dependent antibody responses. The inhibitory effect of Th1 or IFN-gamma affected primarily the hapten-carrier-linked portion of the response. The overall effect was a modulation of the antigen dose-response curve for antibody production, eliminating the sharp increases in dose response mediated by isolated T cell clones. The data suggest that collaborative interactions of Th1 and Th2 cells in antibody production may have important physiological consequences.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.     

Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

Identification of the Schwann cell as a peripheral nervous system cell possessing a differentiation antigen expressed by a human lung tumor.

Recent studies of the plasma membrane antigens of a human lung tumor (oat cell carcinoma) indicated that the tumor expressed at least two normal differentiation antigens undetectable in normal respiratory epithelium. One antigen was characteristic of certain endodermally derived epithelial cells of the digestive system; the other antigen was characteristic of certain neural crest-derived cells in the peripheral nervous system. The present studies were undertaken to identify the reactive cell type in the peripheral nervous system. Since similar cells in the rat peripheral nervous system expressed a cross-reactive form of this antigen, and since pure cultures of different rat nerve cell type were available, the following approach was possible. Cultures of pure neurons, pure Schwann cells, pure fibroblasts, neurons and Schwann cells, and neurons, Schwann cells, and fibroblasts were assayed for this antigen with rabbit anti-oat cell carcinoma plasma membrane antiserum absorbed with normal lung and liver. The indirect immunofluorescence method on both whole, viable cell and fixed cell substrates was used. Only Schwann cells expressed the antigen; Schwann cells in the presence of neurons expressed the antigen much more strongly than did pure Schwann cells. It was concluded that the oat cell carcinoma of the lung expressed a differentiation antigen present on Schwann cells.     

Lipid rafts and B cell signaling

B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secrete pathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process them for presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasma membrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation including B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we review the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.