Confounding B-cell defences: lessons from a staphylococcal superantigen

Studies of microbial superantigens that target large clonal sets of B cells through conserved antigen-receptor-variable-region sites are providing new insights into the mechanisms of B-cell activation-induced cell death. These investigations have shown differences between the clonal regulation of follicular B cells (B2 cells) and the innate-like marginal-zone B cells and B1 cells, and have also shown how B-cell superantigens can affect specialized host defences against infection. Agents designed to emulate the properties of B-cell superantigens might also provide new approaches for the treatment of B-cell-mediated autoimmune and neoplastic diseases.     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

Mechanism of Epstein-Barr virus-induced human B-lymphocyte activation

The mechanism of Epstein-Barr virus (EBV) activation of human B lymphocytes toward Ig synthesis was investigated in a direct anti-sheep red blood cell (SRBC) antibody plaque-forming cell (PFC) system. Exposure of human peripheral blood lymphocytes to EBV in vitro resulted in an anti-SRBC PFC response in 12 of 16 normal donors. The EBV-induced anti-SRBC PFC response did not require the presence of autologous helper T lymphocytes, but was inhibited by the presence of autologous concanavalin A-generated suppressor T cells. Live virus was required for B-cell activation since the EBV-induced PFC response was inhibited by exposure of EBV to ultraviolet light. Using fluorescent techniques which detected simultaneous intracytoplasmic (ICP) Ig production and the presence of EB nuclear antigen, we found that most, if not all, EBV-activated ICP Ig-positive cells were virally infected. Thus, these studies suggest that viral infection of Ig-producing B lymphocytes is required for EBV-induced polyclonal B-lymphocyte activation. Although the participation of T lymphocytes is not required for the induction of EBV-triggered B-lymphocyte Ig production, activated T lymphocytes can serve as modulators of this response.     

An assay for human lymphocyte mitogenic factor: B-lymphocyte stimulation by a T-lymphocyte product.

Human lymphocyte mitogenic factor (LMF) is produced only by E-rosette positive T-lymphocytes when purified cell populations are used. Production is antigen specific and requires the presence of monocytes during the first 24 hr of culture. However, LMF production or regulation of production by cells other than T-lymphocytes may occur as more LMF activity is produced when B-lymphocytes are added to a primary culture of T-lymphocytes. The molecular weight of LMF is estimated by gel filtration to be between 20,500 and 28,000. LMF is heat stable.The response to LMF by B- or T-lymphocytes without monocytes is non-specific and is independent of and unaffected by antigen. The B-lymphocyte response is quantitatively greater that the response of T-lymphocytes. B-lymphocytes are much more sensitive to small concentrations of LMF.     

Telomeric associations in a lymphoblastoid cell line from a patient with B-cell follicular lymphoma

We have established a new Epstein-Barr virus transformed cell line from a patient with B-cell follicular lymphoma. Telomeric fusions were observed in several subclones, with the nonrandom involvement of chromosomes 1, 5, 12, and 17. Centromeric staining with immunofluorescent anti-kinetochore antibodies was positive in both centromeres of the fused chromosomes, suggesting they were both active. Unlike previously reported cases, we were unable to demonstrate telomeric fusions directly in cells from the patient's blood. However, the finding of identical immunoglobulin gene rearrangements in DNA from the patient's blood and cell line suggested that they originated from the same malignant B-cell clone.     

Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen

Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.     

Activation of proacrosin by a locally produced component of human follicular fluid

Components of human follicular fluid were separated on Sepharose 6B columns and the effects of different fractions on the conversion of pig proacrosin to acrosin were examined. A high-molecular-weight fraction (Mr greater than 3,000,000) of follicular fluid was a potent stimulator of this reaction. The proacrosin converting activity was absent in the corresponding fraction of blood serum. The acceleration of proacrosin activation was dependent on the concentration of material with proacrosin converting activity. The results indicate that human follicular fluid contains a high-molecular-weight component of local origin which is capable of accelerating proacrosin in a dose-dependent manner.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Negative modulation of Epstein-Barr virus episomes by a human B-cell growth factor.

To study the effect of T-cell-derived BCGF-12kD on human B-cell autocrine growth-associated functions, we cultured Epstein Barr virus (EBV)-transformed normal B cells (LCL 72285) and Burkitt's lymphoma cells (Raji) in the presence or absence of BCGF-12kD. When cultured in media supplemented only with fetal calf serum, the LCL and Raji cell lines maintained relatively high levels of episomes. Although a similar level of proliferation could be maintained under defined culture conditions in media supplemented with BCGF-12kD, these conditions resulted in a time-dependent reduction of EBV sequences, as detected with EBV nuclear antigen (EBNA-1 and EBNA-2) gene probes. These results suggest that stimulation with T-cell-derived BCGF-12kD can alter a regulatory step which may be involved in the EBV transformation of B cells.     

Molecular characterization of bovine CD26 upregulated by a staphylococcal superantigen

In this report, we describe the cloning, sequencing, and expression of the bovine orthologue of CD26 (BoCD26). Several monoclonal antibodies specific for a molecule, activation molecule 3 (ACT3), aberrantly expressed on superantigen-stimulated bovine CD8(+) lymphocytes, reacted with recombinant BoCD26 expressed in COS-7 and CHO cells. We also showed that human CD8(+) T cells stimulated by a superantigen expressed CD26 at high levels. These results demonstrate that ACT3 is identical to BoCD26 and suggest that CD26 upregulation on CD8(+) T cells is a general phenomenon of superantigens and not limited to their effects on bovine cells.     

Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin

Introduction

B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers.

Methods

A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples.

Results

The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC₅₀, nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels.

Conclusions

Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.     

Glucose-induced positive cooperativity of fructose phosphorylation by human B-cell glucokinase

Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-¹⁴C]fructose, its rate of phosphorylation is increased to 214.3 ± 11.0%, 134.0 ± 4.3% and 116.5 ± 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination. (Mol Cell Biochem 175: 263–269, 1997)     

Molecular mechanisms of B-lymphocyte transformation by Epstein-Barr virus

The B-lymphotropic virus Epstein-Barr virus (EBV) has been implicated in the pathogenesis of B-cell malignancies, particularly in immunodeficient individuals. This review provides a brief overview of the EBV-encoded proteins involved in B-cell transformation, and the current state of knowledge about their roles in this process.     

Characterization and distribution of a new enterotoxin-related superantigen produced by Staphylococcus aureus

Staphylococcal enterotoxins (SEs) are a family of structurally related pyrogenic exotoxins consisting of the five prototypic SEs (types A to E) and three newly characterized SEs (types G to I) produced by Staphylococcus aureus (S. aureus). They also work as superantigens and cause food poisoning and shock symptoms in humans. In this study, we cloned a new variant gene of the seg and characterized its superantigenic properties and distribution among the clinical isolates of S. aureus. The gene encodes a 233 amino acid protein which is highly homologous to SEG (97.7%). The variant SEG (SEGv) expressed by the cloned gene exerted mitogenic activity on human peripheral blood mononuclear cells at the concentration of 100 pg/ml. T cells bearing Vbeta3, 12, 13.1, 13.2, 14 and 15 were preferentially expanded after stimulation with the recombinant protein. The mRNA of the variant seg gene was detected in the total RNA of the organisms bearing this gene. By PCR, 27 out of 48 clinical isolates of S. aureus (56%) possessed either the seg or variant seg gene. These findings suggest that SEG, or SEGv, is one of the most frequently produced superantigen exotoxins by S. aureus and may participate in the inflammatory process of the host by activating a distinct set of Vbeta families of T cells.     

Microparticle-induced release of B-lymphocyte regulators by rheumatoid synoviocytes

Introduction  

In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes.     

Intercellular transfer of a soluble viral superantigen

Mouse mammary tumor virus (MMTV) superantigens (vSAgs) can undergo intercellular transfer in vivo and in vitro such that a vSAg can be presented to T cells by major histocompatibility complex (MHC) class II proteins on antigen-presenting cells (APCs) that do not express the superantigen. This process may allow T-cell activation to occur prior to viral infection. Consistent with these findings, vSAg produced by Chinese hamster ovary (CHO) cells was readily transferred to class II IE and IA (H-2(k) and H-2(d)) proteins on a B-cell lymphoma or mouse splenocytes. Fixed class II-expressing acceptor cells were used to demonstrate that the vSAg, but not the class II proteins, underwent intercellular transfer, indicating that vSAg binding to class II MHC could occur directly at the cell surface. Intercellular transfer also occurred efficiently to splenocytes from endogenous retrovirus-free mice, indicating that other proviral proteins were not involved. Presentation of vSAg7 produced by a class II-negative, furin protease-deficient CHO variant (FD11) was unsuccessful, indicating that proteolytic processing was a requisite event and that proteolytic activity could not be provided by an endoprotease on the acceptor APC. Furthermore, vSAg presentation was effected using cell-free supernatant from class II-negative, vSAg-positive cells, indicating that a soluble molecule, most likely produced by proteolytic processing, was sufficient to stimulate T cells. Because the membrane-proximal endoproteolytic cleavage site in the vSAg (residues 68 to 71) was not necessary for intercellular transfer, the data support the notion that the carboxy-terminal endoproteolytic cleavage product is an active vSAg moiety.     

3C8 antigen is a novel protein expressed by human follicular dendritic cells

Although the pivotal role of follicular dendritic cells (FDCs) in humoral immune responses has been demonstrated, little is known at the molecular level of how FDCs contribute to the organogenesis, B cell differentiation, and regulation of T cell functions in the germinal center. By immunizing with FDC-like cells, we have developed a monoclonal antibody (MAb), which stains the germinal centers in tonsil section. In the current study, the target cell of MAb 3C8 was identified as FDC by confocal scanning fluorescence microscopy. Unlike other MAbs against FDC, MAb 3C8 does not cross-react with bone marrow-derived blood cells. Amino acid sequencing of NH(2)-terminal region of immunoprecipitated 3C8 Ag reveals that 3C8 is a novel FDC protein. Further studies of 3C8 molecule will shed light on the cellular origin of FDC and reveal unknown functions of FDC.