Gamma-interferon is one of several direct B cell-maturing lymphokines

Two classes of molecules often released after the interaction of T lymphocytes, macrophages and antigen are B-cell maturation factors (BMF)1-3 and immune (gamma) interferon (IFN-gamma)4-7. BMFs directly induce the maturation of resting B lymphocytes to the state of active immunoglobulin secretion, while IFN-gamma is defined by the reduction of viral infectivity in vitro. However, interferons have been shown to have a variety of effects and they have also been reported both to increase and decrease B-cell differentiation in intact animals and complex cellular mixtures in vitro. Here we show that murine IFN-gamma produced by recombinant DNA technology shows similar biological effects to BMFs from two other sources. All three preparations induce immunoglobulin secretion by both normal resting murine splenic B cells and the comparable B-cell tumour line WEHI-279.1 (refs 1, 3). IFN-gamma and the other two BMFs are not identical, however, as anti-IFN-gamma antibodies block the effects on B cells of IFN-gamma, but not those of the other two lymphokines. IFN-gamma may be one of several molecules with a direct role in driving the maturation of resting B cells to active immunoglobulin secretion.     

Recombinant interleukin-2 directly augments the cytotoxicity of human monocytes

Interleukin-2 (IL-2), originally described as a growth factor required for sustained proliferation of T cells in vitro is a glycoprotein hormone of known structure which appears to be important for the generation of immune responses in vivo. As well as T lymphocytes, B lymphocytes and large granular lymphocytes with natural killer activity (NK cells) can also respond to IL-2. The action of IL-2 seemed to be limited specifically to lymphocytes, however, and the term 'T-lymphocytotrophic hormone' was used. Here we provide evidence that human monocytes display a substantially increased cytotoxic activity as a direct and rapid response to human recombinant IL-2 but not to human recombinant glycosylated interferon-gamma (IFN-gamma) or lipopolysaccharide. Our results reveal a previously unknown function of IL-2 and suggest its possible involvement in monocyte-T cell interactions.     

Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B

Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.     

绒白乳菇中提取的乳菇菌素-D免疫抑制作用的研究

研究从绒白乳菇菌中分离出的一种新型倍半萜类化合物-乳菇菌素D的免疫抑制作用及机制。运用体外培养法取BALB/C小鼠脾细胞,研究乳菇菌素D对静止脾细胞、Con A(刀豆蛋白A)和LPS(脂多糖)刺激后的脾细胞(含B淋巴细胞和T淋巴细胞)增殖的抑制作用;运用MTT法经酶标仪检测吸光度,并计算刺激指数(stimulating index,SI);体外培养小鼠脾细胞,运用ELISA检测法观察乳菇菌素D对脾脏中T淋巴细胞分泌IL-2的抑制作用。结果显示该化合物对静止的脾细胞无抑制作用;对Con A和LPS刺激后的脾细胞增殖有显著抑制作用(P0.05),最大浓度药物组刺激指数SI低至0.28;高浓度的乳菇菌素D对Con A刺激的小鼠脾细胞产生的IL-2,具有显著抑制作用(P0.05)。结果显示乳菇菌素D通过抑制抗原刺激后的T淋巴细胞和B淋巴细胞的增殖、抑制其分泌细胞因子,从而发挥免疫抑制作用。研究结果将为开发以乳菇菌素D为母体、经结构修饰、安全低毒的免疫抑制剂提供理论基础。     

A new immunomodulating compound (AS-101) with potential therapeutic application

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.     

T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria

Endemic Burkitt's lymphoma, a tumour of children in which B lymphocytes are infected with Epstein-Barr virus (EBV), is common in areas of Africa where malaria is holoendemic. The tumour is characterized by chromosome translocations; usually the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14, near the enhancer of the immunoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lambda light-chain locus of chromosome 22. In vitro, EBV induces B cells to proliferate and secrete immunoglobulin and antibody. However, in vivo the infected B lymphocytes are under immunological control, so that abnormal proliferation is found only in immunosuppressed patients. Such patients are subsequently liable to develop lymphomas. Burkitt believed that the tumour he had described resulted from interaction between a virus(es) and a "reticuloendothelial system altered by chronic and heavy infection by malarial or other parasites". We report here that during an attack of Plasmodium falciparum malaria, T-cell subpopulations are radically altered so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt's tumour and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common.     

A new immunodeficiency disorder in humans involving NK cells

Immunodeficiency disorders have provided much information on the development and interaction of the various B and T lymphoid components in the immune system of man. As the lymphoid system becomes increasingly divided into functional subsets of cells it will be important to find immunodeficiencies affecting newly discovered cell types. Natural killer (NK) cells are a recently described but ill-defined subpopulation of lymphocytes which is thought to play an important part in surveillance against tumour development. Mice homozygous for the beige gene were found to have a selective deficiency in NK function and were more susceptible to transplantation of syngeneic tumours as predicted. We report here that patients carrying the analogous, autosomal recessive Chediak-Higashi (CH) gene have a profound defect in their ability to spontaneously lyse various tumour cells in vitro by either antibody-dependent or independent mechanisms. Since other cell-mediated cytolytic functions were relatively normal, these results suggest that the beige or Chediak-Higashi gene in both man and mouse controls NK function.     

Surface immunoglobulin-negative B-cell precursors detected by formation of antibody-secreting colonies in agar

The development of semi-solid in vitro cloning assays has helped distinguish the different stages in early haematopoietic differentiation. The progenitors of erythrocytes, granulocytes, macrophages and megakaryocytes have been quantitated and characterized in such systems but until now, similar assays for progenitors of antibody-producing B lymphocytes have not been established despite many reports describing the properties of B-cell precursors which proliferate and differentiate either in vivo in adoptive hosts or in in vitro liquid culture systems. A semi-solid agar assay is described here which permits a murine B-cell precursor to develop into a colony containing antibody-secreting cells after 7-11 days in culture. The precursor cells were found in fetal liver and could be clearly distinguished from mature clonable B cells. This assay thus provides a method to quantitate functional B-cell precursors and establish the requirements for the generation of B lymphocytes.     

Peptide sequences of T-cell receptor delta and gamma chains are identical to predicted X and gamma proteins

Although most mature peripheral T lymphocytes express a major histocompatibility complex restricted, CD3-associated, antigen receptor (TCR) which has been well characterized, some T cells carry a different CD3-associated heterodimer on their surface. One of the two disulphide-linked chains of this putative second receptor, which in mice has relative molecular mass (Mr) 35,000 (35K), has been identified as a product of the group of gamma genes. The other chain, termed delta (Mr 45K in mice), is not as well characterized. Although gamma/delta-bearing cells are a minor subset among peripheral T lymphocytes, they are the only CD3+ cells in the thymus early in ontogeny. Taking advantage of these kinetics, we have generated gamma/delta-bearing hybridomas, using a new TCR alpha chain-negative variant of the AKR thymoma BW5147 as tumour parent, fetal thymocytes as normal cell partners, and an anti-CD3 monoclonal antibody (mAb) as screening reagent. Gamma and delta chains from one of these hybrids have been purified and partially sequenced. The sequences obtained indicate that delta is indeed identical to the polypeptide encoded by the recently described gene X, as suggested by Chien et al.     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Lymphokine-induced IgM secretion by clones of neoplastic B cells

The induction of antibody secretion by B cells requires T-cell-derived factors1-5. Such factors have been described1,2,6-12 but the precise relationship among these various factors is not clear, and it has been difficult to demonstrate that these factors act directly on the B cell and do not exert their effect via T cells or macrophages. In this report we describe the direct induction of IgM synthesis and secretion in cloned lines of long-term tissue culture adapted neoplastic B cells (BCL1) by T-cell supernatants from phorbol-12-myristate 13-acetate (PMA)-induced EL-4 cells or concanavalin A (Con A)-induced 7.1.1a cells5,9. We have termed this activity BCDFmu (B-cell differentiation factor for IgM). The supernatants containing BCDFmu induce activated and neoplastic B cells to secrete IgM5 and the factor responsible is distinct from BCGF13, interleukin-2 (IL-2)5, the classical T-cell replacing factor (TRF) described by Schimpl and Wecker5, and immune interferon (IFN gamma)5.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Mitotic EBNA-positive lymphocytes in peripheral blood during infectious mononucleosis

Infectious mononucleosis (IM) is usually a benign lymphoproliferative disease caused by Epstein-Barr virus (EBV). Although EBV induces a state of continuous proliferation in infected B lymphocytes in vitro, the most prominent lymphoproliferation during IM is of activated, or atypical, T lymphocytes presumably responding to the virus or virus-infected cells. However, EBV genome-carrying cells are known to be circulating during IM, as cultured peripheral blood leukocytes from patients with the disease give rise to continuous lymphoblastoid cell lines, each cell of which contains the EBV genome and expresses the EBV determined nuclear antigen (EBNA). The proposal that EBV-infected cells in IM blood are not endowed with enhanced growth potential but are merely latently infected is supported by demonstrations that cells infected in vivo enter a viral replicative cycle when placed in vitro and that most cell lines derived from cultured lymphocytes of IM patients are infected by virus released in vitro. However the cells could also be capable of proliferation in vivo, since virus production and transformation are not mutually exclusive properties of EBV-transformed cells. Recently, EBNA has been detected in a very small fraction of peripheral blood lymphocytes of IM patients after T cells were first removed and this has been interpreted to indicate that cell transformation occurs in vivo during IM. The isolation of colonies of EBNA-positive cells from IM blood leukocytes cultures in soft agar suggests that at least some infected cells are capable of direct outgrowth into transformed cells. We report here direct evidence that circulating EBV-infected cells exhibit increased growth properties during IM.     

Herpesvirus-transformed cytotoxic T-cell lines

Investigations of cellular cytotoxicity of the immune system are hampered by the lack of continuously growing, transformed cell lines which express a cytotoxic potential. Here we describe cytotoxic cell lines from the cotton-topped marmoset monkey, transformed by Herpesvirus Ateles (HVA) or Herpesvirus Saimiri (HVS), which can kill certain target cells in a short-term in vitro test. HVA/HVS-transformed cells have earlier been classified as belonging to the T-cell lineage in contrast to Epstein-Barr virus (EBV)-transformed cells derived from B lymphocytes. We suggest that the HVA/HVS-transformed killer cell lines described here represent an effector population resembling, or corresponding to, marmoset natural killer (NK) cells and that they may be used to define the cytolytic mechanism involved in cellular cytotoxicity and possibly also effector cell receptors and target cell antigens, as well as regulatory mechanisms of general biological interest.     

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.     

Regulated progression of a cultured pre-B-cell line to the B-cell stage

The variable (V) regions of heavy and light immunoglobulin chains are encoded by multiple germline DNA elements which are assembled into complete variable-region genes in precursor(pre-) B lymphocytes. The heavy-chain V region (VH) is assembled from three separate germline DNA elements, the variable (VH), diversity (D) and joining (JH) segments; whereas light-chain variable regions of either the kappa or lambda type are assembled from two elements, the VL and JL. Analysis of tumour cell lines or sorted cell populations which represent early and late pre-B cells has suggested that heavy-chain assembly and expression generally precedes that of light chains; but, primarily because of the lack of appropriate model systems to study the phenomenon, the mechanism and significance of this apparently orderly differentiation process are much debated. Here we describe for the first time a transformed cell line, 300-19, which sequentially undergoes all of the immunoglobulin gene rearrangement and expression events associated with the differentiation of pre-B cells to surface immunoglobulin-positive B lymphocytes. Analysis of the in vitro differentiation of 300-19 cells provides direct evidence for distinct differentiation phases of first VH and subsequently VL assembly during B-cell differentiation. Furthermore, these analyses suggest that the mu heavy chain, resulting from a productive VHDJH rearrangement, has both a positive and a negative regulatory role in mediating this ordered differentiation process, that is, signalling the cessation of VH gene assembly and simultaneously signalling the onset of VL assembly.     

Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.