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.     

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β-mercaptoethanol, L-α-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce Interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).     

Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells.

Transforming growth factor-beta (TGF-beta) is a potent negative regulator of normal human B cell growth mediated by exogenous signals, including IL-2 and low m.w. B cell growth factor 12 kDa (BCGF-12 kDa). In the present study, we investigated the regulatory linkage between viral or nonviral transformation of human B cells and the growth inhibitory effects of TGF-beta 1. A panel of EBV+ and EBV- B cell lines, derived either by in vitro EBV B cell transformation, or from cases of lymphoma was used to quantitate the negative growth effects of TGF-beta 1. The proliferative response of three EBV- B cell lines to rBCGF-12 kDa or serum was inhibited by low concentrations of TGF-beta 1 (0.2-0.5 ng/ml for 50% maximal effect), as measured by tritiated thymidine uptake and viable cellular recovery. In contrast, rBCGF-12 kDa or serum mediated proliferation of three EBV+ B cell lines was refractory to the growth inhibitory effects of TGF-beta 1. In an attempt to understand the mechanism(s) for this differential growth control in EBV+ and EBV- B cells, we studied the expression of TGF-beta 1, c-myc, and TGF-beta 1 receptors. No correlation was observed between the expression of TGF-beta 1 or c-myc gene and growth inhibition by TGF-beta 1 in the cell lines studied. Our results indicate that sensitivity or resistance to TGF-beta 1 correlated with the presence or absence (loss) of high affinity receptors for TGF-beta 1. EBV- B cell lines expressed levels of high affinity receptors similar to those found on activated normal B or T cells. In contrast, EBV+ B cell lines showed no detectable high affinity receptors. Chemical cross-linking studies with a bifunctional reagent, dissuccinimidyl suberate revealed a normal expression of type I (65-70 kDa), type II (85-90 kDa), and type III (280-300 kDa) TGF-beta 1 high affinity receptors on EBV- B cell lines. In contrast, EBV+ B cell lines did not express type I and type II receptors, whereas type III receptors were expressed but could not be inhibited by unlabeled TGF-beta 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

V delta 1+ subset of human gamma delta T cells responds to ligands expressed by EBV-infected Burkitt lymphoma cells and transformed B lymphocytes.

Human gamma delta T cells of peripheral blood can be divided in two groups in terms of their TCR as well as their behavior upon in vitro stimulation. The major subset expresses the TCR V-segments V gamma 9 and V delta 2 and proliferates in response to ligands revealed by various microorganisms, and the cell line Daudi in addition. The minor group is less homogenous on the gamma-chain but is almost completely identified by mAb against the V delta 1 segment; there is no ligand known to promote growth of these cells. Here we demonstrate that gamma delta T cells out of this subgroup are strongly stimulated in vitro by cells from several Burkitt's lymphoma cell lines. EBV infection of the Burkitt's lymphoma cell lines enhanced the stimulatory ability towards the T cells. Although EBV infection influenced the expression of a variety of cell surface molecules including ICAM-1 and LFA-3, no correlation to the gamma delta T cell-stimulating capacity became apparent. We conclude that Burkitt's lymphoma cells and transformed B cells express ligands of cellular origin for a hitherto poorly characterized subgroup of human gamma delta T cells.     

Marker rescue of a transformation-negative Epstein-Barr virus recombinant from an infected Burkitt lymphoma cell line: a method useful for analysis of genes essential for transformation.

A Burkitt lymphoma cell line infected in vitro with a transformation-defective mutant recombinant Epstein-Barr virus (EBV) was used to attempt marker rescue of transformation competence by transfection with cloned wild-type DNA. EBV replication was induced in the transfected cells, and wild-type EBV DNA recombined via flanking homologous sequences adjacent to the deletion, resulting in a virus which transformed primary B lymphocytes in vitro. This strategy should be useful for molecular genetic analysis of the role of part or all of any gene in cell growth transformation.     

Characterization of the cell surface receptors for human B cell growth factor of 12,000 molecular weight

Quiescent normal human B cells have been shown to require an activation step before proliferating in response to B cell growth factor (BCGF) of 12,000 m.w. (12 kd). One effect of cell activation has been the putative acquisition of specific cell surface growth factor receptors. In this report, the existence of such receptors has been confirmed by using purified radioiodinated BCGF-12 kd. BCGF-12 kd receptors on activated B cells have been shown to be distinct form those interacting with IL 2. Scatchard analysis revealed both high affinity receptor sites with an apparent Kd of 28.6 pM and low affinity receptor sites with Kd of 1.2 nM on freshly prepared, anti-IgM activated peripheral blood B cells. Human B cells grown in culture for extended periods of time in the presence of BCGF-12 kd also displayed high affinity receptor sites (Kd, 41.4 pM) and low affinity receptor sites (Kd, 0.9 nM). The action of BCGF-12 kd therefore appears to be mediated by binding to its lineage-specific receptors on the cell surface.     

Interferon-α and interleukin-12 gene therapy of cancer: interferon-α induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.     

Epstein-Barr virus-transformed B lymphocytes produce low molecular mass molecules with autocrine growth factor and competence factor activity

A human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, produces a factor of a molecular mass less than 10 kDa that promotes cell proliferation of both BA-D10-4 cells and other human T or B lymphoid cell lines, either EBV-positive or -negative. The factor synergizes with higher molecular mass autocrine growth factors and makes both BA-D10-4 cells and B cell lines from Burkitt's lymphoma, but not cells from T cell leukemia, more responsive to interleukin-1 and interleukin-6. Therefore, this low molecular mass factor seems to be an autocrine growth factor per se and to have the characteristics of a competence factor.     

Interferon production potentials of various human lymphoblastoid cell lines

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus-induced interferon (IFN). It was found that, in addition to B cells, various T and nonT-nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN-alpha and might become an alternative source of IFN production. Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein-Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN-alpha. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.     

Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta 2/B cell stimulatory factor 2/IL-6

EBV infects human B lymphocytes and induces them to proliferate, to produce Ig, and to give rise to immortal cell lines. Although the mechanisms of B cell activation by EBV are largely unknown, the continuous proliferation of EBV-immortalized B cells is dependent, at least in part, upon autocrine growth factors produced by the same EBV-infected B cells. In the present studies we have examined the influence of monocytes on B cell activation by EBV and found that unlike peripheral blood T cells and B cells, monocytes enhance by as much as 30- to 50-fold virus-induced B cell proliferation and Ig production. Upon activation with LPS, monocytes secrete a growth factor activity that promotes both proliferation and Ig secretion in EBV-infected B cells and thus reproduces the effects of monocytes in these cultures. Unlike a number of other factors, rIFN-beta 2/B cell stimulatory factor 2 (BSF-2)/IL-6 stimulates the growth of human B cells activated by EBV in a manner similar to that induced by activated monocyte supernatants. In addition, an antiserum to IFN-beta that recognizes both IFN-beta 1 and IFN-beta 2 completely neutralizes the B cell growth factor activity of activated monocyte supernatants. These findings demonstrate that IFN-beta 2/BSF-2/IL-6 is a growth factor for human B cells activated by EBV and suggest that this molecule is responsible for B cell growth stimulation induced by activated monocyte supernatants. We have examined the possibility that IFN-beta 2/BSF-2/IL-6 might also be responsible for B cell growth stimulation by supernatants of EBV-immortalized B cells and thus may function as an autocrine growth factor. However, IFN-beta 2/BSF-2/IL-6 is not detectable in supernatants of EBV-immortalized B cells by immunoprecipitation. Also, an antiserum to IFN-beta that neutralizes IFN-beta 2/BSF-2/IL-6 fails to neutralize autocrine growth factor activity. This suggests that autocrine growth factors produced by EBV-immortalized B cells are distinct from IFN-beta 2/BSF-2/IL-6. Thus, the continuous proliferation of EBV-immortalized B cells is enhanced by either autocrine or paracrine growth factors. One of the mediators with paracrine growth factor activity is IFN-beta 2/BSF-2/IL-6.     

A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines

The LSP1 gene is a new lymphocyte-specific gene which is expressed in normal mouse B and T lymphocytes and in transformed B cells but not (or in much smaller amounts) in nine T lymphoma lines tested. No LSP1 mRNA is found in myeloid cells or in liver, kidney, or heart tissue. Inspection of the predicted LSP1 protein sequence reveals the presence of two putative Ca2+-binding domains in the LSP1 protein. Southern blotting analysis of genomic DNA from mouse liver suggests that the LSP1 gene is present as one copy per haploid genome. Similar analysis of genomic DNA extracted from three transformed B cell lines and five transformed T cell lines shows that the absence of LSP1 mRNA in T cell lines is not due to deletion or gross rearrangements of the LSP1 locus. With the use of the mouse LSP1 cDNA as a probe we can detect a cross-hybridizing RNA species in four normal human functional T cell lines but not in three transformed human T cell lines. This suggests that at least part of the DNA sequence and the expression pattern of the LSP1 gene is conserved between mouse and man. These conserved features, together with the particular expression pattern and the protein sequence homologies, suggest that the LSP1 protein is involved in a Ca2+-dependent aspect of normal T cell growth.     

Interleukin-4. A regulatory protein

Since its discovery in 1982, numerous biological activities of interleukin-4 (IL-4) have been described. Like other cytokines, IL-4 is highly pleiotropic, both with respect to the number of different target cells that are responsive to it and with respect to the number of different biological responses it elicits. Interleukin-4 was initially described as a costimulant for the proliferation of B lymphocytes stimulated with anti-IgM antibody. Synonyms for this cytokine are B cell growth factor-1 (BCGF-1) and B cell stimulatory factor-1 (BSF-1). After cloning of both the murine and human IL-4, the use of recombinant IL-4 enabled detailed studies of its biological functions. Many cell types, mainly of hematological origin, express receptors for IL-4. Accordingly, effects of IL-4 have been described on B lymphocytes, T lymphocytes, NK cells, mononuclear phagocytes, mast cells, fibroblasts and hematopoietic progenitor cells. Currently, there are three major areas in which IL-4 appears to play an important role: 1) regulation of B cell growth and of antibody isotype expression. In this context, a possible role for IL-4 in allergic reactions is of special interest. 2) Stimulation of T cell growth and the generation of cytotoxic T lymphocytes. In addition to the suppressive effects on the induction of non HLA-restricted cellular cytotoxicity by natural killer- (NK) and lymphokine-activated killer (LAK) cells, this suggests a role for IL-4 in the regulation of cellular immune responses. 3) Regulation of the growth and differentiation of hematopoietic bone marrow stem cells. IL-4 itself does not induce proliferation of hematological progenitor cells but it can modulate the growth-factor dependent proliferation of these cells. In this review the biological functions of IL-4, reported until present, are discussed.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents

This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or β-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications.     

Regulation of CD44-hyaluronan interactions in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4.

In this study, we investigated the regulation of CD44-hyaluronan (HA) interactions in a panel of EBV+ Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV transformation of normal human B cells. The results show that among B cell mitogens, phorbol 12-myristate 13-acetate (PMA) alone induced strong HA recognition in EBV+ BL-30/B95-8 cells. Among the cytokines that affect B cell growth and differentiation, IL-4 alone induced HA recognition in BL-30/B95-8 cells. Attempts to delineate the molecular mechanism for this increased HA adhesion in BL-30/B95-8 cells revealed an enhanced expression of CD44 H, isoforms containing V3, V6, and V9 exons, alterations in the splicing pattern of the V4 exon, and the increased electrophoretic mobility of the CD44 H protein. In contrast, the ability to recognize HA was not observed in B-LCL cells stimulated with either PMA or IL-4, even though these cells respond to IL-4, as observed by upregulation of CD23 expression. The molecular pathways that regulate CD44 expression and CD44-mediated HA binding may be selectively inactivated in B-LCL cells. These results may have implications with respect to the generation and spread of B cell tumors.     

Transforming growth factor beta 1 (TGF-beta 1) receptor expression on resting and mitogen-activated T cells

Transforming growth factor beta 1 (TGF-beta 1) is a potent autocrine growth inhibitor of lymphocytes. In this study, the expression of TGF-beta 1 binding proteins was characterized on murine splenic T cells. With an affinity cross-linking method and by neutralizing antibodies to TGF-beta 1, [125I] TGF-beta 1 was found to bind to three cell surface-binding proteins (280-200 kD, 95-85 kD, 65 kD) that were differentially expressed on resting and mitogen-stimulated T cells. Freshly prepared (resting) T cells were found to constitutively express the 95-85-kD form of these binding proteins, whereas mitogenic stimulation by either concanavalin-A (Con-A), interleukin-1 (IL-1), interleukin-2 (IL-2), or 12-tetradecanoyl-phorbol-13-acetate (TPA) for 12-72 h induced the appearance of all forms of the TGF-beta 1 binding proteins (280-200 kD, 95-85 kD, and 65 kD). Furthermore, antibodies that neutralized the biologic action of TGF-beta 1 also blocked the binding of [125I] TGF-beta 1 to all three binding proteins, suggesting that these binding proteins are involved with signal transduction. These results suggest that the expression of the TGF-beta 1 receptor on T cells is regulated by T cell mitogenic signals and that a regulatory relationship may exist between T cell growth-promoting cytokines (IL-1 and IL-2) and the T cell growth inhibitor, TGF-beta 1.     

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.