A growth factor different from IL-6 promotes the growth of Epstein-Barr virus-transformed cells at low cell density

Interleukin-6 (IL-6) was recently described to support the growth of Epstein-Barr (EBV) transformed lymphocytes. However no effect was reported on cloning of EBV transformed lymphocytes under limiting dilution conditions. In this paper we demonstrate that the Supernatant of Human Endothelial Cells (HECS) contains a factor(s) different from IL-6 which induces high cloning efficiency of EBV transformed cell lines, cultured under limiting dilution conditions. Furthermore HECS is superior to IL-6 in all the passages of EBV lymphocytes transformation.     

Carboxyethyl gamma-aminobutyric acid, a polyamine derivative, improves the recovery of EBV-transformed lymphocytes

A polyamine derivative, carboxyethyl - Aminobutyric Acid (CEGABA), induces the formation of a large number of Epstein-Barr Virus (EBV) transformed lymphocytes, when added to the culture medium immediately after EBV infection. However, CEGABA shows only a moderate effect on the stability of EBV-transformed cell lines over time, and does not affect the growth of stabilized cell lines. It is possible that CEGABA acts on cells other than EBV transformed lymphocytes (in fact, after EBV infection all types of mononuclear cells from the blood are present in the culture) and indirectly promotes the growth of EBV transformed lymphocytes.     

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.     

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.     

An Epstein-Barr virus that expresses only the first 231 LMP1 amino acids efficiently initiates primary B-lymphocyte growth transformation

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.     

Epstein Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin delivery system

Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable.     

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions.     

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.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

Phosphonoacetic acid: inhibition of transformation of human cord blood lymphocytes by Epstein-Barr virus.

Phosphonoacetic acid disodium salt (PAA) inhibited the transformation of human cord blood lymphocytes by Epstein-Barr virus (EBV) at concentrations of 50-100 microgram/ml. At these concentrations, PAA had no effect on the multiplication of EBV transformed human lymphoblastoid cells or on the survival of human cord blood lymphocytes. The transformation of human cord blood lymphocytes by the B95-8 strain of EBV was measured by 3H-thymidine uptake, 5 days or more after infection. The degree of inhibition of transformation was correlated with the relation between the input of EBV and the concentration of PAA in the experiment. PAA inhibited the transformation even when added 24 h after EBV infection, but had no effect when added 48 h after EBV infection. The inhibitory effect of PAA could be overcome by its removal and normal 3H-thymidine uptake was restored even after 6 days of inhibition. The specificity of the inhibitory effect on EBV induced transformation of human cord blood lymphocytes is discussed.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

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.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells.

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.     

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).     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

含HTNV S基因重组痘苗病毒在HFRS患者B淋巴母细胞系中的表达

采用Ficoll密度梯度离心法(淋巴细胞分离液)分离肾综合征出血热(HFRS)患者外周血单个核细胞（PBMC),并用EB病毒(EBV)感染B淋巴细胞,建立永生化的B淋巴母细胞系(B—LCL)。然后,用含汉滩病毒(Hantaan virus,HTNV)S基因的重组痘苗病毒感染B—LCL,应用问接免疫荧光检测核衣壳蛋白的表达。结果表明,B淋巴细胞经EBV感染4周左右,可形成永生化B—LCL。成功转化后的B—LCL,体积增大,且增殖的淋巴细胞积聚成团。汉滩病毒S基因在B—LCL中能有效表达核衣壳蛋白。含S基因的重组痘苗病毒感染的B—LCL可用作HTNV核衣壳蛋白特异性CTL活性研究的靶细胞。     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

Induction of cytolytic activity of lymphocytes from carcinomatous pleural effusion by IL-2 and autologous tumor cells

We established a cell line (STKM-1) from tumor cells obtained from carcinomatous pleural effusion of a gastric cancer patient. The lymphocytes separated from her peripheral blood or pleural effusion were cryopreserved and immunological experiments were performed after the establishment of the cell line. They were treated with IL-2 or with both IL-2 and mitomycin C (MMC)-treated autologous STKM-1 cells. The cytolytic activity against STKM-1 cells was elevated in lymphocytes cultured with IL-2, and was more prominently augmented in lymphocytes cultured with both IL-2 and MMC-treated STKM-1 cells. The elevation in cytolytic activity was more marked with pleural effusion lymphocytes than with the peripheral blood lymphocytes. The results suggest that the lymphocytes obtained from the pleural effusion would be an excellent source for adoptive immunotherapy.Abbreviations IL-2 interleukin-2 - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - MMC mitomycin C - MoAbs monoclonal antibodies - TIL tumor infiltrating lymphocytes