Lymphoblastoid cell-induced suppression of human peripheral blood leukocyte mitogenic responses

Two lymphoblastoid tumor cell lines, the Burkitt lymphoma derived BJAB cell line which is free of Epstein-Barr virus (EBV) and B95-8 cells, which are marmoset lymphocytes transformed by EBV isolated from an infectious mononucleosis patient, were studied in regards to their effects on the blastogenic responsiveness of normal human peripheral blood leukocytes stimulated in vitro with mitogens. Mitomycin C treated tumor cell suspensions, when cocultured with normal human blood leukocytes, markedly depressed the expected blastogenic responses in vitro to concanavalin A, pokeweed mitogen, and phytohemagglutin. In addition, cell-free sonicates from the cell lines also depressed blastogenic responsiveness of the leukocytes in vitro. Heating the sonicates for 10 min at 100 degrees C markedly diminished the suppressive properties of the sonicates, as did ultraviolet light irradiation. The suppressive activity of the B95-8 sonicates was pelleted by high speed centrifugation as compared to the activity of sonicates derived from the BJAB cells. Further studies are warranted to determine the nature and mechanism of suppression of blastogenic responsiveness of normal human leukocytes by soluble components derived from such lymphoblastoid cell lines.     

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.     

X-Linked Agammaglobulinemia Patients Are Not Infected with Epstein-Barr Virus: Implications for the Biology of the Virus

Epstein-Barr virus (EBV) infects both B lymphocytes and squamous epithelial cells in vitro, but the cell type(s) required to establish primary and persistent infection in vivo has not been definitively elucidated. The aim of this study was to investigate a group of individuals who lack mature B lymphocytes due to the rare heritable disorder X-linked agammaglobulinemia in order to determine the role of the B cell in the infection process. The results show that none of these individuals harbored EBV in their blood or throat washings. Furthermore, no EBV-specific memory cytotoxic T lymphocytes were found, suggesting that they had not undergone infection in the past. In contrast, 50% of individuals were found to carry human herpesvirus 6, showing that they are infectible by another lymphotropic herpesvirus. These results add weight to the theory that B lymphocytes, and not oropharyngeal epithelial cells, may be required for primary infection with EBV.     

EB病毒转化Hu－TLC－SCID小鼠脾细胞的实验研究

利用ＥＢ病毒转化可产生较高水平人ＩｇＧ和特异性抗２型登革病毒人抗体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞,通过免疫组化、免疫荧光和ＰＣＲ法检测转化细胞的人Ｂ细胞表面标志、ＥＢ病毒抗原和ＥＢ病毒基因。结果表明,被转化的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞能继续产生抗２型登革病毒的特异性人抗体,并具有人Ｂ细胞的、ＳｍＩｇＧ标志及ＥＢ病毒潜伏膜蛋白－１（ＬＭＰ－１）基因,可表达ＬＭＰ－１和ＥＢ病毒核抗原（ＥＢＮＡ）。     

An Epstein-Barr virus isolated from a lymphoblastoid cell line has a 16-kilobase-pair deletion which includes gp350 and the Epstein-Barr virus nuclear antigen 3A

Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.     

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.     

Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.     

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.     

B lymphocyte regulation of human hematopoiesis

Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) were derived from seven different individuals. The ability of BLCL supernatants to stimulate hematopoietic colony formation in vitro was tested in a conventional stem cell assay system. Supernatants promoted the growth of single (GM, E, MK) as well as multi-lineage (GEMM) colonies in bone marrow cultures. Our results indicate that EBV-transformed B lymphocytes produce cytokines that affect in vitro stem cell proliferation and differentiation. These studies demonstrate the regulatory potential of activated B lymphocytes in human hematopoiesis.     

Inhibition of the in vitro outgrowth of Epstein-Barr virus-infected lymphocytes by TG lymphocytes.

Human T lymphocytes subpopulations from subjects not acutely infected with EBV have been selected and examined for ability to inhibit the outgrowth of autologous B lymphocytes that have undergone in vitro infection with EB virus. The results show that only TG lymphocytes are inhibitory. TG lymphocytes from subjects who are EBV antibody-negative inhibit as well as those from subjects who have EBV antibody. TG lymphocyte populations, as well as other T cell fractions obtained from neonatal subjects, fail to inhibit the outgrowth of infected, autologous lymphocytes under the conditions tested. We propose that NK cells are responsible for the inhibitory effects described in this report.     

Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56(bright)CD16(-) NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-gamma (IFN-gamma) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-gamma concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

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.     

Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.     

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.     

Infectious Epstein-Barr virus lacking major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands

The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.     

Regulated expression of the RB "tumor suppressor gene" in normal lymphocyte mitogenesis: elevated expression in transformed leukocytes and role as a "status quo" gene.

Expression of the RB retinoblastoma tumor suppressor gene product is regulated early during the stimulation of normal human peripheral blood lymphocytes, suggesting a regulatory role for the amount of this protein in mitogenesis of normal cells. When normal human peripheral blood lymphocytes were mitogenically stimulated with pokeweed mitogen, bivariate flow cytometric measurements of cellular DNA and RB protein content showed an early decrease in the amount of RB protein per cell, anteceding onset of S phase. A subsequent increase in the amount of RB protein per cell occurred with cell proliferation. Thus the amount of RB protein relative to the total cell mass underwent a biphasic response with mitogenesis. The resulting proliferating cells had a slightly elevated level of RB protein per cell compared to the unstimulated cells. Comparison of other proliferating leukocytes to normal lymphocytes showed that both EBV virally transformed lymphocytes and human promyelocytic leukemia cells (HL-60) had elevated levels of RB protein per cell compared to normal peripheral blood lymphocytes. Mitogenic stimulation or transformation by other means thus is associated with regulation of the amount of RB protein per cell, suggesting a regulatory role for the RB protein in normal cell growth control.     

Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors.

Since Epstein-Barr virus (EBV) infection of Burkitt's lymphoma (BL) cells in vitro reproduces many of the activation effects of EBV infection of primary B lymphocytes, mRNAs induced in BL cells have been cloned and identified by subtractive hybridization. Nine genes encode RNAs which are 4- to > 100-fold more abundant after EBV infection. Two of these, the genes for CD21 and vimentin, were previously known to be induced by EBV infection. Five others, the genes for cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, and the myristylated alanine-rich protein kinase C substrate (MARCKS), are genes which were not previously known to be induced by EBV infection. Two novel genes, EBV-induced genes 1 and 2 (EBI 1 and EBI 2, respectively) can be predicted from their cDNA sequences to encode G protein-coupled peptide receptors. EBI 1 is expressed exclusively in B- and T-lymphocyte cell lines and in lymphoid tissues and is highly homologous to the interleukin 8 receptors. EBI 2 is most closely related to the thrombin receptor. EBI 2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. EBI 2 is also expressed at lower levels in a promyelocytic and a histiocytic cell line and in pulmonary tissue. These predicted G protein-coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection.     

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-��

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56^brightCD16⁻ NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.