Long-term cultivation of plasma cell leukemia cells and autologous lymphoblasts (LCL) in vitro: a comparative study.

Two long-term cell lines were established in vitro from the peripheral blood of a patient with plasma cell leukemia: one line with plasma cell proliferation, the other with lymphoblastoid cell proliferation (LCL). The 9-month-old plasma cell line showed the typical morphology of plasmoblasts. The cells neither had B- nor T-lymphocyte characterisitics, were EBV negative, and showed aneuploidy with various marker chromosomes, including the 14 q+ marker. The cytogenetic findings indicate a monoclonal proliferation of the plasmacells. No tumor growth in thymusless nude mice could be induced upon intracranial inoculation with these cells. In contrast, the autologous LCL, cultured after addition of exogenous EBV, showed the characteristic markers of lymphoblastoid cells, with the typical morphology of pear- and handmirror-shaped lymphoblasts, growing in clumps. They had C3- and Fc-receptors, surface-Ig, E-rosette-negativity, a diploid karyotype, and EBV dependent macromolecule synthesis. They lymphoblastoid cells produced intracranial tumors in nude mice in 8 out of 8 attempts.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.     

Cell-mediated immune response of marmosets to herpesvirus-associated antigens in vitro

Summary Immune responses in vitro of some species of marmosets to herpesvirus-associated antigens expressed on virus-transformed lymphoblastoid cell lines (LCL) were studied by determining lymphocyte proliferation, interferon production, and the induction of cytotoxic effector cells in mixed lymphocyte-LCL cultures (MLLC). Autologous Epstein-Barr virus (EBV)-transformed B-cell lines induced neither lymphocyte proliferation nor interferon production in MLLC, while autologous Herpesvirus ateles (HVA)-transformed T-cell lines stimulated responder cell DNA synthesis and interferon production. Both EBV-LCL and HVA-LCL failed to induce cytotoxic effector cells in autologous MLLC responder cells. These findings differ markedly from the human immune response to autologous EBV-LCL in vitro and may have implications for the unusual susceptibility of marmosets to the induction of lymphoproliferative disease following inoculation with oncogenic herpesviruses.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

Regulation of the growth of Epstein-Barr virus-infected B cells. I. Growth regression by E rosetting cells from VCA-positive donors is a combined effect of autologous mixed leukocyte reaction and activation of T8+ memory cells

Regression of B cell proliferation in co-cultures of EBV-infected B cells (BEBV) and autologous T cells at 1:4 ratio was studied. 3H-TdR incorporation was used to measure proliferation by the participating lymphocyte populations and a 51Cr release assay was used to document the generation of cells capable of killing autologous EBV-transformed B lymphoblastoid cell lines (LCLEBV). EBV-infected B cells cultured alone transformed to blasts by culture day 10, and continued to proliferate throughout the 22 day observation period. When EBV-infected B cells were co-cultured with E rosetted cells from VCA-positive donors, there was a characteristic proliferative response on day 10 (an augmented autologous mixed lymphocyte reaction; AMLR), followed by the development of T8+ cells capable of killing autologous LCLEBV, as well as over 90% suppression of EBV growth by day 22 as assessed by 3H-TdR incorporation, and confirmed in a visual outgrowth assay. Negative and positive selection techniques were used to define the regulatory components in the T cell population. Depletion of T8+ cells from the blood lymphocytes of VCA-positive donors did not significantly reduce the 10 day proliferative response, but the subsequent development of cytotoxic cells and the regression of BEBV outgrowth was not observed. Thus, the circulating T8+ cells are required for the subsequent appearance of autologous LCLEBV cytotoxicity and BEBV growth regulation. However, when the responder population consisted only of T8+ cells, the augmented AMLR response was absent, cytotoxic cell development was weak or absent, and there was no regression of EBV outgrowth. Therefore, the cells participating in the AMLR, as well as T8+ memory cells from VCA-positive donors, are necessary for the control of the in vitro EBV infection. Growth regression is dependent on the proliferation of the regulatory T cells. Mitomycin C treatment of fresh E rosetting cells or those exposed to BEBV for up to 10 days in culture abrogates growth regression and the subsequent appearance of LCLEBV killer cells. However, E rosetting cells exposed to BEBV for 14 days or more already have developed the ability to kill LCLEBV and no longer need to proliferate to induce growth regression when cultured with newly infected BEBV. These results lend additional support to the view that the control of EBV-induced B cell expansion requires a AMLR-dependent clonal amplification of EBV-specific, T8+ cytotoxic cells.     

Epstein-Barr virus recombinants with specifically mutated BCRF1 genes.

Epstein-Barr virus (EBV) recombinants with specifically mutated BCRF1 genes were constructed and compared with wild-type BCRF1 recombinants derived in parallel for the ability to initiate and maintain latent infection and growth transformation in primary human B lymphocytes. A stop codon insertion after codon 116 of the 170-codon BCRF1 open reading frame or deletion of the entire gene had no effect on latent infection, B-lymphocyte proliferation into long-term lymphoblastoid cell lines (LCLs), or virus replication. LCLs infected with the stop codon recombinant were indistinguishable from wild-type recombinant-infected LCLs in tumorigenicity in SCID mice. However, mutant BCRF1 recombinant-infected cells differed from wild-type recombinant-infected cells in their inability to block gamma interferon release in cultures of permissively infected LCLs incubated with autologous human peripheral blood mononuclear cells. This is the first functional assay for BCRF1 expression from the EBV genome. BCRF1 probably plays a key role in modulating the specific and nonspecific host responses to EBV infection.     

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.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Capacity of robot handling for Epstein-Barr virus transformation

Objectives:  Epstein-Barr virus (EBV) transformation has been described as a routine method to establish human B lymphoblastoid cell lines. Each established lymphoblastoid cell line represents one unique genetic information carrier and can produce unlimited quantities of DNA materials available for downstream applications and research. Undoubtedly, it is of great value to human clinical and experimental genetic studies. However, the current process of EBV transformation requires much manpower in the routine renewal of medium, which is time-consuming. This situation can become a serious problem especially when establishing a human B lymphoblastoid cell bank. A modified and cost-effective protocol for EBV transformation should be considered.
Materials and methods:  In the present study, process in EBV transformation was modified to fit the requirements of robot handling.
Results:  1 mL of whole blood was demonstrated to be sufficient to perform EBV transformation. Additionally, EBV transformation can performed in 96-deep-well plates that are directly and widely used with automatic work platforms.
Conclusions:  Based on these facts, a process of EBV transformation can be modified to fit the requirements of robot handling.     

C3 synthetic peptides support growth of human CR2-positive lymphoblastoid B cells

The nature of CR type 2 (CR2)-ligand interactions which leads to the activation of human B cells was analyzed by using synthetic peptides and CR2-positive cell lines. The third component of C (C3) supported the growth of human lymphoblastoid B cells in serum-free medium containing human transferrin. This effect was inhibited by an antibody to C3d (mAb 130) which specifically inhibits C3d binding to CR2, but not by other anti-C3 mAb. Synthetic peptides corresponding to the CR2-binding site on C3d, P28 (residues 1187-1214) or multivalent P13 [1202-1214)4-template), supported the proliferative response of CR2-positive human lymphoblastoid lines in a similar way as C3 and this response could be inhibited by the anti-CR2 mAb OKB7. The proliferative response to C3 or peptides was dose dependent and a 60-fold higher concentration of P28 peptide was required to induce the same level of proliferation as C3. This stimulation of growth was observed only on CR2 expressing cell lines Raji and Daudi, and not on the CR2-negative Burkitt lymphoma cell line Rael and the monocytic cell line U937. In contrast to the stimulatory effect of P28 and P13-template, monomeric P14 (1201-1214) was not able to support the growth of these cell lines. This peptide, however, inhibited the proliferative response of the CR2-positive lines to C3, P28, and multivalent-P13, thus indicating that cross-linking of the CR2 receptor is necessary for B cell proliferation. Another peptide, E12 (from glycoprotein (GP)350, the major EBV outer membrane GP) which shows a high degree of similarity with P14, also inhibited the proliferative response of Raji cells, suggesting that this segment on GP350 is involved in the interaction of EBV with CR2. The possibility of using the above peptides as well as other peptides with "tailor-made" structure in studying the multifunctional role of C3 is discussed.     

Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Development and characterization of a human B cell line that responds to B cell growth factor but not interleukin 2

The human lymphoblastoid cell line we present here proliferated in response to a 14,000 m.w. B cell growth factor (BCGF), and not to interleukin 2 (IL 2). This cell line, designated B-A3, was established by Epstein Barr virus (EBV) transformation of Staphylococcus aureus Cowan I (SAC)-activated spleen B cells, and has been maintained in RPMI 1640 medium complemented with 15% fetal calf serum (FCS) without the addition of other exogenous growth factors. A proliferative response, as measured by [3H]thymidine uptake of B-A3 cells was significantly induced by either commercial IL 2-free human BCGF preparations, or phytohemagglutinin-stimulated mixed lymphocyte culture supernatant at all FCS concentrations used in the assay. The most marked proliferation due to BCGF, however, was observed in the absence of FCS. This BCGF-induced proliferation was not influenced by IL 2 or interferon-gamma (IFN-gamma), because both recombinant IL 2 and IFN-gamma failed to induce proliferation. The response of B-A3 cells to a specific BCGF was additionally indicated by the responsiveness of this cell line to BCGF purified by a series of chromatographic steps. The BCGF to which B-A3 cells responded had a m.w. of 14,000 and was similar to low m.w. BCGF reported from other laboratories. Surface characterization of B-A3 cells, analyzed by flow cytometry with a panel of monoclonal antibodies, demonstrated that the majority of B-A3 cells were stained positively with Leu-12, HLA-DR, and surface IgG markers, whereas staining with surface IgM, IgD markers, pan T cell markers (Leu-4 and Leu-9), and IL 2 receptor (Tac) were consistently negative. Taken together, the human lymphoblastoid cell line we present here responded specifically to a low m.w. BCGF. This cell line may be of value in the purification of BCGF to homogeneity, in studies of the interactions of BCGF with human B cells, and in the identification of the BCGF receptor.     

Immortalization of EBV-infected B cells is not influenced by exogenous signals acting on B cell proliferation. Effects of mutant EL-4 thymoma cells and transforming growth factor-beta

We have recently developed a culture system in which 90% of B cells from human peripheral blood or spleen are induced to strongly proliferate and generate short-term clones of a mean of about 400 antibody-secreting cells. B cells are stimulated by mutant EL-4 thymoma cells in conjunction with T cell supernatant. In the present study, we first investigated whether the frequency of B cell immortalization by EBV would be higher in this system than in a conventional system by using PBMC as fillers. The results showed that the EBV-dependent cloning frequency (0.7%) was not increased compared with the system with the use of PBMC (2.1%). However, the short term proliferation of EBV-infected B cells was 20 times increased in the EL-4 system and EBV nuclear Ag-positive cells participated in this response. Recent reports showed that transforming growth factor-beta (TGF-beta) inhibited the growth of normal B cells, whereas the growth of EBV-immortalized (lymphoblastoid) cells was not inhibited. We have studied the effects of this cytokine in the EL-4 system. TGF-beta (200 pg/ml) was found to inhibit the proliferative response of normal B cells by greater than 95% and the short term response of EBV-infected B cells by 78%. In contrast, the EBV-dependent long term cloning frequency, as well as the proportion of clones producing IgM, IgG, or IgA, was not influenced at all by TGF-beta. In conclusion, potent modulation of the early proliferative response of an EBV-infected B cell population by either stimulatory or inhibitory exogenous signals did not influence the number of cells that subsequently became immortalized.     

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.     

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis¹. LCL have been used to present antigens in a variety of immunologic assays^{2, 3}. In addition, LCL can be used to generate human monoclonal antibodies^{4, 5} and provide a potentially unlimited source when access to primary biologic materials is limited^{6, 7}.A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide⁸, and pokeweed mitogen⁹ to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells^{7, 10-12}.The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23^hiCD58⁺ cells observed as early as three days post-infection indicates a successful outcome.     

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.     

Regression of Epstein-Barr virus-induced B-cell transformation in vitro involves virus-specific CD8+ T cells as the principal effectors and a novel CD4+ T-cell reactivity

T-cell memory to Epstein-Barr virus (EBV) was first demonstrated through regression of EBV-induced B-cell transformation to lymphoblastoid cell lines (LCLs) in virus-infected peripheral blood mononuclear cell (PBMC) cultures. Here, using donors with virus-specific T-cell memory to well-defined CD4 and CD8 epitopes, we reexamine recent reports that the effector cells mediating regression are EBV latent antigen-specific CD4+ and not, as previously assumed, CD8+ T cells. In regressing cultures, we find that the reversal of CD23+ B-cell proliferation was always coincident with an expansion of latent epitope-specific CD8+, but not CD4+, T cells; furthermore CD8+ T-cell clones derived from regressing cultures were epitope specific and reproduced regression when cocultivated with EBV-infected autologous B cells. In cultures of CD4-depleted PBMCs, there was less efficient expansion of these epitope-specific CD8+ T cells and correspondingly weaker regression. The data are consistent with an effector role for epitope-specific CD8+ T cells in regression and an auxiliary role for CD4+ T cells in expanding the CD8 response. However, we also occasionally observed late regression in CD8-depleted PBMC cultures, though again without any detectable expansion of preexisting epitope-specific CD4+ T-cell memory. CD4+ T-cell clones derived from such cultures were LCL specific in gamma interferon release assays but did not recognize any known EBV latent cycle protein or derived peptide. A subset of these clones was also cytolytic and could block LCL outgrowth. These novel effectors, whose antigen specificity remains to be determined, may also play a role in limiting virus-induced B-cell proliferation in vitro and in vivo.     

Modulation of Epstein-Barr virus-induced B-cell activation by concanavalin A

Direct addition of the T-cell mitogen, concanavalin A (Con A), to cultures of Epstein-Barr virus (EBV)-stimulated peripheral blood mononuclear cells (PBMC) resulted in a dose-dependent inhibition of immunoglobulin M (IgM) secreted in the supernatant, as measured by an enzyme-linked immunosorbent assay. Furthermore, Con A inhibited IgM secretion of isolated T-depleted cells stimulated with EBV, and both the proliferation and IgM secretion of EBV-driven lymphoblastoid cell lines. T-Enriched cells, precultured for 48 hr with Con A, were also able to suppress the IgM response of fresh autologous PBMC stimulated with EBV. This suppression was radiation sensitive (2000 rad), a procedure which resulted in enhancement of the IgM secretion of the responder cells in two out of three experiments. Studies on the long-term effects of Con A showed that the early suppression of IgM secretion was transient and that the mitogen prevented the development of the cytotoxic T-cell response normally seen with lymphocytes from EBV-seropositive donors after 5 weeks of culture. Thus, Con A appears to modulate human lymphocyte responses to EBV by multiple mechanisms.