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 induces normal B cell responses but defective suppressor T cell responses in patients with systemic lupus erythematosus

Epstein-Barr virus (EBV) is a polyclonal B cell activator, independent of helper T cells, which induces the generation of suppressor T cells in vivo and in vitro. Given the complexity of the immunologic abnormalities in patients with systemic lupus erythematosus (SLE), we used EBV as a tool to examine the following questions: a). Are SLE B cells primarily defective? and b). Does EBV stimulate the generation of suppressor activity from SLE T cells? It was found that B cells from SLE patients infected with EBV in vitro generate plaque-forming cell (PFC) responses that are similar to those raised by normal B cells infected with EBV within the first 14 days of culture. T cells from SLE patients, in contrast to T cells from normal individuals, cultured with autologous B cells plus EBV fail to develop the expected normal decrement of PFC during the late phase of the in vitro culture (day 14). However, B cells from SLE patients are susceptible to suppression as mixed cultures of SLE B cells and normal allogeneic T cells showed a pattern of PFC response to EBV similar to that of the co-culture of normal B cells with normal T cells. T cells from SLE patients, in analogous mixed cell cultures, failed to suppress either normal B cells or allogeneic SLE B cells. The above experiments indicate that the B cells are not intrinsically defective in SLE patients; rather, a specific T cell abnormality contributes to the lack of normal immunoregulation of certain B cell responses in SLE.     

Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

T-lymphocyte subset interactions in the cell-mediated immune response to Epstein-Barr virus

The T-lymphocyte subset interactions in the cell-mediated response to Epstein-Barr virus (EBV) were studied in an in vitro system in which the ability of T lymphocytes to inhibit outgrowth of autologous EBV-transformed B lymphocytes is assessed. Inhibition could be demonstrated within lymphocytes of both the T4 and T8 surface phenotypes. Outgrowth inhibition was observed more frequently when the effector T-cell population contained cells of both surface phenotypes. In blocking experiments the OKT3 antibody completely prevents development of outgrowth inhibitory activity; the OKT4 and OKT8 antibodies were less effective in interfering with outgrowth inhibitory function. Maximal blocking activity occurred when antibody addition occurred in the early phase of generation of suppressor function. Pharmacologically achievable concentrations of interferon-alpha restored outgrowth inhibitory activity after blocking with monoclonal antibody. EBV reactivation was easily demonstrable in a group of 10 renal allograft recipients who received OKT3 antibody for treatment of acute rejection. These studies suggest that the immunoregulatory control of proliferation of EBV-transformed B lymphocytes is complex, and involves a collaborative interaction of lymphocytes of both the T4 and T8 surface phenotypes.     

Natural killer cells inhibit outgrowth of autologous Epstein-Barr virus-infected B lymphocytes

To determine whether natural killer (NK) cells are the cells responsible for inhibition of outgrowth of Epstein-Barr virus (EBV)-infected autologous B lymphocytes, NK-enriched or NK-depleted populations were prepared by Percoll density gradient fractionation and complement lysis depletion of cells reacting with NK-specific monoclonal antibody HNK-1. These cells were then examined in parallel for NK activity and inhibition of outgrowth. NK-enriched low density cells inhibited outgrowth whereas NK-depleted high density cells did not. Low density cells treated with monoclonal antibodies HNK-1 and DR plus complement had little NK activity and failed to inhibit EBV-induced outgrowth, whereas these same cells treated with monoclonal antibodies OKT3 and DR plus complement had strong NK activity and caused marked inhibition of outgrowth. These findings indicate that NK cells rather than mature T cells, monocytes, or B cells, are responsible for inhibition of EBV-induced B cell outgrowth.     

The cellular basis for viral-induced immunodeficiency: analysis by monoclonal antibodies

Viral infections are often associated with immunodeficiency states. Although T lymphocytes have been thought to suppress the host's response, the precise etiology remains unclear. Therefore, we characterized T lymphocytes from six patients during both acute and convalescent phases of infectious mononucleosis (IM) with monoclonal antibodies (titer, 10(-5) to 10(-7) to antigens restricted to the TH2- helper (T4) and TH2 suppressor (T5) T cell subsets as well as to a common T cell antigen (T3) and HLA-D related Ia antigens. It was found that during acute infectious mononucleosis, there is both activation and increase of suppressor T cells (T5+, Ia+ phenotype). Fuctionally, the acute IM lymphocytes suppress autologous T cell proliferation to antigens as well as pokeweed mitogen driven B cell immunoglobulin production. In contrast, convalescence is associated with a return to normal of T cell subsets and immune function. These results demonstrate that viral infections can preferentially activate a specific T cell subset and suppress the overall human immune response.     

Epstein-Barr病毒特异性T细胞对重组痘苗病毒表达LMP和EBNA2的靶细胞的识别

本文用EB病毒转化自体淋巴细胞所建立的类淋巴母细胞系(LCL),以及用EB病毒潜伏感染膜蛋白(LMP)基因和核蛋白-2(EBNA2)基因与痘苗病毒重组的重组病毒(Vac-LMP和Vac-EBNA2)感染的自身纤维母细胞,同时作为刺激细胞和靶细胞,以~(51)Cr释放法检测5例血清中EB病毒VCA—IgA抗体阳性者及1例阴性健康者外周血单个核细胞(PBMC)的特异性T细胞杀伤效应。结果表明,用自身LCL激活的EB病毒特异性T细胞杀伤效应高峰出现在第14～28天;参与杀伤性细胞免疫反应的T细胞亚群主要是T3、T8阳性的细胞毒性T细胞,其对靶细胞的识别及杀伤受HLA-I的限制。用重组牛痘病毒感染的纤维母细胞作靶细胞或刺激细胞,有1例供者可接受LMP,另1例可接受EBNA2的刺激,并对相应的靶细胞产生特异性T细胞杀伤反应,表明EB病毒-LMP和EBNA2可能既是EB病毒特异性T细胞的刺激抗原,又是其识别的靶抗原。     

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.     

Human B lymphocytes activated by Epstein-Barr virus (EBV) or by mitogens suppress mitogen-induced immunoglobulin production

Polyclonal activation of human B lymphocytes by LPS or protein A, alone or in combination or by Epstein-Barr virus (EBV), generates suppressive conditions that inhibit the response of human B lymphocytes to pokeweed mitogen (PWM), measured by the induction of immunoglobulin-secreting cells (PFC). Moreover, EBV-transformed B cell lines of normal or neoplastic (Burkitt lymphoma) origin also suppressed the PWM-induced immunoglobulin production of normal B cells. Cell separation experiments have shown that mitogen activated autologous B cells stimulate suppressor T cells in a similar way as B cell-derived lymphoblastoid cell lines. The significance of this phenomenon is considered in relation to the escape of the activating microorganism or virus from immune control and the occurrence of network interactions within the immune system.     

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.     

Cell-mediated amplification and down regulation of cytotoxic immune response against autologous human cancer

The cytotoxic host immune response toward autologous human cancer may be regulated by the immunoregulatory network. Here we show that helper T cells, cloned from peripheral blood lymphocytes that were sensitized in vitro against an autologous human malignant paraganglioma, proliferated against and made interleukin 2 when cocultured with the tumor-associated antigen in the presence of autologous accessory cells. Furthermore, the helper cell clones amplified cytotoxic immune response by peripheral blood lymphocytes against the paraganglioma cells in coculture with the blood lymphocytes and the paraganglioma cells. An autologous T cell line bearing suppressor phenotype, established from a lymph node that had been infiltrated with the paraganglioma tumor cells, in contrast to the helper cells, selectively suppressed the cytotoxic immune response by the blood lymphocytes against the paraganglioma cells in identical coculture. These results, therefore, demonstrate the existence of cell-mediated immunologic regulations of the cytotoxic immune response (concurrent amplification and suppression in the same host) against an autologous human tumor.     

Human cytotoxic T lymphocytes (CTL) against Epstein-Barr virus (EBV) infected cells: EBV specificity and involvement of major histocompatibility complex determinants in the lysis exerted by anti-EBV CTL toward HLA-compatible and allogeneic target cells

Human peripheral blood lymphocytes (PBL), from anti-Epstein-Barr virus (EBV)-seropositive donors, were stimulated by EBV and were shown to be cytotoxic toward autologous, HLA-compatible, and fully allogeneic EBV-transformed target cells. The lysis was not due to natural killer (NK) cells since the target cells used were resistant to lysis by fresh PBL and by virus-stimulated PBL-depleted of AET-SRBC-rosetting T cells (the latter being still fully cytotoxic on K562 NK-susceptible target cells). Conversely only E-rosette-purified (T) lymphocytes killed EBV-transformed HLA-compatible and allogeneic target cells. Moreover, anti-MHC antibodies inhibited the cytotoxicity exerted by EBV-induced cytotoxic T lymphocytes (CTL) on both autologous and allogeneic target cells. Finally the lysis was EBV specific since PHA blasts were not killed and since only EBV-transformed cells could compete for lysis with the EBV-positive target cells. Efficient competition was achieved by EBV-transformed cells autologous or allogeneic to the targets, even when effector and target cells were fully allogeneic. All together, the data suggest that human anti-EBV CTL may recognize nonpolymorphic HLA determinants on the target cells in association with the virus-induced antigens.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Epstein-Barr virus antibodies in patients with ataxia-telangiectasia and other immunodeficiency diseases

An unusual antibody response to the Epstein—Barr virus (EBV) has been noted in patients with ataxia—telangiectasia. Of a group of 16 such patients 8 were found to have antibodies in their serum to the EBV viral capsid antigen (VCA), and 4 of them also had antibodies to the EBV early antigen (EA); antibodies to the nuclear antigen (EBNA), however, were absent in 3 of the 8. The antibody pattern persisted for more than 2 years in the patients available for follow-up study. In comparison, of 24 patients with various other immunodeficiency syndromes 9 were found to have EBV-VCA antibodies in their serum, but none of the 9 had EA antibodies and 3 lacked EBNA antibodies. Two other groups of subjects, all of whom had EBV-VCA and EBNA antibodies in their serum late after an EBV infection, were also studied; 82 had infectious mononucleosis and 55 were healthy and had no such history. EA antibodies were detected in 45 of the first group during the acute phase of the illness but persisted in only 6 of the 68 who were followed up for more than 2 years, and they were detected in only 7 of the second group.All eight lymphoblastoid cell lines established from the peripheral blood of the four patients with ataxia—telangiectasia who are still available for follow-up study express EBV-VCA, whereas most similar cell lines established from normal individuals express only EBNA. In two of these patients cell-mediated immunity, as assessed from lymphocyte transformation induced by mitogens, was markedly decreased but autologous cell-mediated immune regression of EBV-induced transformation of B (bone-marrow-derived)-lymphocytes was normal. The percentage of T (thymus-derived)-helper cells was greatly decreased in two of the three patients in whom it was measured, and the percentage of T-suppressor cells was greatly increased in one of them, but the percentage of total T-lymphocytes was within normal limits in all three.The possible significance of these findings — in particular, the persistence of EA antibodies and the diminished restriction of expression of EA — in the late development of tumours after an EBV infection in patients with ataxia-telangiectasia deserves careful attention. Finally, the apparent correlation between immunoglobulin deficiency and poor or absent EBNA antibody response warrants further study.     

Characterization of thymus-derived lymphocyte subsets in acute Epstein-Barr virus-induced infectious mononucleosis.

Changes in thymus-derived (T) lymphocyte subpopulation numbers were studied in patients with acute and convalescent Epstein-Barr virus (EBV)-induced infectious mononucleosis (LM). T cell subsets were characterized by the presence of Fc receptors for IgG (TG), for IgM (TM) or by the absence of either receptor (Tnon-M, non-G). We found that in acute IM, total numbers of T and B lymphocytes were elevated (p less than 0.01). Of the T lymphocyte subsets, the total number of Tnon-M, non-G lymphocytes was increased six fold compared to normal subjects (p less than 0.001) and included the majority of the atypical T lymphocytes. The number of total TG and TM lymphocytes was moderately increased (p less than 0.05). In convalescent IM patients, the number of total T cells remained slightly elevated (p less than 0.02) whereas proportions and absolute numbers of B lymphocytes and T cell subsets returned to near normal levels. Thus, acute Epstein-Barr virus-induced IM is associated with a T lymphocytosis which is composed predominantly of atypical T cells which lack detectable Fc receptors for IgG or IgM.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.     

Heat shock protein 90 expression in Epstein-Barr virus-infected B cells promotes gammadelta T-cell proliferation in vitro

The aim of this study was to elucidate the in vitro response of gammadelta T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as gammadelta T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after 125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that gammadelta T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of alphabeta T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited gammadelta T-cell expansion by 92%. The inhibition of gammadelta T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of gammadelta T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of gammadelta T cells during acute EBV infection.     

Reactivity of Paul-Bunnell type heterophile antibody in sera from infectious mononucleosis patients with the surface of lymphoid cells carrying Epstein-Barr virus genomes.

The possible presence of Paul-Bunnell (PB) antigen on Epstein-Barr virus (EBV)-transformed lymphocytes were investigated. Of 23 EBV genome-positive lymphoblastoid cell lines tested all but one absorbed PB type antibody from the serum of an infectious mononucleosis patient. The one EBV-negative B cell line tested did not absorb the heterophile antibody. PB antibody, purified by an immunoadsorbent procedure using beef cell antigen, reacted with the EBV producer P3HR-1 cell line in an indirect membrane immunofluorescence test and was shown to be IgM antibody. Titers of heterophile agglutinin and reactivity with the cell surface were reduced to the same degree by absorption with beef cell antigen but not with guinea pig kidney antigen. PB antibody was distinct from IgM antibody against the EBV-determined membrane antigen, since the latter was not absorbed by beef cell antigen. PB antibody was also reactive with other EBV-positive B cell lines (QIMR-WIL, NC-37, and Raji) which were free of surface IgM. No reaction occurred with the nonproducer P3HR-1 line, a null cell line, and two T cell lines. The results suggest the presence of PB antigen on most EBV-transformed B lymphocytes, and its appearance in each of the transformed lymphocytes of patients with acute infectious mononucleosis.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.     

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.