Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.     

Differential interleukin-2 and interferon-gamma production by human lymphocyte cultures exceptionally resistant to Epstein-Barr virus immortalization

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. However, during the past several years, we found that PBL from two exceptional EBV-seropositive healthy adult individuals were refractory to immortalization by EBV. We report here a study aimed at learning about the immunobiological features which differentiate these EBV-resistant (R) PBL from others which are susceptible (S) to EBV immortalization. Results of this investigation indicate that: (a) Following EBV infection, R-PBL produced significantly higher amounts of interferon gamma (IFN-gamma) than S-PBL. There were however no differences in regard to interferon alpha production between these two types (R and S) of EBV-infected cultures. (b) R-PBL had a maximal interleukin-2 (IL-2) production by S-PBL occurred at least 48 hr later, i.e., at Day 7. (c) The percentage of non-B cells expressing the IL-2 receptor was also higher in EBV-infected R-PBL than S-PBL. (d) In contrast, expression of IL-2 receptors after EBV infection was higher on B cells from S-PBL than on B cells from R-PBL. Interestingly, no differences were noted in regard to IL-2 receptor expression between R-PBL and S-PBL treated with mitogens (i.e., phytohemagglutinin and pokeweed mitogen). (e) Finally, using anti-IL-2 and anti-IFN-gamma antibodies in EBV-infected R-PBL cultures, we were able to obtain EBV-induced immortalization of these cultures. Taken together, these results suggest that an early IL-2 synthesis and high IFN-gamma production by EBV-infected PBL play an important role against lymphocyte immortalization by EBV.     

Suspension cultures of human malignant lymphoma in the leukocyte migration test

The effects of supernatants of primary and secondary malignant human lymphoma cell cultures were analyzed as parameters of spontaneous secretion of factors by these cells using the leukocyte migration test (LMT). Spontaneous cultivation for up to five weeks was successful in four cases. The postulated production of mediators, i.e. the inhibitory and stimulating effects on leukocyte migration were characterized by testing the influence of (a) concentration, (b) temperature and (c) absorption with normal blood leukocytes on the effect. Reproducible stimulatory and inhibitory effects on the migration of normal leukocytes were dependent on concentration and temperature and were apparently mediated by one or more factors. The supernatants of a lymphoblastic lymphosarcoma of the T-cell type and of a lymphoblastic lymphosarcoma clearly revealed congruous and reproducible inhibitory effects. A further case of lymphoblastic lymphosarcoma that could not exactly be defined with immunological methods either and a case of centroblastic/centrocytic lymphosarcoma exhibited stimulating effects which could be reduced in a time-dependent manner through preincubation with blood leukocytes. The results of these studies support the assumption that malignant lymphoma cells are capable not only of secreting immunoglobulin, but also of other biologically effective secretion. The effects of such secretion are differentiated into stimulating and inhibitory ones. They might be important for the spreading of a tumor or for resistance of the organism to the disease.     

Occurrence of 1st division metaphases in human lymphocyte cultures

Summary Human lymphocytes were cultured for 40, 42 and 44h and analysed for the number of 1st and 2nd division metaphases using the BrdU-Giemsa technique. The most suitable time to obtain the majority of cells in their 1st division concomittant with an adequate mitotic index was 42h. It is recommended that laboratories use sister chromatid differential staining to determine the most suitable culture time for the analysis of 1st division metaphases. This will permit accurate comparisons between individuals and laboratories to be made.     

Exhausted cytotoxic control of Epstein-Barr virus in human lupus

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8⁺ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8⁺ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8⁺ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8⁺ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8⁺ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.     

Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers

Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.     

Effect of levamisole on interferon production by PHA-stimulated human leukocyte cultures

Mononuclear leukocytes of 10 normal blood donors were cultured in vitro and treated with phytohemagglutinin (PHA-P) and/or levamisole. Interferon-like activity was investigated in the supernatant fluids of the cultures, using VSV as challenge virus. In most of the cases the PHA-stimulated interferon-like activity was slightly but significantly enhanced by levamisole. The antiviral activity produced in the supernatant fluids was characterized as interferon since it was trypsin sensitive, species specific and inhibited by specific antiserum. This interferon has the characteristic sensitivity to PH2 of immune interferon.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.     

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.     

Replication of Epstein-Barr virus primary infection in human tonsil tissue explants

Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95–8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12–15 days through 24 days post-infection in tissue models infected with B95–8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP⁺ cells were CD3⁻ CD56⁻ CD19⁺ HLA-DR⁺, and represented both naïve (immunoglobulin D⁺) and memory (CD27⁺) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents.