Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

Mechanisms of enhancement of Epstein-Barr virus-induced transformation of peripheral blood lymphocytes by a tumor promoter, TPA, with special reference to lowering of cytotoxicity of T cells

The mechanism of the previously observed enhancement of Epstein-Barr virus (EBV)-induced cell transformation of human lymphocytes by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was studied. A concentration of TPA (0.5 ng/ml) was used. In cultures of human cord blood lymphocytes infected with EBV in the presence of TPA, a larger number of EBV-associated nuclear antigen (EBNA)-positive and/or DNA synthesizing cells was observed than in the absence of TPA. In the virus-infected lymphocyte cultures of EBV-seropositive adult donors, in vitro regression of transformation, which is known to be caused by T lymphocytes, was suppressed by TPA, EBV-specific and -non-specific cytotoxicity of T cells generated in mixed cultures of peripheral blood lymphocytes (PBL) of seropositive adults and autologous lymphoblastoid cell line (LCL) cells was markedly lowered by the presence of TPA in the cultures, However, TPA had little effect on proliferation of T cells in stimulated cultures, and addition of TPA to the reaction mixture for the cytotoxicity test did not lower the cytotoxicity. These results suggest that TPA has an inhibitory effect on T cells which suppress EBV-transformation. THe effect on T cells, together with the direct promoting effect on proliferation of EBV-transformed cells, may explain the outcome of enhancement of EBV-induced LCL establishment from PBL of seropositive donors by TPA previously observed.     

Monoclonal antibody to chicken fetal antigen on Marek's disease lymphoblastoid cell line MDCC-MSB1

A monoclonal antibody (2H3) to chicken fetal antigen (CFA) expressed on the cell surface of the Marek's disease lymphoblastoid cell line MDCC-MSB1 was generated. 2H3 reacted specifically with various cells from chicken embryos, especially with fetal red blood cells, fetal bursa-derived lymphocytes, fetal liver cells, and embryo fibroblasts but not with fetal peripheral blood lymphocytes. 2H3 reacted also with all lymphoblastoid cell line cells tested: Marek's disease, avian leukosis, and reticuloendotheliosis line cells. On the basis of 2H3 specificity, CFA detected by 2H3 seemed to be similar to chicken fetal red blood cell antigen previously reported, but not to chicken alpha-fetoprotein. A transient increase of reactivities with 2H3 was observed in lymphocytes prepared from thymus, spleen, and bursa of chickens inoculated with Marek's disease virus or herpesvirus of turkeys from 7 to 21 days postinoculation. The average percentage of CFA-positive cells in lymphoid cells from Marek's disease virus-infected chickens at the peaks was higher than that of the cells in Marek's disease tumor cells from 146- and 156-day-old chickens in the field. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that the monoclonal 2H3 is recognized a Mr 50,000 oncodevelopmental cell surface antigen present on MDCC-MSB1 cells. The possible differences between CFA detected by 2H3 and chicken fetal red blood cell antigen expressed on normal chicken fetal erythrocytes are discussed.     

Generation in vitro of EBV-induced specific cytotoxic T cells in autologous serum avoids complications due to self-preferred foetal calf serum-specific T-cell cytotoxicity

A previous report has established that in cultures of human mononuclear leukocytes, foetal calf serum (FCS) is capable of generating high levels of T cells preferentially cytotoxic for the autologous lymphoblastoid cell line (LCL). The present study compared the capacity of Epstein-Barr virus (EBV) to generate cytotoxic T cells in cultures of mononuclear cells grown in FCS in this system. Five EBV-seropositive and three seronegative donors were used and cultures were harvested at 14 days. With cultures from seropositive donors, whether grown in FCS or in autologous serum, EBV infection generated T cells cytotoxic for the autologous LCL; the response in uninfected control cultures was markedly lower. With seronegative donor cultures grown in FCS, there was virtually no difference in the capacity of T cells generated in infected or uninfected cultures to lyse the autologous LCL. Moreover, cells from seronegative donors cultured in human serum gave no detectable lysis of autologous LCL in either infected or uninfected cultures, clearly showing the absence of a response to EBV. This evidence shows that it is possible to distinguish the generation of specific cytotoxic T cells by FCS from generation by EBV, and with certain donors the apparently EBV-induced response may actually include a significant component induced by FCS in the medium. The cytotoxicity patterns of EBV-induced and FCS-induced T cells for autologous and allogeneic LCL targets showed a degree of parallelism, stressing the need for caution in interpretation of data obtained from cultures using FCS.     

Lymphokine-activated killer cells in rats: analysis of tissue and strain distribution, ontogeny, and target specificity

The coculture of lymphoid cells from Fischer 344 rats with recombinant human interleukin 2 (rIL-2) resulted in the generation of lymphokine-activated killer (LAK) cells. Maximal LAK activity was obtained between 200 and 1000 units/ml rIL-2. Lymphoid cells from spleen, thymus, bone marrow, peripheral blood, and lymph nodes were able to generate LAK activity although the kinetics and magnitudes of the responses were appreciably different among these tissues. Thus, while spleen and blood lymphocytes responded quickly (by day 3) and gave the highest level of LAK activity in response to rIL-2, bone marrow and thymus cells responded only by 7 to 9 days in culture. LAK activity could be generated from a variety of rat strains regardless of whether there were high or low levels of endogenous splenic natural killer (NK) activity, but the early (day 3) response was lower in the strains with low levels of NK activity. Cells with LAK activity could lyse a variety of tumor targets including fresh ascites or fresh syngeneic solid tumor explants but could not lyse fresh normal cells including syngeneic fibroblasts, peripheral blood lymphocytes, bone marrow cells, thymocytes, or T,B blasts. The generation of LAK activity required a concomitant proliferative response and could be completely abrogated by mitomycin C, actinomycin D, or X-irradiation above 500 rads. These treatments, however, did not affect natural killer activity or short-term (4 h) IL-2-boosted NK activity. LAK activity could be generated from spleen cells obtained from rats as early as 10 days of age but could not be generated from unfractionated neonatal spleen, neonatal liver, or peritoneal macrophages. The ontogeny of the development of splenic LAK activity correlated closely to the development of concurrent natural killer activity. When mixed with an NK-resistant mammary adenocarcinoma (MADB106) and adoptively transferred to normal syngeneic recipients in standard Winn-type assays, LAK cells were effective at inducing complete tumor inhibition.     

Lymphoblastoid transformation and kinetics of appearance of viral nuclear antigen (EBNA) in cord-blood lymphocytes infected by Epstein-Barr Virus (EBV).

Human cord-blood lymphocytes were infected with B95.8 Epstein-Barr virus (EBV) before and after separation into B- and T-cell populations. Lymphoblastoid cells exhibiting B-cell characteristics appeared after 2 to 3 days of culture in the total population and in the separated B-cell subpopulation but not in the T-cell subpopulation. EBV nuclear antigen (EBNA) was detected concurrently with the appearance of lymphoblastoid cells. The proportion of EBNA-positive cells corresponded to that of lymphoblastoid cells, and reached 50% after 4 days. EBNA was present only in cells with B-cell markers. These observations indicate that only B-cells are susceptible to EBV infection, that the transformation occurs within a few days and that EBNA is a valid early marker for susceptibility to EBV transformation.     

EL-4 metastases in spleen and bone marrow suppress the NK activity generated in these organs

The relationship between metastatic cells in the spleen and bone marrow of tumor-bearing mice and the NK activity generated in vitro by cells obtained from these organs was investigated. EL-4 lymphoma and B16 melanoma cells injected intraperitoneally into syngeneic mice (10(6) cells/animal) killed the recipients in 16 days. These tumors had a different metastatic profile: EL-4 metastasized to the spleen and bone marrow while B16 did not. The number of metastatic cells was evaluated by plating spleen or bone-marrow cells of tumor-bearing mice in agarose cultures; in parallel, the ability of spleen and bone-marrow cells to generate NK activity in vitro was assessed. The presence of 10(5) EL-4 cells/10(6) spleen or bone-marrow cells correlated with a total lack of NK activity in these organs; in contrast, no decrease in NK activity was evident in the spleen or bone marrow of B16-bearing mice. The removal of metastatic EL-4 cells (by antibody and complement) from the spleen or bone marrow did not rescue the NK activity. The lack of NK activity in spleen and bone marrow colonized by metastatic cells was not due to induction of a suppressor cell in the host. Metastatic EL-4 cells appeared to have a direct and irreversible suppressive effect on the generation of NK activity by spleen or bone marrow. A possible cause-effect relationship between metastatic colonization of lymphoid organs and suppression of local NK activity is considered.     

EBV-transformed lymphoblastoid cell lines down-regulate ebna in parallel with secretory differentiation

Four monoclonal and one polyclonal lymphoblastoid cell line (LCL) were studied with regard to cytoplasmic immunoglobulin (clg) expression, presence of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) and DNA synthesis. Each line was found to consist of two subpopulations, with only minimal overlap. Proliferating, EBNA-positive, clg-negative cells formed the majority. The minority were EBNA-negative, contained abundant clg and were largely non-proliferating. This suggests the continuous occurrence of a maturation process within each LCL. The concomitant downregulation of EBNA raises the interesting question whether continued synthesis of the nuclear antigen is incompatible with differentiation for epigenetic reasons, or, alternatively, whether differentiation takes place when the viral genomes are suppressed or lost.     

The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV

Continuous human lymphoblastoid cell lines (LL), derived from lymphoid tissue or peripheral blood of 20 adults without histories of recent infectious mononucleosis at a frequency close to 100 %, were examined for the presence of Epstein-Barr virus (EBV) using immunofluorescence methods for the detection of EBV-dependent cell membrane and EB viral capsid antigens. All lines except one were found to be EBV carriers in the initial tests, but the antigens gradually disappeared in most during the course of an observation period of 4 months. Only eight lines maintained the initial percentage of antigen-containing cells. The results of the two immunofluorescence tests, performed simultaneously on each cell pool, were concordant in all instances. Sera were available from 16/20 donors. All cell donors, except one, possessed antibodies to EBV as an indication of prior EBV infections. The growth-promoting role of EBV in the establishment of LL was supported by studies with fetal lymphoid tissue cultured by the same grid method which yielded the high frequency of LL from adults. In sharp contrast to the results obtained with adult lymphoid tissue, no lines were established from 20 fetuses aged 13-20 weeks. However, when such tissue was exposed to a cell-free filtrate prepared from an EBV-carrying LL lymphoblastoid cell, lines were established in some instances. Filtrate from an EBV-negative line inoculated into parallel cultures failed to promote the establishment of LL. The results indicate that EBV infection in vivo or in vitro may be a prerequisite for the indefinite growth of lymphoblastoid cells in vitro and that EBV infections, as a rule, are not vertically transmitted.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

Expression of Epstein-Barr virus-encoded proteins and B-cell markers in fatal infectious mononucleosis

We assessed 33 lymphoid tissues from 15 patients, including 7 with X-linked lymphoproliferative disease (XLP) and 8 patients with sporadic fatal infectious mononucleosis (IM), to determine whether the cellular infiltrate had the immunophenotype and expressed Epstein-Barr virus (EBV)-encoded proteins characteristic of either EBV-immortalized lymphoblastoid cell lines (LCL) or EBV-carrying Burkitt lymphoma (BL) cells. The results of these studies revealed that in 13 cases the proliferating B cells were polyclonal, LCL-like, and in 2 cases they were monoclonal, malignant lymphoma-like.     

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.     

Nature of the delayed graft-versus-host reactivity of fetal liver cell transplants in mice.

Experiments were designed to determine which actual differences in the cellular composition between fetal liver and bone marrow account for the distinct types of graft-versus-host (GvH) disease. The assay of reactive lymphocytes (by in vitro mitogenic stimulation) in fetal liver transplants in mice, the purification of hemopoietic stem cells (HSC) of the transplants, and the quantitation of HSC numbers in the grafts traced the basis for the distinctly weak type of GvH disease after fetal liver cell grafts. It was found that transplantation of purified HSC concentrates did not modify the severity of GvH mortality. The moderate character of the delayed GvH disease was shown to depend on the presence of an HSC population in fetal liver with different qualities and not on numerical differences between the HSC in fetal liver and bone marrow. Data collected also demonstrated that when GvH disease occurred in the recipients of transplants of fetal liver, it shared the characteristic histologic features of the bone marrow GvH syndrome. The recovery of mitogen responsiveness of spleen cells may have been delayed in fetal liver allotransplantation as compared to syngeneic grafting. By supportive infusion of lymphoid cells, it was suggested that the immunodeficiency coinciding with GvH disease represented a secondary manifestation of the disease rather than a primary impairment in lymphoid differentiation.     

B cells from leukemic patients are relatively resistant to in-vitro EBV transformation

Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation.     

Enhanced AKR leukemogenesis by the dual tropic viruses. I. The time and site of origin of potential leukemic cells

The occurrence of potential leukemia cells (PLC) among bone marrow, spleen, and thymus of AKR mice during the preleukemic period was tested by an in vivo transplantation bioassay. The presence of PLC in 30- and 75-day-old AKR mice was demonstrated mostly among bone marrow cells, less in spleen, and was lacking in thymus. Occurrence of PLC in young AKR mice was shown to be thymus independent. However, progression of PLC from young donors (14-80 days old) into overt leukemia following transplantation into F1 recipients was shown to be dependent on specific host conditions including an intact thymus and an Fv-1nn allele. In contrast, PLC from 7-9-month-old AKR mice or frank leukemic cells when transplanted grew in any intact or thymectomized histocompatible host, thereby indicating their autonomous growth state. Infection of 2-week-old AKR mice with the dual-tropic virus DTV-70 induced characteristic changes in the thymus and accelerated leukemia development. DTV-70 inoculation into 14-day-old AKR mice did not change the spontaneous PLC distribution pattern in the tested host organs within 30 days postinfection, nor did it change PLC-specific host requirements for further progression into leukemic cells; however, it enhanced PLC transition to autonomous leukemic cells. The preferential cell tropism of DTV-70 for target cells (prothymocytes) among bone marrow and young spleen cells rather than for thymocytes was also demonstrated in an in vitro-in vivo test. The dual tropic virus may act as a promoter on preexisting PLC (present mostly among bone marrow cells) by enhancing their ability to progress into autonomous leukemic cells.     

Role of Hepatocyte Growth Factor in Hemopoiesis

Hepatocyte growth factor (HGF) is a polypeptide that stimulates proliferation, motility, and morphogenesis of various cells, particularly epithelial cells. There is considerable evidence that HGF is a regulator in hemopoiesis not only in mice but also in humans. In mice, HGF and c-met (its receptor) mRNA are coexpressed in the fetal liver in the middle and late stages, when hemopoiesis is most active. HGF and c-met mRNA are also expressed in the stromal cells of both fetal liver and bone marrow. Human HGF (2 to 20 ng/ml) enhances colony-forming units in culture (CFU-C) counts and cobblestone colony counts in the long-term cultures of the fetal liver and bone marrow, although HGF has no effect on freshly isolated bone marrow or fetal liver cells in the CFU-C assay. However, when the bone marrow or fetal liver cells are cocultured with HGF in the presence of IL-3, CFU-C counts increase. In humans, it has also been shown that HGF in the presence of erythropoietin induces the formation of erythroid burst-forming unit (BFU-E) colonies from CD34⁺ cells purified from the bone marrow, peripheral blood, or cord blood. This review discusses the role of HGF as a regulator in hemopoiesis.