Individual tumors of multifocal EB virus-induced malignant lymphomas in tamarins arise from different B-cell clones.

Cotton-top tamarins were inoculated with sufficient Epstein-Barr virus to induce multiple tumors in each animal within 14 to 21 days. The tumors consisted of large-cell lymphomas that contained multiple copies of the Epstein-Barr virus genome and generated Epstein-Barr virus-carrying cell lines showing no detectable consistent chromosomal abnormality. Hybridization of tumor DNA with immunoglobulin gene probes revealed that each lymphoma was oligo- or monoclonal in origin and that individual tumors from the same animal arose from different B-cell clones. Thus the virus induced multiple transformation events in tamarins in vivo to cause malignant tumors resembling the Epstein-Barr virus-associated lymphomas of patients with organ transplants.

Endemic Burkitt's lymphoma: phenotypic analysis of tumor biopsy cells and of derived tumor cell lines.

Tumor cells from 10 patients with Epstein-Barr virus-positive endemic Burkitt's lymphoma (BL) have been examined for cell surface phenotype, both at the biopsy stage and during BL cell line outgrowth in vitro, the cultures being followed for up to 150 passages. In all 10 cases, the biopsy cells showed coexpression of the common acute lymphoblastic leukemia antigen (CALLA) and of the BL-associated glycolipid antigen (BLA) with no accompanying expression of several "lymphoblastoid" cell surface markers defined by selected monoclonal antibodies. During cell line establishment and in vitro passage, the individual BL cell lines showed different degrees of progression toward a more "lymphoblastoid" cell surface phenotype, some even losing CALLA and BLA expression while retaining the chromosomal translocations indicative of their malignant origin. This differential capacity for phenotypic progression in vitro explains much, if not all, of the heterogeneity of the BL cell phenotype apparent from many previous studies with panels of long-established lines. Such heterogeneity in vitro belies the true homogeneity of the tumor cell phenotype in vivo.

Specific cytogenetic abnormalities in two new human colorectal adenoma-derived epithelial cell lines.

Two new epithelial cell lines from sporadic human colorectal adenomas designated S/AN and S/RG are reported. S/AN was from a villous adenoma and S/RG from a tubular adenoma. Both cell lines have extended growth capacities in vitro reaching passages 18 and 15, respectively, so far and show no signs of senescence. S/AN and S/RG have retained in vitro the ability to form mucin-producing goblet-like cells. Every cell of S/AN has a deletion on the short arm of chromosome 1 and one normal copy of chromosome 1. S/AN is also monosomic for chromosome 18. The majority of cells of S/RG only have one normal copy of chromosomes 6, 7, 14, 17, 18, and 22. S/RG also has several marker chromosomes. Although aneuploid S/AN and S/RG are nontumorigenic in athymic nude mice, these cytogenetic abnormalities are insufficient for the fully tumorigenic phenotype. The common abnormality for S/AN and S/RG is monosomy for chromosome 18, indicating that this is a central and important step in colorectal carcinogenesis. Our cytogenetic analysis of the adenoma cell lines suggests at least two possible routes by which premalignant colonic cells can develop and progress to malignancy. S/RG, unlike most other adenoma cell lines, is clonogenic. Aneuploidy, clonogenicity, and extended in vitro growth capacity may therefore be useful in vitro markers for adenoma cell lines with a relatively high malignant potential.

Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus oedipus oedipus (cotton top tamarin).

32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the beta chain of LFA-1 (CD18) cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the alpha chain of CD11a. Two monoclonals tested against ICAM-1(CD54) were negative.

Transcriptional expression of the viral genome in the Epstein-Barr virus-induced tamarin lymphoma and the corresponding lymphoblastoid tumour lines.

Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) invariably gives rise to mono- or oligoclonal large cell lymphoma occurring at multiple sites, and which resembles to a certain extent B cell lymphoma that occurs in the immunodeficient patient. The viral transcriptional pattern in tamarin tumour biopsies and in the corresponding tumour cell lines was investigated by means of the synthesis of radioactive single-stranded cDNA. It was found that the EBV transcripts came mainly from the fragments BamH1-H, BamH1-S, BamH1-A and EcoR1-Dhet. Transcripts from a few other early or late genes, namely BARF1, BSLF1/BMLF1, BBLF-4, BLLF1 and BXLF2, were also detected in one of the three biopsies tested. It would be important to characterize the transcripts that originate from the region where viral latent expression has not previously been observed. Our results also revealed that there is a sharp increase in EBV transcription in the tumour cell lines derived from the tamarin lymphomas. Simultaneously, the copy number of the viral genome was found to be amplified. Such a significant change in viral activity might be indicative of a close virus-host cell interaction in vivo.

Mucosal infection and vaccination against feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8+ cells and greater levels of IL-2, IL-6, IL-10 and gamma-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats.

Immortalization of a human colorectal adenoma cell line by continuous in vitro passage: possible involvement of chromosome 1 in tumour progression.

A non-tumorigenic epithelial cell line designated PC/AA, derived from a large pre-malignant colorectal adenoma from a patient with familial polyposis coli (also referred to as hereditary adenomatosis of the colon and rectum) has become immortal in vitro. PC/AA has been passaged in vitro continuously for over 4 years and shows no signs of senescence. At early passage, PC/AA has a normal diploid karyotype but with late passage is showing signs of progression, becoming aneuploid and displaying signs of morphological transformation. Every cell examined of late-passage PC/AA has an isochromosome (1q), and one other marker chromosome which is probably derived from an additional chromosome 8. The majority of cells examined have 48 chromosomes. Despite showing signs of progression in vitro, late-passage PC/AA has remained non-tumorigenic in athymic nude mice and retained morphological differentiation characteristics of colonic cells, in particular the ability to synthesize and secrete mucin. Two other cell lines derived from small adenomas did not become immortal in vitro and were also non-tumorigenic in athymic nude mice. The isolation of an immortal pre-malignant human epithelial cell line could prove invaluable in studies on human carcinogenesis and tumour progression. Our results, showing that only a large adenoma and no small adenomas have given rise to immortal cell lines, raise the possibility that the acquisition of in vitro immortality is associated with a relatively late stage in the adenoma-carcinoma sequence. The possible involvement of chromosome 1 in tumour progression is discussed.

Interaction of Epstein-Barr virus with leukaemic B cells in vitro. I. Abortive infection and rare cell line establishment from chronic lymphocytic leukaemic cells.

Leukaemia B cell populations, each with an individual pattern of monoclonal surface immunoglobulin expression, were obtained from 23 patients with chronic lymphocytic leukaemia (CLL) and, following exposure to a potent dose of Epstein-Barr (EB) virus in vitro, were monitored for expression of the virus associated nuclear antigen EBNA, for activation of immunoglobulin synthesis and for virus-induced transformation to an established cell line. Although possessing the EB virus receptor, CLL cells were generally refractory (vis-à-vis normal adult B cells) to the full effects of the viral infection. All the leukaemic populations tested developed a small proportion of EBNA positive cells within a few days post-infection, but in most instances this disappeared with no subsequent evidence of viral activity. In certain cases, however, the EBNA staining became more intense, involving a larger fraction of the population and persisting for some weeks, but again this was not accompanied by virus-induced immunoglobulin synthesis or transformation. In contrast, the leukaemic cells from a single patient, tested on three separate occasions, regularly responded to EB virus infection with the rapid establishment of an EBNA positive B cell line in which the restricted pattern of surface and cytoplasmic immunoglobulin expression (gamma lambda) exactly matched that present on the original leukaemic cells.

Demonstration in vitro of cell mediated immunity to Epstein-Barr virus in cotton-top tamarins.

In the course of developing an effective Epstein-Barr (EB) virus vaccine, the immune responses in cotton-top tamarins to a tumourigenic dose of EB virus were studied. Cell mediated responses were measured using a tissue culture 'growth inhibition' assay where peripheral blood lymphocytes were tested for their ability to inhibit the outgrowth of autologous EB virus transformed lymphoblastoid cells. This system has previously been recognized as a very sensitive assay for detecting cell-mediated responses to EB virus in man. Using this assay no cell-mediated immunity was detected up to the time of death in two tamarins following injection with a tumourigenic dose of EB virus. However, two other animals which had recovered from tumours induced by a first dose of EB virus 18 months previously when subsequently re-stimulated with a second tumourigenic dose did exhibit cell-mediated responses. These latter animals remained healthy following the re-challenge and did not show evidence of EB virus-induced disease.

Mechanism of the establishment of Epstein-Barr virus genome-containing lymphoid cell lines from infectious mononucleosis patients: studies with phosphonoacetate.

A concentration of disodium phosphonoacetate (PA) has been defined which will reduce the synthesis of infectious EB virus in a producer cell line to 1% of control values but which will not affect the growth of EB virus-transformed cells in a 12-week colony-forming assay. When total mononuclear cells or T-lymphocyte-depleted mononuclear cells from the blood of acute IM patients were cultured in the presence of PA at the above concentration, the regular establishment of EB virus genome-containing cell lines seen in control cultures was almost totally abolished. In further experiments, when T-lymphocyte-depleted IM mononuclear cells were co-cultivated with foetal cells of the opposite sex in the presence and absence of PA, cell lines of mixed or of exclusively foetal origin were obtained not only from control co-cultures but also on those rare occasions when transformed foci developed in PA-treated co-cultures. The results suggest that all cell lines derived from the blood of IM patients are initiated in culture by a two-step process of virus release and secondary infection, and argue against the occurrence of any direct outgrowth of IM cells transformed by the virus in vivo.

Comparative studies on adult donor lymphocytes infected by EB virus in vivo or in vitro: origin of transformed cells arising in co-cultures with foetal lymphocytes.

Co-cultures were set up between equal numbers of mononuclear cells from the blood of EB virus-infected individuals, either acute IM patients or healthy seropositive adult donors, and foetal cord blood mononuclear cells of the opposite sex. The cell lines arising in the co-cultures were of mixed origin, with foetal cells predominating in many cases. In contrast, when mononuclear cells from seronegative adult donors were first infected with EB virus in vitro and then 5 to 12 days later co-cultured with a large excess of foetal cells of the opposite sex, the cell lines which arose were almost exclusively derived from the adult donor despite the fact that a small minority of the virus-infected adult cells released infectious virus capable of transforming the co-cultivated foetal cells. The experiments suggest that EB virus-infected cells present in the blood of IM patients and seropositive donors do not possess the capacity for unlimited in vitro growth shown by seronegative adult donor lymphocytes experimentally infected with the virus.

Possible involvement of chromosome 1 in in vitro immortalization: evidence from progression of a human adenoma-derived cell line in vitro.

We have previously reported that continuous in vitro passage in the presence of 3T3 feeders of a non-tumorigenic adenoma-derived epithelial cell line, designated PC/AA, resulted in its becoming immortal. At early passage PC/AA was normal diploid, whereas every cell of PC/AA late passage had an isochromosome 1(q) which led us to suggest that abnormalities of chromosome 1 may be involved in tumour progression. We now report the isolation of a 3T3-feeder-independent variant of early-passage PC/AA, designated PC/AA/FI, which was immortal in vitro and remained non-tumorigenic. Each cell of PC/AA/FI again has an isochromosome 1(q), like the late-passage PC/AA. However, with PC/AA/FI it is the other chromosome 1 of the homologous pair which is involved in the formation of the isochromosome 1(q). This is possible to determine because of the polymorphic centromeric heterochromatin on chromosome 1 of the early-passage PC/AA. With the late-passage PC/AA (grown with 3T3 feeders) the homologue with the large C-band has given rise to an isochromosome 1(q) whereas with PC/AA/FI it is the other homologue with the smaller C-band which has given rise to this isochromosome. Both the immortal PC/AA/FI and the immortal PC/AA late passage, therefore, have independent abnormalities involving chromosome 1. These results indicate that chromosome 1 may be involved in in vitro immortalization.

Replication-defective recombinant adenovirus expressing the Epstein-Barr virus (EBV) envelope glycoprotein gp340/220 induces protective immunity against EBV-induced lymphomas in the cottontop tamarin.

A replication-defective recombinant adenovirus (Ad) expressing the full length Epstein-Barr virus (EBV) major envelope glycoprotein gp340/220 was tested for its ability to protect against EBV-induced lymphoma in the cottontop tamarin. Antibody responses against Ad capsid proteins and EBV gp340/220 were observed but these antibodies did not neutralize EBV in vitro. However, all immunized animals were protected against challenge following three intramuscular doses of the recombinant Ad. These data indicate that the recombinant Ad is potentially a useful vector for vaccination.

Analysis of Epstein-Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy.

Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein-Barr virus (EBV).

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro restimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4- CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.

Not all potently neutralizing, vaccine-induced antibodies to Epstein-Barr virus ensure protection of susceptible experimental animals.

Cottontop tamarins have been immunized with a high molecular weight, Epstein-Barr (EB) virus membrane antigen (MA) glycoprotein (gp340) separated by monoclonal antibody immunoaffinity chromatography (MCABgp340). Specific antibody production was monitored by immunofluorescence, a highly sensitive enzyme-linked immunosorbent assay (ELISA), and virus neutralization tests, and was found to reach high titre after 4/5 inoculations. The animals were challenged with a 100% lymphomagenic dose of EB virus but despite possessing powerful neutralizing antibodies were not protected against tumour causation by the virus. This result contrasts with that of earlier experiments in which tamarins with neutralizing antibodies induced by gp340 prepared by a molecular weight-based method (MWgp340) were protected. The reasons for this difference in protection associated with vaccine molecules prepared in different ways are discussed together with the need for parameters other than neutralizing antibody for use in the assessment of subunit immunogens against EB virus.

Protection of cottontop tamarins against Epstein-Barr virus-induced malignant lymphoma by a prototype subunit vaccine.

Epstein-Barr (EB) virus is one of the five herpesviruses of man. Strong links between this agent and the chain of events causing two human cancers, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, have long been evident (reviewed in ref. 1). Because of this, and because of the very high incidence of nasopharyngeal carcinoma in certain large populations, it was suggested in 1976 that a vaccine should be developed against EB virus to prevent infection and thereby reduce tumour incidence amongst those at risk. The virus-determined membrane antigen (MA) was proposed as immunogen because it was known to elicit naturally occurring virus-neutralizing antibodies in man and because analogous antigens had been shown to act as effective experimental vaccines for preventing the herpesvirus-induced lymphomas of Marek's disease in chickens. Progress has been achieved in defining, quantifying and preparing MA molecules, and in enhancing their immunogenicity; a sensitive assay for antibodies to MA has been elaborated. Here we report that isolated cell membranes expressing MA, or purified MA glycoprotein of relative molecular mass (Mr) 340,000 (gp340), have been used to vaccinate cottontop tamarins (Saguinus oedipus oedipus), and that animals receiving either preparation were protected against the effects of a 100% tumour-inducing challenge dose of EB virus.

Epstein-Barr (EB) virus genome-containing, EB nuclear antigen-negative B-lymphocyte populations in blood in acute infectious mononucleosis.

Experiments have been performed to identify the type and size of cell infected by EB virus in the blood of acute infectious mononucleosis (IM) patients, and to investigate the nature of the infection. Virus-infected cells, recognized by their ability to give rise to lymphoblastoid cell lines when co-cultivated with foetal lymphocytes, were shown to be restricted to the B-lymphocyte population. Samples of this population from each of eight IM patients were found to be negative for EB nuclear antigen (EBNA) staining. Thereafter, fractions of IM B-lymphocytes prepared on the basis of cell size were assayed either by co-cultivation, for the incidence of virus-infected cells, or by immunofluorescence staining for the presence of cells expressing EBNA. The great majority of virus-infected cells were found in the fractions of normal sized B-lymphocytes and yet these fractions were unequivocally EBNA-negative B-cell populations in IM blood is discussed in terms of the type of infection established by EB virus in the circulation of IM patients.

Interaction of Epstein-Barr virus with leukaemic B cells in vitro. II. Cell line establishment from prolymphocytic leukaemia and from Waldenström's macroglobulinaemia.

Leukaemic B-cell populations were prepared from six patients with high-count prolymphocytic leukaemia (PLL) as well as from one patient with Waldenström's macroglobulinaemia (WM) in frankly leukaemic phase, and their response to in vitro Epstein-Barr (EB) virus infection was monitored in terms of expression of the virus-associated nuclear antigen EBNA and of virus-induced transformation to continuous cell lines. The individual leukaemic populations, tested on several occasions, gave reproducibly different responses one from another which were not obviously related to differences either of surface immunoglobulin phenotype or of immunoglobulin secretor status in vivo. After infection, four out of six PLL populations showed either transient or a more persistent expression of EBNA, always involving a minority of the cells, with no evidence of any virus-induced transformation up to six weeks. In contrast, two out of six PLL samples as well as the WM sample rapidly gave rise to EBNA-positive cell lines which, on the evidence both of restricted immunoglobulin class expression and of abnormal marker chromosomes, were clearly derived from the leukaemic cells. Further comparative studies of such leukaemic B-cell populations may help to define host cell components necessary for the triggering of EB-virus-induced cellular transformation.