Autologous Lymphoblastoid Cell Lines Research Articles

Rapid Determination of Epstein-Barr Virus–Specific CD8+T-Cell Frequencies by Flow Cytometry

AbstractWe have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8+ T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon γ (IFNγ) is detected by multiparameter flow cytometric analysis. EBV-specific CD8+ T-cell frequencies ranged between 0.63% and 1.29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme-linked immunospot assay detecting IFNγ-producing T cells. The class I restriction of IFNγ production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV-specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8+ T-cell frequency has significant advantages for evaluation of EBV-specific CD8+ T-cell responses in PBMCs of patients with EBV-related diseases.

Rapid Determination of Epstein-Barr Virus–Specific CD8+T-Cell Frequencies by Flow Cytometry

We have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8+ T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon γ (IFNγ) is detected by multiparameter flow cytometric analysis. EBV-specific CD8+ T-cell frequencies ranged between 0.63% and 1.29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme-linked immunospot assay detecting IFNγ-producing T cells. The class I restriction of IFNγ production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV-specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8+ T-cell frequency has significant advantages for evaluation of EBV-specific CD8+ T-cell responses in PBMCs of patients with EBV-related diseases.

Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease.

The treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (PTLD) in EBV seronegative solid organ transplant recipients who acquire their EBV infection after engraftment poses a considerable challenge because of underlying immunosuppression that inhibits the virus-specific cytotoxic T cell (CTL) response in vivo. We have developed a protocol for activating autologous EBV-specific CTL lines from these patients and show their potential use for immunotherapy against PTLD in solid organ transplant patients. Peripheral blood mononuclear cells from a panel of solid organ transplant recipients with and without active PTLD were used to assess EBV-specific memory CTL responses. The activation protocol involved cocultivation of peripheral blood mononuclear cells with an autologous lymphoblastoid cell line under conditions that favored expansion of virus-specific CTL and hindered the proliferation of allospecific T cells. These CTL consistently showed (i) strong EBV-specificity, including reactivity through defined epitopes in spite of concurrent immunosuppressive therapy, and (ii) no alloreactivity toward donor alloantigens. More importantly, adoptive transfer of these autologous CTLs into a single patient with active PTLD was coincident with a very significant regression of the PTLD. These results demonstrate that a potent EBV-specific memory response can be expanded from solid organ recipients who have acquired their primary EBV infection under high levels of immunosuppressive therapy and that these T cells may have therapeutic potential against PTLD.

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.

The major Epstein-Barr virus (EBV) envelope glycoprotein gp340 when incorporated into Iscoms primes cytotoxic T-cell responses directed against EBV lymphoblastoid cell lines

A recombinant form of the EBV envelope glycoprotein and vaccine candidate gp340, lacking its hydrophobic transmembrane region, was incorporated into Iscoms after coupling to phosphatidyl ethanolamine via carbohydrate residues. Coupling by partial oxidation of gp340 carbohydrate with sodium periodate partly denatured the incorporated gp340 as indicated by its reduced reactivity with monoclonal antibodies that recognise the major neutralising epitope. Immunisation of cottontop tamarins with these Iscoms elicited antibody responses to gp340, but these antibodies only poorly recognised the major neutralising epitope in a competition ELISA and were unable to neutralise EBV in vitro. Despite the lack of neutralising antibody, immunisation with these Iscoms primed significant in vitro proliferative responses to soluble gp340 in lymphocytes from the draining lymph nodes and spleen. T-cell lines were raised from both immunised and control animals by in vitro stimulation of peripheral blood lymphocytes or spleen cells with autologous EBV-transformed lymphoblastoid cell lines. The T-cell lines from control animals had higher numbers of CD4+ T-cells than CD8+ T-cells and were not cytotoxic for autologous lymphoblastoid cell lines (LCL). In contrast the lines from immunised animals contained more CD8+ T-cells than CD4+ T-cells and had marked cytotoxicity for autologous LCL.

Accessing Epstein-Barr virus-specific T-cell memory with peptide-loaded dendritic cells.

The conventional means of studying Epstein-Barr virus (EBV)-induced cytotoxic T-lymphocyte (CTL) memory, by in vitro stimulation with the latently infected autologous lymphoblastoid cell line (LCL), has important limitations. First, it gives no information on memory to lytic cycle antigens; second, it preferentially amplifies the dominant components of latent antigen-specific memory at the expense of key subdominant reactivities. Here we describe an alternative approach, based on in vitro stimulation with epitope peptide-loaded dendritic cells (DCs), which allows one to probe the CTL repertoire for any individual reactivity of choice; this method proved significantly more efficient than stimulation with peptide alone. Using this approach we first show that reactivities to the immunodominant and subdominant lytic cycle epitopes identified by T cells during primary EBV infection are regularly detectable in the CTL memory of virus carriers; this implies that in such carriers chronic virus replication remains under direct T-cell control. We further show that subdominant latent cycle reactivities to epitopes in the latent membrane protein LMP2, though rarely undetectable in LCL-stimulated populations, can be reactivated by DC stimulation and selectively expanded as polyclonal CTL lines; the adoptive transfer of such preparations may be of value in targeting certain EBV-positive malignancies.

Definition of unique and shared T-cell defined tumor antigens in human renal cell carcinoma.

From peripheral blood mononuclear cells of a patient with renal cell carcinoma (RCC), we isolated several T-cell clones, which efficiently lyse the autologous RCC cell line (LE-8915-RCC), but not the autologous Epstein Barr virus-transformed lymphoblastoid cell line. Most of the cytotoxic T lymphocyte (CTL) clones recognize HLA-A1-positive allogeneic RCC cell lines, indicating that HLA-A1 is the restricting element for these T cells. One CTL clone exclusively recognizes the autologous tumor cells. The HLA-A1-restricted CTL clones can be divided further into two subsets of T-cell clones, one blocked by an HLA-A1-specific monoclonal antibody, the other not. The reactivity of HLA-A1-restricted T-cell clone 6/135 was studied in greater detail. This T-cell clone also recognizes a number of melanoma cell lines, indicating that expression of the antigen seen by this CTL clone is not restricted to RCC. Strikingly, the antigen is not exclusively expressed by tumor cell lines, because primary cultures of proximal tubulus epithelium cells, adult mesangial cells, and normal breast epithelium cells are also lysed. These results corroborate the notion that renal carcinoma cells are immunogenic by virtue of a broadly distributed antigenic structure that may serve as a target for cytotoxic T cells and may be a potential candidate for tumor vaccine development.

Assessment and characterization of the cytolytic T lymphocyte response against Epstein-Barr virus in patients with non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation.

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.

Recognition of B-CLL cells experimentally infected with EBV by autologous T lymphocytes

We compared 5-day-old cultures of two B-CLL clones experimentally infected with EBV for their interaction with autologous T lymphocytes. The clone which was strongly activated by the virus stimulated autologous T cells. It was also damaged by the cytotoxic T cells which were generated in mixed cultures with autologous lymphoblastoid cell lines (LCL). Cultured, non-infected CLL cells were not lysed by these effectors. The other B-CLL clone, which was activated to considerably lesser extent by the virus, did not stimulate the autologous T lymphocytes. While, also in this case cytotoxic function was generated in the mixed T cell—LCL culture, the effectors did not damage the EBV-infected CLL cells. The results with B-CLL cells can be regarded as a model for the EBV genome carrier normal B lymphocytes. They substantiate the current concept that such cells persist in seropositive healthy individuals undisturbed by the specific immune response as long as they maintain the phenotype of resting cells. However, after activation they can be recognized and eliminated by T cells.

Dominant cytotoxic T lymphocyte response to the immediate-early trans-activator protein, BZLF1, in persistent type A or B Epstein-Barr virus infection.

Five healthy human leukocyte antigen-B8 (HLA-B8)-positive virus carriers were studied to investigate the CD8+ cytotoxic T lymphocyte (CTL) response to an HLA-B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early trans-activator protein, BZLF1. Of the 5 virus carriers, 4 were infected with type A and 1 with type B EBV. Using limiting-dilution analysis of peripheral blood mononuclear cells, a high RAKFKQLLQ-specific CTL precursor frequency was demonstrated after specific peptide or autologous lymphoblastoid cell line stimulation in both type A and type B EBV carriers. The RAKFKQLLQ-specific CTL precursor frequencies in all 5 persons were at least as dominant as those observed with two other EBV-associated, HLA-B8-restricted latent epitopes, FLRGRAYGL and QAKWRLQTL. These findings show that healthy virus carriers maintain a high frequency of BZLF1-specific memory T cells, potentially to control virus spread from lytically infected cells.

Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro.

CD8+ T cells play a crucial role in surveillance against EBV-associated malignancies. EBV-specific T cells traditionally have been identified by their ability to kill autologous EBV-transformed B lymphoblastoid cell lines (LCL). Here we report CD8+ cloned and bulk T cells that specifically recognize EBV nuclear Ag EBNA-3C, but do not efficiently kill autologous or HLA-matched LCL. The low cytolysis of these T cells was due to the extremely low density of the antigenic epitope (LDFVRFMGV, EBNA-3C amino acids 285-293) on autologous LCL. The T cells efficiently killed target cells in the presence of < 1 pM synthetic EBNA-3C peptide and, therefore, recognize peptide/HLA complexes with high avidity. Donor T cells with this phenotype were stimulated by autologous LCL and dominated the in vitro EBV-specific response. This indicates that low abundance viral peptides can induce a dominant T cell response.

Use of Epstein-Barr virus-transformed, autologous B-lymphoblastoid cells as antigen-presenting cells for establishment and maintenance of dengue virus-specific, human cytotoxic T lymphocyte clones

We have been maintaining dengue virus specific CD8+ cytoxic T lymphocyte (CTL) clones by repeated stimulation using autologous peripheral blood mononuclear cells (PBMC) as antigen presenting cells (APCs). In the present study, Epstein-Barr virus (EBV)-transformed autologous lymphoblastoid cell lines (LCL) were compared with autologous PBMC as APCs for long term culture of a dengue virus-specific, HLA class I-restricted CD8+ CTL clone CB2.8. We substituted autologous LCL for autologous PBMC and maintained CB2.8 for several months. CB2.8 cultured using LCL as APCs maintained antigen specific cytolytic activity. No demonstrable difference in the specificity or in the level of cytolytic activity against a panel of target cells was noted between the CB2.8 maintained with LCL and those maintained with PBMC. Lysis of the target cells was blocked by the anti-HLA-class I antibody indicating that HLA class I-restriction was also maintained. We then compared autologous LCL with autologous PBMC in the establishment of CD4+ CTL clones from the PBMC of a dengue-1 immune donor. Dengue 1-specific clones were derived from limiting dilution cultures using either type of APCs. Similar numbers of dengue virus-specific CD4+ CTL clones were established using LCL or PBMC as APCs. These results indicate that autologous LCL act as APCs for long term culture of virus-specific CTL clones and represent a cost effective alternative to repeated collection of PBMC.

Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia

AbstractThe existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia

The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vbeta21+ T-cell clone that was most dominant among Vbeta21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti-HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

EXPANSION OF EPSTEIN-BARR VIRUS (EBV)-SPECIFIC CYTOTOXIC T CELLS (CTLs) IN GAS PERMEABLE CELL CULTURE BAGS

EBV-specific CTLs (CD8 effector T cells) cultured in 24-well plates have been used to treat post-transplant lymphoproliferative disease or prevent its development in childhood recipients of bone marrow transplants. Expansion in"open" systems, particularly in multiwell plates, is cumbersome and associated with risks of contamination. Therefore we have investigated the feasibility of expansion of EBV CTL's in closed gas-permeable bags. Methods: Lymphoblastoid cell lines (LCL) were produced by immortalizing peripheral blood lymphocytes with EBV. Autologous mononuclear cells collected by apheresis were incubated with irradiated LCL's. These incubations were set up in parallel in 24 well plates and in gas-permeable polyolefin-polystyrene bags (PL2417) from Baxter. After 3 weeks of stimulation with weekly addition of irradiated LCL's. IL-2 (25 units/ml) was added to the media. On day 28 the expanded products were evaluated. The T cell products expanded in bags were administered to patients in a pilot study. Results: Expansion of cells in 24 well plates required 19 and 27 plates versus 2 bags. EBV-specific cytolytic activity was evaluated by chromium release assay using autologous LCL's as targets. At various effector to target ratios lytic activity against autologous LCLs were similar. Flow cytometric analysis also showed similar profiles with >95% of cells marking with CD3 and >80% of cells marking with CD8. Two patients were treated with weekly CTL infusions(107/m2) for four consecutive weeks without adverse effects. Conclusions: Gas-permeable polyolefin polystyrene bags are suitable for use in the expansion of EBV-specific CTLs for clinical purposes.

Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays

To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumsporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8 + T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8 + T-cell responses against Plasmodium sp. proteins.

Defect of cell-mediated immune response against hepatitis B virus: an indication for pathogenesis of hepatitis-B-virus-associated membranous nephropathy.

To elucidate the questions of why not all patients with hepatitis B virus (HBV) infection develop HBV membranous nephropathy (HBVMN), we first measured serum HBe circulating immune complex (CIC) during the acute nephrotic phase of HBVMN and in HBV carriers. We found that the level of HBe CIC was low in the HBVMN patients and absent either in HBsAg+/HBeAg+ patients without HBVMN or HBsAg+/HBeAg- asymptomatic carriers. Second, we needed to characterize the cellular immune response to HBV in patients with HBVMN. However, lack of a suitable autologous effector/target cell system makes a precise study of HBVMN pathogenesis difficult. In the present study, we established a model system by using autologous HBcAg-expressing Epstein-Barr-virus-immortalized lymphoblastoid cell lines (LCL) as stimulator/target cells. Both proliferative response after stimulation with HBcAg and cytotoxic activity against autologous HBcAg-expressing LCL of the peripheral blood T cells obtained from the HBVMN patients and HBsAg carriers could be measured. Using autologous HBcAg-expressing LCL as stimulator/target cells for the study of HBcAg-specific cytotoxic T lymphocytes, we found that HBVMN patients had lower cytotoxic activity than did both HBV carriers and HBsAg-/HBsAb+, HBeAg-/HBeAb+ children. From the in vitro cytokine production study of peripheral blood T cells after stimulation with HBcAg, we found that T-helper-cell-1-related IL-2 and IFN-gamma productions were very low in HBVMN patients but T-helper-cell-2-related IL-10 production was higher in HBsAg+/HBeAg+ patients with HBVMN than in those without HBVMN. Based on these findings, we conclude that HBVMN children seem to have an inadequate cellular immune response to HBcAg.

Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability

Several lymphomas are associated with Epstein-Barr virus (EBV) infection. However EBV is not detectable in 100% of cases using standard staining techniques. It still remains an open question whether in these EBV-negative cases EBV has never infected the cell, whether it has infected the cell and escapes conventional screening methods, or whether it has been lost again after initial infection. The physical status of EBV in the Burkitt's lymphoma cell line BL60-P7 as well as in three somatic cell hybrids between BL60-P7 and its autologous EBV-immortalized lymphoblastoid cell line IARC 277 was analyzed using conventional cytogenetics, Southern blotting, and fluorescence in situ hybridization (FISH). Integration of EBV into the host genome of the lymphoma cell line BL60-P7 leads to an achromatic gap which causes a 'vulnerable site'. In hybrid cells, loss of integrated EBV, together with an adjacent chromosomal fragment, occurs during long-term cultivation. The integrated EBV genome, including genes encoding for LMP and EBER, is partly deleted. We assume that integration of EBV into the host cell genome could be a more common event in lymphoma cells. Partially deleted EBV might escape standard detection assays. The integration might constitute a chromosomal region prone to break events akin to the phenomenon of fragile sites, leading to the loss of viral DNA as well as chromosomal DNA. This observation makes it tempting to speculate that under certain conditions EBV can act in lymphomagenesis by a so-called hit-and-run mechanism.

Identification of type B-specific and cross-reactive cytotoxic T-lymphocyte responses to Epstein-Barr virus.

Persistent Epstein-Barr virus (EBV) infection is primarily controlled by HLA class I-restricted memory cytotoxic T-cell (CTL) responses which can be reactivated in vitro by stimulation of peripheral blood lymphocytes with autologous lymphoblastoid cell lines. During an investigation of a donor infected by both type A and type B EBV, CTL specific for type B EBV were isolated. The CTL were found to recognize an epitope encoded by the EBNA-6B gene. The minimal epitope sequence was identified as QNGALAINTF, corresponding to residues 213 to 222 in the EBNA-6B protein, and presentation of this epitope was shown to be via HLA B62 (B15). This is the first report of the characterization of an epitope that is EBV type B specific. CTL recognizing sequences common to type A and type B EBV were identified as well. A cross-reactive epitope recognized by these CTL was encoded within the EBNA-6 gene of both type A and type B. This minimal sequence for this epitope was LLDFVRFMGV (residues 284 to 293 in both types), and the epitope was restricted through HLA A*0201. This second epitope sequence overlaps with a published EBV B44-restricted epitope (EENLLDFVRF). The implications of these findings are discussed with respect to the design and efficacy of epitope-based vaccines.

Loss of an HLA haplotype in pancreas cancer tissue and its corresponding tumor derived cell line.

A combination of immunohistochemical, biochemical, and recombinant DNA techniques were used to investigate class I expression in 26 pancreatic adenocarcinomas and 6 autologous tumor-derived cells. The prevalence of HLA losses was found to be comparable to that observed in other tumor types (> 35%), using monomorphic and locus-specific antibodies. In one patient, the original tumor tissue, a tumor derived cell line (IMIM-PC-2), and EBV-transformed lymphocytes were available for study. The patient's phenotype was A25, A30, B18, B18. However, A30 allele product could not be detected in the original tumor not in the cultured tumor cells. In addition, A30 allele could not be isolated from cDNA or genomic clones from the cultured tumor cells whereas it was isolated from the autologous lymphoblastoid cell line. Using isoelectric focusing analysis a significant reduction in the B18 heavy chain product was also observed in the tumor cell line, IMIM-PC-2, suggesting the absence of expression of one allele. Further studies revealed loss of heterozygosity at DR and other loci of chromosome 6 and cytogenetic data strongly suggested deletion of a full chromosome 6. This work indicates for the first time that loss of a full HLA haplotype occurs in tumor tissue and suggests that this mechanism may contribute to the progression of human cancer.