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Recognition of the Epstein-Barr Virus-Encoded Nuclear Antigens EBNA-4 and EBNA-6 by HLA-A11-Restricted Cytotoxic T Lymphocytes: Implications for Down-Regulation of HLA-A11 in Burkitt Lymphoma
Abstract
Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-A11. To gain some insight into the role of HLA-A11 down-regulation in pathogenesis of BL, we have investigated the target specificity of HLA-A11-restricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBV-transformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-A11-restricted CTLs in all three donors. A less pronounced and less regular EBNA-6-specific cytotoxic component was found in two of the donors.
... Thus, EBV-specific CTL precursors can be reactivated in relatively large numbers from the T-cell pool of EBV-seropositive donors by challenging in vitro with autologous virus-infected B cells (22,27). Recent studies have shown that in each individual such CTL responses are composites of several virus-induced reactivities (3,11,18). Each reactivity is directed against one or another of the transformation-associated viral proteins presented in the context of particular HLA class I restricting determinants. ...
... The HLA All allele provides a particularly clear example of this phenomenon, since CTLs restricted through this determinant are often dominant in polyclonal CTL cultures reactivated in vitro from HLA All-positive individuals (17,26). Using recombinant vaccinia virus vectors, this All-restricted response was shown to be directed largely to the transformation-associated viral antigen EBNA4 (3,18). Here, we show that the EBNA4 protein contains multiple HLA All-restricted epitopes with different immunogenicities. ...
... However, these studies did not seek to determine the relative importance of these multiple epitopes within the context of the overall response. Work on the EBV system conducted to date has, likewise, identified certain alleles (for example, HLA B8) which can present epitopes from several different EBV target proteins, while other alleles show a strict preference for one particular protein (for example, the preference of HLA All for presentation of EBNA4) (3,11,18). The latter combination is particularly interesting because All-restricted EBNA4-specific responses tend to be the major component in polyclonal CTL cultures reactivated from HLA All-positive individuals (17,26). ...
... Cells expressing viral antigens are eliminated primarily by cytotoxic T lymphocytes (CTLs) in an MHC class I-restricted manner . Among EBV latent infection genes, EBNA-2, -3, -4 and -6 and LMPs can induce efficient CTL responses in combination with HLA-A2 or -A11 (Murray et al., 1988Gavioli et al., 1992;Khanna et al., 1992;Lee et al., 1993). Thus, chronic pyothorax patients with these alleles might show efficient CTL responses to transformed B-cells or to lymphoma cells, which would suppress the development of overt lymphoma. ...
... We assumed that chronic pyothorax patients with HLA-A2 or -A11, which could efficiently induce EBV latent antigen-specific CTL responses (Murray et al., 1988Gavioli et al., 1992;Khanna et al., 1992;Lee et al., 1993), would not develop overt lymphoma. We thus report the distribution of HLA-A alleles in patients with EBV-positive malignancy in the latency III form of infection. ...
Pyothorax‐associated lymphoma (PAL) is an Epstein‐Barr virus (EBV) latent antigen‐positive lymphoma resembling EBV‐transformed lymphoblastoid cell lines (LCLs) and develops in non‐immunocompromised patients. Thus, deficient anti‐viral‐antigen immune responses might be involved in the development of PAL. As MHC class I–restricted cytotoxic T lymphocytes (CTLs) are the major constituent of anti‐viral immune responses, the HLA allele type and its expression may affect the development of PAL. Flow‐cytometric analyses of PAL cell lines and LCLs using the W6/32 monoclonal antibody revealed that expression of HLA class I varied among cell lines. Although one PAL cell line, OPL‐2, exhibited low expression, an LCL and another PAL cell line, OPL‐1, strongly expressed HLA class I. Among the EBV latent infection genes, EBV nuclear antigens 2, 3, 4 and 6 and latent membrane proteins can induce efficient CTL responses in combination with HLA‐A2 or ‐A11. HLA‐A alleles of PAL patients were determined using low‐resolution PCR‐based typing with HLA‐A locus sequence‐specific primer combinations. The antigen frequencies of HLA‐A2 and ‐A11 in PAL patients were not significantly different from those in the normal Japanese population. Although HLA class I antigen should be expressed during the course of lymphomagenesis, no HLA‐A alleles influenced the development of overt PALs. Int. J. Cancer 82:630–634, 1999. © 1999 Wiley‐Liss, Inc.
... Human tumors and tumor cell lines exhibiting defects in transport of class I molecules and in antigen presentation have been recently described (30)(31)(32)(33)(34). A large number of Burkitt's lymphoma cell lines are unable to process intracellular antigenic determinants, and are highly resistant to class I-restricted CTL-mediated lysis (33,35). Khanna et al. (33) demonstrated that both peptide transporters (TAP1 and 500 TAP2) are downregulated in the tumor cells, resulting in inefficient transport of peptides to the ElK. ...
... Cromme et al. (32,34) demonstrated that in neoplastic cervical ceils that had lost their class l MHC surface expression, the peptide transporter TAP1 was no longer expressed by some or all of the tumor cells (36). Some DNA viruses are associated with both human and murine tumors that are deficient in class I molecule transport; cervical carcinomas harbor human papilloma virus sequences (34), Burkitt's lymphomas contain EBV sequences (35), and in Ad12-transformed rodent cells the early region oncogenes (E1A and E1B) of Adl2 are essential and sufficient for Ad12-mediated transformation (37). We previously showed that reexpression of the TAP2 gene in an Ad12-transformed cell line, which is downregulated for the expression of TAP genes, partially restored PD1 (a miniature swine class I transgene) and H-2D u expression, but did not reconstitute that of the endogenous H-2K b (31). ...
Expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the in vivo oncoge-nicity of this virus. In primary embryonal fibroblasts (H-26) that express a transgenic swine class I antigen (PD 1), Ad12-mediated transformation results in inhibition in transport of newly synthesized class I molecules, as well as significant reduction in transporter associated with antigen presentation (TAP) gene expression. In this report we show that reexpression of TAP molecules either by stable transfection of mouse TAP genes or by infection with recombinant vac-cinia viruses expressing human TAP genes, only partially reconstitutes the expression and transport of the class I molecueles. Further analysis of Ad12-transformed cells revealed that the expression of both LMP2 and LMP7, but not of other proteasome complex components, was downregulated, resulting in altered proteolytic activities of the 20S proteasomes. Reconstitu-tion of both TAP and LMP expression resulted in complete restoration of PD1 cell surface expression and enhanced expression of the endogenous H-2D 6 molecules. Despite high expression of TAP, LMP, and class I MHC molecules encoded by recombinant vaccinia viruses, in reconstituted Ad12-transformed cells, efficient transport of H-2 class I molecules could only be achieved by treatment of the cells with ~/-interferon. These data suggest that an additional fac-tor(s) that is interferon-regulated plays a role in the biosynthetic pathway of the class I complex, and that its function is deficient in this cell system. Thus, Ad12 viral transformation appears to suppress the expression of multiple genes that are important for antigen processing and presentation , which allows such transformed cells to escape immune surveillance. This coordinate downregnlation of immune response genes must likely occur through their use of common regulatory elements.
... EBNA3B is a transformation-associated EBV nuclear antigen that has been shown to contain multiple HLA-A11- restricted epitopes with different immunogenicities. A high prevalence of EBNA3B (EBNA-4) mutations has been proposed as mechanism of escaping the CTL response in certain HLA types (Gavioli et al., 1992 and 1993; de Campos-Lima et al., 1993). EBNA3B (EBNA-4) induces expression of vimentin and CD40 (Silins et al., 1994). ...
... EBNA3B (EBNA-4) is a transformation-associated EBV nuclear antigen that has been shown to contain multiple HLA- A11-restricted epitopes with different immunogenicities (Chu et al., 1999). A high prevalence of EBNA3B (EBNA-4) mutations has been proposed as a mechanism of escaping the CTL response in certain HLA types (Gavioli et al., 1992 and 1993; de Campos-Lima, 1993; Chu et al., 1999). ...
Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection. Epstein-Barrin virus (EBV) aiheuttaa nuoruusiässä yleisen mononukleoosin, mutta liittyy myös pahanlaatuisiin tauteihin. Perusterveillä infektiopotilailla EBV-infektio voidaan osoittaa luotettavasti modernien virusvasta-ainetutkimusten eli serologian avulla. Primaarissa EBV-infektiossa kehittyy IgM- ja IgG-vasta-aineita viruksen kapsidiantigeenille (viral capsid antigen, VCA). Toipumisvaiheessa VCA-IgM- vasta-aineet häviävät, kun taas VCA-IgG-vasta-aineet säilyvät koko eliniän. EBV voi aktivoitua elimistössä, ja tuolloin serologia yksinään ei riitä taudinmääritykseen. Määrittämällä VCA-IgG:n aviditeetti eli sitoutumisvoima voidaan erottaa toisistaan tuore infektio ja elimistössä piilevän viruksen aktivoituminen. Mononukleoosin diagnostiikassa nähdään toisinaan, paradoksaalisesti, VCA-IgM samanaikaisesti korkea-aviidisen VCA-IgG:n kanssa. Selvitimme tämän ilmiön syytä ja totesimme, että primaari sytomegalovirus (CMV) -infektio aiheuttaa joka neljännellä potilaalla EBV:n aktivoitumisen tai VCA-IgM:n ilmaantumisen. Primaarinen EBV-infektio sen sijaan ei aktivoinut CMV:ta vastaavalla tavalla. Elimistön immuunipuolustuksen heikentyessä, kuten elinsiirtojen yhteydessä tai AIDS-potilailla, EBV:n aktivoituminen voi saada elimistön valkosolut lisääntymään hallitsemattomasti. Tätä EBV:n aiheuttamaa lymfoproliferatiivista syndroomaa (post-transplant lymphoproliferative disease, PTLD) esiintyy erityisesti luuytimen, mutta myös kiinteiden elinten (maksa, munuainen, sydän), siirtojen yhteydessä. PTLD:n taudinkuva vaihtelee diffuusista muodosta B-solulymfoomaan ja vaikeusaste lievästä henkeä uhkaavaan; fataalit muodot ovat hyvinkin nopeasti progredioivia. Pääasiallisena hoitona on käytetty immunosuppression keventämistä, mikä edellyttää taudin varhaistoteamista. Lupaavan tehokas uusi hoitomuoto perustuu solutyyppispesifisten CD-vasta-aineiden käyttöön (myös Suomessa). PTLD:n diagnostiikka on ollut ongelmallista immunosuppression, siirtoverituotteiden ja viruksen piilevyyden vuoksi. Olemme vuonna 1999, ensimmäisenä Suomessa minkään mikrobin osalta, pystyttäneet EBV-tautien, ml. PTLD, diagnostiikkaan kvantitatiivisen real-time tyyppisen geenimonistusmenetelmän. Lisäksi pystytimme in situ hybridisaatio -menetelmän, jonka avulla EBV:n RNA voidaan osoittaa kudoksista. Yhteistyössä Meilahden sairaalan hematologien kanssa määritimme takautuvasti fataalin PTLD:n esiintyvyyden luuydin-kantasolusiirroissa (LYS) vuosina 1994-1999. Post-morten -analyysi paljasti 18 PTLD-tapausta. Tutkimme suuren aineiston pakastettuja seeruminäytteitä osasta PTLD-tapauksia (N=12) ja vastaavista verrokkipotilaista (N=36) uuden, kvantitatiivisen EBV-qPCR:n avulla: Kaikilla PTLD-potilailla havaittiin seeruminäytteissä EBV-DNA:ta, jonka määrä lisääntyi nopeasti taudin vaikeutuessa. EBV-DNA ilmaantui verenkiertoon keskimäärin 70 päivää luuydinsiirron jälkeen. EBV DNA oli havaittavissa uudella menetelmällä jo ennen oireiden puhkeamista (kuume, imusolmukkeiden suurentuminen) tai verenkuvan muutoksia. Verrokkiryhmässä EBV-DNA:ta näkyi vain satunnaisesti ja sen määrä jäi matalaksi. EBV-qPCR:n herkkyys ja tarkkuus olivat erinomaiset. Etsimme ja löysimme luuydinsiirtopotilasaineistosta myös PTLD-taudille altistavia riskitekijöitä. Selvitimme yhteistyössä elinsiirtokirurgiryhmän kanssa niin ikään EBV-infektioiden yleisyyttä ja vakavuutta maksansiirtopotilailla; tutkimme EBV-qPCR-menetelmän avulla 1284 plasmanäytettä 105:ltä aikuiselta maksansiirtopotilaalta. EBV-DNA:ta löydettiin 14 siirrepotilaalta (13%) ensimmäisten 12 kuukauden aikana. Kolmellatoista oireettomalla maksasiirtopotilaalla EBV-DNA:n määrät jäivät mataliksi (<6600/ml), ja EBV-DNA hävisi verenkierrosta nopeasti. Yhdelle potilaalle kehittyi fataali PTLD. Halusimme vielä selvittää erityyppisten EBV-infektioiden kokonaismäärän suurella, retrospektiivisellä aineistolla luuydinsiirtopotilaita: analysoimme EBV-qPCR:n avulla 5479 seeruminäytettä 406 potilaalta. EBV-DNA:ta löytyi kaikkiaan 57 potilaalta (14.0 %), joista 22:lla EBV-DNA määrä nousi korkealle (keskiarvo 54 milj./ml). Oireeton EBV-infektio todettiin 19 potilaalla (4.7 %), joilla nähtiin EBV-DNA:ta ohimenevästi (keskiarvo 6300/ml). Näin ollen matala EBV-positiivisuus näyttää olevan varsin yleistä luuydinsiirron jälkeen, ja EBV-DNA voi hävitä ilman toimenpiteitä. Kuitenkin EBV-DNA:n suuri pitoisuus (>50 000/ml) viittaa vahvasti hengenvaaralliseen EBV-infektioon. Kehitimme lopuksi EBV-DNA-tasojen matemaattisen analyysikaavan, jonka avulla voidaan ennustaa henkeä uhkaavan EBV-infektion kehittymistä.
... The same decreased risk of developing the disease was reported for Hodgkin lymphoma patients with HLA-A11 [28,62]. HLA-A*11 was also found by Gavioli et al. [63] to have an important down-regulating role in the case of Burkitt lymphoma. A study among Koreans noted the protective role of HLA-A*33 against DLBCL [24]. ...
Background and Objectives: The implications of the genetic component in the initiation and development of chronic lymphoproliferative disorders have been the subject of intense research efforts. Some of the most important genes involved in the occurrence and evolution of these pathologies are the HLA genes. The aim of this study is to analyze, for the first time, possible associations between chronic lymphoproliferative diseases and certain HLA alleles in the Romanian population. Materials and Methods: This study included 38 patients with chronic lymphoproliferative disorders, diagnosed between 2021 and 2022 at Fundeni Clinical Institute, Bucharest, Romania, and 50 healthy controls. HLA class I and class II genes (HLA-A/B/C, HLA-DQB1/DPB1/DRB1) were investigated by doing high resolution genotyping using sequence specific primers (SSP). Results: Several HLA alleles were strongly associated with chronic lymphoproliferative disorders. The most important finding was that the HLA-C*02:02 (p = 0.002, OR = 1.101), and HLA-C*12:02 (p = 0.002, OR = 1.101) have a predisposing role in the development of chronic lymphoproliferative disorders. Moreover, we identified that HLA-A*11:01 (p = 0.01, OR = 0.16), HLA-B*35:02 (p = 0.037, OR = 0.94), HLA-B*81:01 (p = 0.037, OR = 0.94), HLA-C*07:02 (p = 0.036, OR = 0.34), HLA-DRB1*11:01 (p = 0.021, OR = 0.19), and HLA-DRB1*13:02 (p = 0.037, OR = 0.94), alleles have protective roles. Conclusions: Our study indicates that HLA-C*02:02 and HLA-C*12:02 are positively associated with chronic lymphoproliferative disorders for our Romanian patients while HLA-DRB1*11:01, HLA-DRB1*13:02, and HLA-B*35:02 alleles have a protective role against these diseases.
... 39 Among EBV latent infection genes, EBNA-2, 3, 4 and 6 and LMPs can induce efficient CTL responses in a manner restricted to HLA-A-2, -11, B-7, -8, -27 and -44. 4,5,35 In the present cases, there was a significant difference in the frequency of HLA class I subtype between T-and B-cell cases. All 8 patients with T-cell type had HLA-A-2 or -11, while 44% of B-cell type had these subtypes. ...
Post‐transplantation lymphoproliferative disorders (PT‐LPD) are characterized by a clinically and morphologically heterogeneous group of lymphoid proliferation occurring after organ or bone marrow transplantation. The immunodeficient state provides a basis for lymphomagenesis probably through activation of oncogenic viruses. Twenty‐four patients in whom PT‐LPD developed after renal transplantation in Japan were analyzed. They received hemodialysis for 4 to 226 (median 13) months before transplantation. In situ hybridization was performed to detect Epstein‐Barr virus (EBV). Polymerase chain reaction and Southern hybridization with primers in the tax and pol regions of human T‐cell leukemia virus type I (HTLV‐1) were performed on DNA extracted from paraffin‐embedded specimens. Immunohistochemical analysis revealed that 12 cases were B‐cell type, 10 cases (42%) T‐cell type and 2 NK‐cell type. Five of the T‐cell cases were classified as adult T‐cell lymphoma with proven HTLV‐1 genome in the tumor and seropositivity for the virus. These cases were classified as adult T‐cell lymphoma (ALT). More than 80% of B‐cell, 30% of T‐cell and both NK/T‐cell lymphomas were EBV‐positive. Co‐infection of EBV and HTLV‐1 was found in 2 cases with ATL. These findings showed that ATL is common among Japanese renal transplant patients, which might be due to transmission of HTLV‐1 via blood transfusion during hemodialysis. © 2001 Wiley‐Liss, Inc.
... The immunological elimination of EBV-infected B lymphocytes depends on MHC class I-restricted presentation of processed EBV-derived peptides (Khanna et al., 1992;Murray et al., 1992;Gavioli et al., 1992). Therefore, we tested with FACS whether the expression of MHC surface molecules of the EBV-infected cells was reduced (Fig. 3) as described for BL. ...
Primary infection with the Epstein‐Barr virus (EBV) results in fatal infectious mononucleosis in up to 70% of males affected by the X‐linked lymphoproliferative syndrome (XLP). This rare disease is often associated with diverse natural killer (NK)‐, B‐ and T‐cell deficiencies. We describe experiments testing whether the B lymphocytes of affected males play a role in the pathogenesis of XLP due to a low susceptibility to T‐cell‐mediated immunity. Using reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemistry we detected in these B cells the expression of viral proteins EBNA‐1, EBNA‐2, EBNA‐3A, EBNA‐3C, LMP‐1 and LMP‐2A, which provide targets for cytotoxic T cells. Major histocompatibility complex (MHC) class I, MHC class II and the B7 costimulatory molecule were present on the cell surface. Accordingly, the EBV‐infected B cells were lysed in ⁵¹Cr‐release assays by T lymphocytes sharing MHC determinants with the targets. This MHC‐restricted and specific lysis was confirmed in competition experiments using MHC‐specific monoclonal antibodies (MAbs) and synthetic peptides. XLP‐derived LCLs could also induce MHC class I‐restricted memory and cytotoxic T lymphocytes. Thus, these XLP‐derived B cells resembled normal LCIs in vitro with respect to induction of EBV‐specific cytotoxic T cells (CTL), the ability to present EB viral antigens and the susceptibility to EBV‐specific and MHC‐restricted CTL‐mediated killing. The failure of the immune system to eliminate these virus‐infected B cells in XLP is clearly not caused by a B‐cell‐specific defect. Int. J. Cancer 76:694–701, 1998.© 1998 Wiley‐Liss, Inc.
... CTL activity was evaluated by a standard 4-h 51 Cr-release assay. Effectors were HLA-A11-restricted EBV-specific CTL obtained by stimulation of lymphocytes from one EBVseropositive donor with autologous B95-8 virus-transformed lymphoblastoid cells, according to Gavioli et al. [31], and expanded in RPMI 1640/FBS 10% medium supplemented with 100 U/ml rIL-2. Targets were autologous EBVtransformed B cells that were labeled with 100 ? ...
... Here we examine one such agent, EBV, a lymphotropic herpesvirus with growth transforming ability, which is widespread in human populations and which elicits strong CTL responses. Studies on healthy virus carriers have shown that EBV-specific CTL memory is predominantly HLA class I-restricted and directed against the limited number of viral pro-teins that are constitutively expressed in virus-transformed lymphobhstoid cell lines (LCLs) 1 (8)(9)(10). Using recombinant vaccinia viral vectors and synthetic peptides in CTL sensitization assays, a number of immunodominant CTL epitopes have now been identified within these EBV products, each presented in the context of a specific I-1LA class I molecule (9,(11)(12)(13)(14)(15). ...
Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.
... 25,26 The deficiency in the HLA class I-mediated stimulation of CD8 + T cells by BL has been well studied and stems from the poor immunogenicity of EBV nuclear antigen 1 (EBNA-1). [27][28][29] However, it remains unclear why the immune system is unable to mount an effective HLA class II-restricted response against BL. It has been suggested that one EBV gene product, gp42, is involved in blocking the HLA class II/T-cell receptor interaction. ...
While Burkitt Lymphoma (BL) has a well-known defect in HLA class I-mediated Ag presentation, the exact role of BL-associated HLA class II in generating a poor CD4+ T cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T cell recognition was not associated with low levels of costimulatory molecules on BL cells, as addition of external costimulation failed to elicit CD4+ T cell activation by BL. Further, the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II-peptide complexes were formed at acidic pH 5.5, which restored immune recognition. Acidic buffer (pH 5.5) eluate from BL cells contained molecules which impaired class II-mediated Ag presentation and CD4+ T cell recognition. Biochemical analysis showed that these molecules were greater than 30 kDa in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 kDa enolase-like molecule which enhances class II-mediated Ag presentation in B cells, macrophage and dendritic cells, but not in BL. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated Ag presentation and immune recognition, which may be exploited for future immunotherapies.This article is protected by copyright. All rights reserved.
... Cytotoxic activity was tested in standard 5-h 51 Cr-release assay [49]. PHA-blasts were labeled with 0.1 ? ...
The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN-α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.
... All NPC express EBNA 1 which is essential for maintenance of the viral episome. It has been shown that EBNA1 is not imunogenic, primarily because of its lack of processing of CTL epitopes (Gavioli et al., 1992; Murray et al., 1992; Khanna et al., 1992). While EBNA 1 is the only EBV protein present in Burkitt lymphoma, which contributes to why these EBV carrying tumor cells are not eliminated by the host antiviral immunity (Rowe et al., 1987). ...
... The immunological elimination of EBV-infected B lymphocytes depends on MHC class I-restricted presentation of processed EBV-derived peptides (Khanna et al., 1992;Murray et al., 1992;Gavioli et al., 1992). Therefore, we tested with FACS whether the expression of MHC surface molecules of the EBV-infected cells was reduced (Fig. 3) as described for BL. ...
Primary infection with the Epstein-Barr virus (EBV) results in fatal infectious mononucleosis in up to 70% of males affected by the X-linked lymphoproliferative syndrome (XLP). This rare disease is often associated with diverse natural killer (NK)-, B- and T-cell deficiencies. We describe experiments testing whether the B lymphocytes of affected males play a role in the pathogenesis of XLP due to a low susceptibility to T-cell-mediated immunity. Using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry we detected in these B cells the expression of viral proteins EBNA-1, EBNA-2, EBNA-3A, EBNA-3C, LMP-1 and LMP-2A, which provide targets for cytotoxic T cells. Major histocompatibility complex (MHC) class I, MHC class II and the B7 costimulatory molecule were present on the cell surface. Accordingly, the EBV-infected B cells were lysed in 51Cr-release assays by T lymphocytes sharing MHC determinants with the targets. This MHC-restricted and specific lysis was confirmed in competition experiments using MHC-specific monoclonal antibodies (MAbs) and synthetic peptides. XLP-derived LCLs could also induce MHC class I-restricted memory and cytotoxic T lymphocytes. Thus, these XLP-derived B cells resembled normal LCIs in vitro with respect to induction of EBV-specific cytotoxic T cells (CTL), the ability to present EB viral antigens and the susceptibility to EBV-specific and MHC-restricted CTL-mediated killing. The failure of the immune system to eliminate these virus-infected B cells in XLP is clearly not caused by a B-cell-specific defect. Int. J. Cancer 76:694–701, 1998.© 1998 Wiley-Liss, Inc.
... The EBV-encoded nuclear antigens (EBNA) were detected by anti-complement immunofluorescence (Reedman and Klein, 1973). LMP was detected by indirect immunofluorescence as described (Gavioli et al., 1992). (ii) Immunoblotting. ...
In studies concerning the interaction of B-CLL cells and Epstein-Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B-cell markers, the CLL cells expressed the exclusive T-cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl-2 and Igλ loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T-cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus-infected normal B cells may be coupled to the high expression of the bcl-2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA-positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T-lymphocyte cultures which acted on autologous EBV-infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T-cell response. © 1997 Wiley-Liss, Inc.
... However, the role of HLA class II-mediated Ag presentation in generating an immune response to BL has not been fully elucidated. The class I defect has been well studied and is understood to result from the weak immunogenicity of EBV nuclear Ag 1 (EBNA1), which is poorly processed and presented through the class I pathway161718. Another EBV gene product, gp42, has a role in mediating virus binding through interaction with HLA class II and it has been speculated to block the interaction between class II and the T-cell receptor [19, 20]. ...
While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.
... Studies have shown that EBV can elicit strong CTL responses which direct against a limited number of viral proteins [31][32][33]. Focus has been on the mutations of CTL epitopes in EBV latent-expressing proteins for their important roles in the associated malignancies, while little is known about the proteins which are expressed in the lytic phase. In the present study, we found 3 of 16 identified CTL epitopes of Rta were affected by AA mutations (Table 3). ...
Epstein-Barr virus (EBV) has a biphasic infection cycle consisting of a latent and a lytic replicative phase. The product of immediate-early gene BRLF1, Rta, is able to disrupt the latency phase in epithelial cells and certain B-cell lines. The protein Rta is a frequent target of the EBV-induced cytotoxic T cell response. In spite of our good understanding of this protein, little is known for the gene polymorphism of BRLF1.
BRLF1 gene was successfully amplified in 34 EBV-associated gastric carcinomas (EBVaGCs), 57 nasopharyngeal carcinomas (NPCs) and 28 throat washings (TWs) samples from healthy donors followed by PCR-direct sequencing. Fourteen loci were found to be affected by amino acid changes, 17 loci by silent nucleotide changes. According to the phylogenetic tree, 5 distinct subtypes of BRLF1 were identified, and 2 subtypes BR1-A and BR1-C were detected in 42.9% (51/119), 42.0% (50/119) of samples, respectively. The distribution of these 2 subtypes among 3 types of specimens was significantly different. The subtype BR1-A preferentially existed in healthy donors, while BR1-C was seen more in biopsies of NPC. A silent mutation A/G was detected in all the isolates. Among 3 functional domains, the dimerization domain of Rta showed a stably conserved sequence, while DNA binding and transactivation domains were detected to have multiple mutations. Three of 16 CTL epitopes, NAA, QKE and ERP, were affected by amino acid changes. Epitope ERP was relatively conserved; epitopes NAA and QKE harbored more mutations.
This first detailed investigation of sequence variations in BRLF1 gene has identified 5 distinct subtypes. Two subtypes BR1-A and BR1-C are the dominant genotypes of BRLF1. The subtype BR1-C is more frequent in NPCs, while BR1-A preferentially presents in healthy donors. BR1-C may be associated with the tumorigenesis of NPC.
... In one study, BL lines derived from five HLA-A11-positive donors (both EBV positive and negative) were shown to have decreased expression levels of HLA- A11 and were resistant to lysis by HLA-A11-restricted CTLs generated by stimulation with autologous B-LCL [106]. Other groups have investigated this aspect of BL immune evasion and reported similar observations, with HLA-A11 being the most commonly reported down-regulated form of HLA class I molecules [107, 108]. Based on the observations that a CTL response is capable of controlling outgrowth of B-LCL but is ineffective against EBV-positive BL cells, Jilg et al. [109] found that B-LCL expressed considerably higher levels of HLA class I molecules than those of BL. ...
B-cell lymphomas arise at distinct stages of cellular development and maturation, potentially influencing antigen (Ag) presentation and T-cell recognition. Burkitt lymphoma (BL) is a highly malignant B-cell tumor associated with Epstein-Barr Virus (EBV) infection. Although BL can be effectively treated in adults and children, leading to high survival rates, its ability to mask itself from the immune system makes BL an intriguing disease to study. In this paper, we will provide an overview of BL and its association with EBV and the c-myc oncogene. The contributions of EBV and c-myc to B-cell transformation, proliferation, or attenuation of cellular network and immune recognition or evasion will be summarized. We will also discuss the various pathways by which BL escapes immune detection by inhibiting both HLA class I- and II-mediated Ag presentation to T cells. Finally, we will provide an overview of recent developments suggesting the existence of BL-associated inhibitory molecules that may block HLA class II-mediated Ag presentation to CD4+ T cells, facilitating immune escape of BL.
... Here we examine one such agent, EBV, a lymphotropic herpesvirus with growth transforming ability, which is widespread in human populations and which elicits strong CTL responses. Studies on healthy virus carriers have shown that EBV-specific CTL memory is predominantly HLA class I-restricted and directed against the limited number of viral pro-teins that are constitutively expressed in virus-transformed lymphobhstoid cell lines (LCLs) 1 (8)(9)(10). Using recombinant vaccinia viral vectors and synthetic peptides in CTL sensitization assays, a number of immunodominant CTL epitopes have now been identified within these EBV products, each presented in the context of a specific I-1LA class I molecule (9,(11)(12)(13)(14)(15). ...
Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.
MHC class I (MHC-I) molecules present a blueprint of the intracellular proteome to T cells allowing them to control infection or malignant transformation. As a response, pathogens and tumor cells often downmodulate MHC-I mediated antigen presentation to escape from immune surveillance. Although the fundamental rules of antigen presentation are known in detail, the players in this system are not saturated and new modules of regulation have recently been uncovered. Here, we update the understanding of antigen presentation by MHC-I molecules and how this can be exploited by tumors to prevent exposure of the intracellular proteome. This knowledge can provide new ways to improve immune responses against tumors and pathogens.
In contrast to other neoplasms, antigen-specific autologous cytolytic T cells have not been detected in patients with human pre-B–cell leukemias. The absence of efficient B7 family (B7-1/CD80; B7-2/CD86) -mediated costimulation has been shown to be a major defect in tumor cells' capacity to function as antigen-presenting cells. We show here the generation of autologous anti–pre-B–cell leukemia-specific cytolytic T-cell lines from the marrows of 10 of 15 patients with pre-B–cell malignancies. T-cell costimulation via CD28 is an absolute requirement for the generation of these autologous cytolytic T cells (CTL). Although costimulation could be delivered by either bystander B7 transfectants or professional antigen-presenting cells (indirect costimulation), optimal priming and CTL expansion required that the costimulatory signal was expressed by the tumor cell (direct costimulation). These anti–pre-B–cell leukemia-specific CTL lysed both unstimulated and CD40-stimulated tumor cells from each patient studied but did not lyse either K562 or CD40-stimulated allogeneic B cells. Cytolysis was mediated by the induction of tumor cell apoptosis by CD8+ T cells via the perforin-granzyme pathway. Although we were able to generate anti–leukemia-specific CTL from the bone marrow, we were unable to generate such CTL from the peripheral blood of these patients. These studies show that antigen-specific CTL can be generated from the bone marrow of patients with pre-B–cell leukemias and these findings should facilitate the design of adoptive T-cell–mediated immunotherapy trials for the treatment of patients with B-cell precursor malignancies.
Purpose of Review
While most adults are infected with Epstein-Barr virus (EBV), 3–5% remain uninfected. The human leukocyte antigen (HLA) complex, which controls infection by many pathogens, may influence infection and disease associated with EBV.
Recent Findings
Numerous EBV proteins and miRNAs downregulate HLA class I and II expression on the cell surface. HLA class II functions as a receptor for EBV entry into B cells. Specific HLA class II alleles correlate with the susceptibility of B cells to EBV infection in vitro and with EBV seropositivity or seronegativity of humans. HLA class I polymorphisms correlate with development and severity of EBV infectious mononucleosis and with the risk of several virus-associated malignancies including nasopharyngeal carcinoma, Hodgkin lymphoma, and post-transplant lymphoproliferative disease.
Summary
These findings indicate that while EBV has evolved to use MHC class II as a receptor for virus entry, polymorphisms in MHC class II, and class I influence virus infection and disease.
Virally induced tumors provide the strongest case of host surveillance against neoplastic cells and their precursors. There is now substantial evidence that Epstein-Barr virus (EBV), hepatitis-B and C viruses (HBV and HCV), several types of papilloma viruses (HPV), Human Herpesvirus type 8 (HHV8) and human T-cell leukemia-lymphoma virus type I (HTLV I) and type II are responsible for approximately 15–20% of the total cancer incidence in the world. These viruses are widespread in populations where the associated diseases are seen at the highest incidence. In the vast majority of cases primary infection is either asymptomatic or is accompanied by benign proliferations of virus infected cells that often appear in concomitance with disturbances of the host immune responses and tend to regress spontaneously once full immunocompetence is restored. This, together with the observation that progression to malignancy occurs after long latency periods, and that the tumors are usually monoclonal, indicates that none of these viruses is by itself tumorigenic. An important aspect of viral oncogenesis is, therefore, the establishment of persistent asymptomatic infection where the transforming potential of the virus is controlled by a combination of cellular control mechanisms that regulate the expression of viral genes, and by strong immune responses that prevent the proliferation of virus infected cells.
Following the identification of the first tumor antigens recognized by CTL on melanoma cells, concern was expressed that tumors of other histological types might be less likely to express such antigens. However, the results obtained now with other types of tumors suggest that there is no fundamental difference in this respect between melanoma and other tumors. As new antigens are discovered, an increasing proportion are found to be the result of mutations, several of which may play a role in tumoral transformation or progression. Some of these tumor antigens are currently in the early stages of clinical study. There can be little doubt that the coming years will witness a large number of clinical trials involving peptides, proteins, DNA, and recombinant defective viruses, while increasingly sensitive tools are developed to measure the induction of CTL responses in immunized patients.
Not too long ago, it was still a mystery how the immune system was able to recognize the infinite number of viruses, bacteria, and other parasites and foreign elements. It was thought that antigens served as a template around which antibodies molded themselves to gain a complementary form. In 1955, Niels K. Jerne was the first to suggest that the immune response was selective rather than instructive, i e , that the immune system has the capacity to synthesize billions of different antibodies and that encountering antigen only increases production of the antibody that makes the best fit. Soon afterwards, Macfarlane Burnet and David Talmage suggested that each lymphocyte bears only one particular antibody as a receptor and that, after encounter with antigen, it divides to start mass production of the relevant antibody. Later, Susumu Tonegawa elucidated the molecular mechanism that generates the enormous diversity of antibodies, and discovery of the genes coding for the T cell receptor (TCR) by Tak W. Mak revealed that TCR diversity is generated in a similar way.
Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/ peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.
Transplantation experiments have demonstrated that most mouse tumors express antigens that can constitute targets for rejection responses mediated by syngeneic T lymphocytes. For human tumors, autologous cultures mixing tumor cells and blood lymphocytes or tumor-infiltrating lymphocytes have produced CD8(+) and CD4(+) cytolytic T cell (CTL) clones that recognize tumor cells specifically. Attempts to identify the target antigens by biochemical fractionation of tumor cells up to now have failed, with the important exception of the identification of underglycosylated mucins present on breast and pancreatic carcinomas. Gene transfection approaches have proved more successful. A gene family named MAGE codes for antigens recognized by autologous CTL on a melanoma tumor. These genes are not expressed in normal tissues except for testis. They are expressed in many tumors of several histological types. Differentiation antigens coded by genes such as tyrosinase are also recognized on human melanoma by autologous CTL. The identification of human tumor rejection antigens opens new possibilities for systematic approaches to the specific immune therapy of cancer.
Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
A strategy, based on using V beta family-specific oligonucleotides, was developed for specific amplification and direct sequencing of human TCR V beta genes. With this strategy, it was possible to undertake a structural analysis of TCRs from human T cell clones in specific responses. 12 HLA-B27-specific cytotoxic clones were examined. The results reveal a nonrandom use of V beta gene diversity in this alloreactive response in that: (a) the clones express a restricted number of V beta segments, including a subset of V beta families that are significantly more related to one another than to most other V beta families; (b) five of seven clones having a particular reaction pattern with HLA-B27 subtypes possess Alanine at the D-J junction; and (c) identical J beta segments are found associated in several instances with identical or highly homologous V beta gene segments. In addition, two new V beta 13 members are reported.
Tumor cells have an altered regulation of their cell cycle and commence to proliferate uncontrollably owing to mutations in their genetic material. Furthermore in addition to the altered proliferation there are genetic modifications which alter the expression of proteins in the malignant cell. This is manifested in the overexpression of some genes or activation which in normal tissue are not normally expressed. These genes encode for proteins which can be recognized as aberrant by the immune system thus generating an anti-tumor response. Recently in studies of animal models and in patients it has been shown that the principal anti-tumor effect is caused by the cellular immune response. In this situation it is the T lymphocyte which play a principal role, by way of their antigen receptors which have been processed and presented in association with the molecules of the major histocompatability complex (MHC). The majority of tumor associated antigens and recognized by CD8+ cytotoxic T lymphocytes are peptides derived from proteins expressed by the tumor cells and furthermore are encountered in the normal tissue of origin. For example in human melanoma exist antigens derived from proteins involved in the synthesis of melanin and expressed both in the tumor and normal melanocytes. There are other MHC restricted antigens which are common to various tumor types. These are embryologically derived proteins and normally are not expressed in the somatic tissues. The identification of various TAA has permitted the production of modern anti-tumor vaccines which are in the experimental stage. These vaccines based on descrete antigens which could be of peptide or DNA origen and would come to replace immunological therapies of less specificity, such as cytokines or adaptive therapy. Pre-clinical and clinical studies in the last two years indicate that the form of immunization essential in order to produce an effective immune response and avoid anergia or tolerance. Here play an important role the dendrite cells in their function as presenting cells of antigens and some preinflammatory citokines. The parody exists between the presence of anti-tumor cells in the patients with cancer and the systemic progression of the disease, this suggests the existence of mechanisms mediated by the tumor to evade the immune response. This strategies range from the secretion of factors immuno-inhibitory to mutations in the molecules related to the presenting antigen. An understanding of the immunological mechanisms involved in the anti-tumor response permits the development of immunotherapy as an alternative or complementary therapy to those already established in the fight against cancer
During the past years a wide array of immunologic tools has become available to study antigen-specific T cell immunity by flow cytometry. By means of these assays progress has been made in our understanding of virus-specific T cell immunity and the role of virus-specific CD4+ and CD8+ T-cell responses in protection against disease. In this review we describe advances made in understanding Epstein Barr-virus (EBV) and cytomegalovirus (CMV) specific T-cell immunity in healthy individuals and immunocompromised patients, such as HIV infected individuals and stem cell transplant (SCT) recipients, using flow cytometry based assays. EBV and CMV are both herpesviruses, which are characterised by a life-long persistence of the virus in the human body. It now becomes clear that the functional capacity, and more specifically poly-functionality, is the most important feature of virus-specific T-cells to protect against disease progression. However, responses directed to different proteins may have different effects, in relation to viral control and subsequent protection against disease. Improved knowledge on these factors may be translated to clinical applications.
Malignant lymphomas frequently develop in the pleural cavity of patients with long-standing pyothorax. Thus, the term pyothorax-associated lymphoma (PAL) has been proposed for this type of tumor. Most PAL are of the type diffuse lymphoma of B cell and contain Epstein-Barr virus (EBV) DNA. This article reviews the mechanism for the development of PAL. The possible contribution of EBV infection, inflammatory cytokines and other genetic lesions, including the p53 gene, towards the growth advantage of neoplastic cells is described. Although the presence of EBV is focused in PAL cells, the contribution of EBV-mediated growth promotion in PAL Is limited. Another important Characteristic of PAL is that the virus antigen-positive lymphoma develops in patients with pyothorax, in whom the systemic immunodeficiency is unlikely to be present. Therefore, in the course of the development of PAL, the mechanism for the evading host immune survelliance must be obvious. In this context, the production of an immunosuppressive cytokine from PAL cells, human histocompatibility leukocyte antigen class 1 alleles of patients with PAL, and the mutations of cytotoxic T lymphocyte epitopes in an EBV-latent antigen are described. These mechanisms could be involved in the development of PAL, an EBV-positive lymphoma developing in an immunocompetent host, and also shed light on the tumorigenesis of other EBV-positive neoplasms and on the lymphomagenesis of other inflammatory lesions.
EBNA2, EBNALP, EBNA3A, EBNA3B, and EBNA3C and to a lesser extentEBNA1 have evolved in the LCV lineage to mediate LCV effects on lymphocytegrowth and survival, probably at the earliest stage of primary infection. Thesegenes work coordinately to regulate c-myc, TCL1, and other cell genes, andLCVs have evolved similar regulatory mechanisms to enable these EBNAproteins to regulate viral promoters for EBNAs and LMPs, the other principalcontributors to cell outgrowth and survival. The EBNA proteins have alsoevolved to be readily recognized as foreign. Thereby, latency III EBV-infectedB-lymphocyte growth is sufficient for successful seeding into lymphoid compartments,but inadequate for lymphoma development in almost all humanswith normal immune function. © Springer Science-Business Media, LLC 2009. All rights reserved.
We have investigated the presentation and CTL recognition of an HLA A∗1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)3, derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A∗0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A∗6801 molecule as efficiently as to A∗1101. The A∗6801:AVF complex is recognized by some A∗1101-restricted AVF- specific CTL clones. However, A∗6801-positive (A∗6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A∗6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A∗6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A∗1101+ individuals recognizes the A∗6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A∗6801+ individuals.
Le virus Epstein-Barr (EBV) persiste chez les sujets qu'il infecte dans les lymphocytes B mémoires quiescents CD27+. Dans les ganglions, l'expression du programme de latence II de l'EBV permet la survie à long terme des lymphocytes B mémoires infectés par l'EBV. Il peut aussi s'y produire des proliférations cellulaires associées au programme de latence III couplé à un cycle lytique. Cet échappement est normalement contrôlé par le système immunitaire et ne se manifestera chez l'immunocompétent que par une excrétion virale asymptomatique. Le contrôle de l'infection EBV est assuré principalement par la réponse cellulaire spécifique. Les lymphocytes cytotoxiques T CD8 (CTL) ont été les premiers étudiés et leur importance clinique a été démontrée chez les patients transplantés en prévenant l'apparition de lymphomes associés à l'EBV ou en permettant leur résolution.
The pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients and more than 5000 controls using a PCR-based sequence-specific oligonucleotide probe hybridization approach. HLA-A68 and HLA-DR11 (5) were significantly increased in the cHL patient population compared with the controls. Three class II associations were observed in the EBV(-) cHL population with an increase of HLA-DR15 (2) and a decrease of HLA-DR4 and HLA-DR7. Allele frequencies of HLA-A1, HLA-B37, and HLA-DR10 were significantly increased in the EBV(+) cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in whites. The allele frequency of HLA-A2 was significantly decreased in the EBV(+) cHL population. Sequence-specific oligonucleotide probe analysis revealed significant differences between EBV(+) and EBV(-) cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV(+) cHL. Furthermore, several new protective and predisposing HLA class I and II associations for the EBV(+), the EBV(-), and the entire cHL population were identified.
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV-associated tumours and is therefore an interesting target for immunotherapy. However, evidence for the recognition and elimination of EBV-transformed and Burkitt's lymphoma (BL) cells by cytotoxic T lymphocytes (CTLs) specific for endogenously presented EBNA1-derived epitopes remains elusive. We confirm here that CTLs specific for the HLA-B35/B53-presented EBNA1-derived HPVGEADYFEY (HPV) epitope are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes, thereby demonstrating that the GAr domain does not fully inhibit the class I presentation of the HPV epitope. In contrast, BL cells are not recognized by HPV-specific CTLs, suggesting that other mechanisms contribute to providing a full protection from EBNA1-specific CTL-mediated lysis. One of the major differences between BL cells and lymphoplastoid cell lines (LCLs) is the proteasome; indeed, proteasomes from BL cells demonstrate far lower chymotryptic and tryptic-like activities compared with proteasomes from LCLs. Hence, inefficient proteasomal processing is likely to be the main reason for the poor presentation of this epitope in BL cells. Interestingly, we show that treatments with proteasome inhibitors partially restore the capacity of BL cells to present the HPV epitope. This indicates that proteasomes from BL cells, although less efficient in degrading reference substrates than proteasomes from LCLs, are able to destroy the HPV epitope, which can, however, be generated and presented after partial inhibition of the proteasome. These findings suggest the use of proteasome inhibitors, alone or in combination with other drugs, as a strategy for the treatment of EBNA1-carrying tumours.
Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for many hematological malignancies and genetic disorders. The majority of patients do not have a human leukocyte antigen (HLA) identical sibling donor, and alternative stem cell sources include HLA-matched or mismatched unrelated donors and haploidentical related donors. However, alternative donor HSCT are associated with three major complications i) graft rejection; ii) graft-versus-host disease (GvHD); and iii) delayed immune reconstitution leading to viral infections and relapse.
Graft rejection and the risk of GvHD can be significantly reduced by using intensive conditioning regimens, including in vivo T cell depletion as well as ex vivo T cell depletion of the graft. However, the benefits of removing alloreactive T cells from the graft are offset by the concomitant removal of T cells with anti-viral or anti-tumor activity as well as the profound delay in endogenous T cell recovery post-transplant. Thus, opportunistic infections, many of which are not amenable to conventional small-molecule therapeutics, are frequent in these patients and are associated with significant morbidity and high mortality rates. This review discusses current cell therapies to prevent or treat viral infections/reactivations post-transplant.
The reader will gain an understanding of the current state of cell therapy to prevent and treat viral infections post-HSCT, and will be introduced to preclinical studies designed to develop and validate new manufacturing procedures intended to improve therapeutic efficacy and reduce associated toxicities.
Reconstitution of HSCT recipients with antigen-specific T cells, produced either by allodepletion or in vitro reactivation, can offer an effective strategy to provide both immediate and long-term protection without harmful alloreactivity.
Nasopharyngeal carcinoma (NPC) is a virally associated cancer which is highly prevalent in Southeast Asia and North Africa. Several linkage analysis studies suggested the association of susceptibility HLA (Human Leukocyte Antigen) alleles and haplotypes with NPC development. The HLA system is very polymorphic and according to the ethnic group studied, it has been found to have the capacity to confer susceptibility or resistance to NPC. Our aim was to review the most important described genetic associations of HLA class I in NPC and to comment on the inconsistent associations found in the different NPC incidence areas. We believe that the mechanisms of these associations may involve HLA genes through the differential capacity of each allele to present antigens. However, because HLA genes contain various linked candidate genes, HLA-NPC associations should be carefully interpreted.
Epstein-Barr virus (EBV) positive Burkitt's lymphoma (BL) cells are markedly less sensitive to EBV-specific cytotoxic T cell (CTL) recognition than EBV-transformed lymphoblastoid cell lines of normal B cell origin. Three features of the BL cell phenotype might contribute to this reduced susceptibility: (i) low expression of cell adhesion molecules, (ii) low expression of HLA class I and selective down-regulation of particular alleles, and (iii) down-regulation of all transformation-associated EBV antigens except EBV-encoded nuclear antigen (EBNA)-1. This study assesses the individual importance of each of these features for immune escape. For this purpose the WW1-BL cell line was used which expresses all the known transformation-associated EBV antigens (EBNA-1 to -6 and latent membrane protein-1 and -2) but which is negative for HLA A11 and for the adhesion molecule leukocyte function associated antigen-3 (LFA-3). Using recombinant vectors, these deficiencies have been sequentially corrected and the cells have been tested for sensitivity to EBV (B95.8 strain)-induced CTL preparations recognizing epitope(s) of EBNA-4 in the context of HLA A11. Expression of HLA A11 alone or in combination with LFA-3 did not sensitize WW1-BL cells to these effectors. Lysis was only achieved when HLA A11 was co-expressed with the B95.8 virus-encoded EBNA-4 protein, and in these circumstances sensitization did not require LFA-3. These results indicate that reconstitution of the relevant HLA-EBV epitope target complex on the cell membrane is sufficient to render BL cells sensitive to virus-specific cytolysis. The requirement for EBNA-4 reconstitution to achieve lysis of the WW1-BL/A11 transfectant suggested that the resident WW1 virus-encoded EBNA-4 protein did not contain the relevant target epitope for HLA A11-restricted recognition. This was confirmed by transferring the WW1 virus isolate into another A11-positive B cell background.
An influence of cytotoxic T lymphocyte (CTL) response over Epstein-Barr virus (EBV) evolution was first suggested by the finding that virus isolates from highly HLA-A11-positive Oriental populations were specifically mutated in two immunodominant A11-restricted CTL epitopes. Here we turn to a second HLA allele, B35.01 and show that B35.01-restricted CTL responses in Caucasian donors reproducibly map to a single peptide epitope, YPLHEQHGM, representing residues 458-466 of the type 1 EBV nuclear antigen 3A protein (B95.8 strain). In this case, however, most EBV isolates from a highly B35.01-positive population (in The Gambia) either retained the CTL epitope sequence or carried a mutation (P-->S at position 2) which conserved antigenicity; changes leading to reduced antigenicity (Y-->N at position 1) were found in only a minority of cases. Furthermore, CTL recognizing the YPLHEQHGM epitope could be reactivated from the blood of some B35.01-positive Gambian donors by in vitro stimulation with the synthetic peptide, indicating that epitope-specific immunity does exist in this population. Possible differences between the A11-based and B35.01-based studies are discussed.
It has been known for many years that cytolytic T lymphocytes that specifically recognize the tumor cells of the same patient can be derived from the blood of melanoma patients. Several of the antigens recognized by these antitumor T lymphocytes have now been completely identified. Some of them are sufficiently tumor-specific to envision their use as antitumor vaccines in selected cancer patients.
Tumor heterogeneity is the presence of intercellular differences, either from clonal origin or present within subpopulations of tumor cells. Recent advances in immuno-histology, flow cytometric analysis, molecular biological techniques, and tissue culture methods makes it possible to investigate tumor heterogeneity in detail. In this review data are presented to document that this hallmark of neoplastic disease results from DNA-instability and the interactions of tumor cells with their environment. The present inability to treat most patients effectively with immunotherapy may partly be due to the occurrence of tumor heterogeneity. Therefore, the heterogeneity of the tumor phenotype is discussed in conjunction with the various immunotherapeutic treatment modalities. In addition to local cytokine production by immune cells and tumor cells, and limited access of either antibodies or immune cells into the tumor, tumor heterogeneity is an important factor that determines the progress of immunotherapy of cancer. Therefore, accurate quantitative methods using antibodies and molecular probes to identify HLA-associated target peptides, tumor-associated antigens and accessory molecules, to predict which patients will have a high probability of responding to treatment, are needed.
Epstein-Barr virus (EBV)-infected cells may sustain three distinct forms of virus latency. In lymphoblastoid cell lines, six EBV-encoded nuclear antigens (EBNA1, 2, 3A, 3B, 3C, -LP), three latent membrane proteins (LMP1, 2A, 2B), and two nuclear RNAs (EBERs) are expressed. This form of latency, termed latency III, is also encountered in some posttransplant lymphoproliferative disorders. In EBV-positive cases of Hodgkin's disease, the EBERs, EBNA1, and the LMPs are expressed (latency II), whereas in Burkitt's lymphoma (BL) only the EBERs and EBNA1 have been detected (latency I). We have studied the expression of EBV proteins in 17 cases of EBV-positive endemic BL by immunohistology. Expression of LMP1 was seen in variable proportions of tumor cells in two cases and EBNA2 was detected in some tumor cells in three other cases. Also, the BZLF1 trans-activator protein was expressed in a few tumor cells in 6 cases, indicating entry into the lytic cycle. A phenotypic drift from latency I to latency III has been observed previously in some BL cell lines. Our results suggest that a similar phenomenon may occur in BL in vivo and indicate that the operational definition of EBV latencies is not easily applied to human tumors.
Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's disease (HD). At present, however, it is not clear whether a defective immunity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in standard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the A11 and B44 HLA alleles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a Th0 phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.
Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-encoded latent membrane proteins LMP1 and LMP2 that could serve as rejection targets in Hodgkin's disease (HD). To examine whether EBV-triggered reactivities can be detected in the tumor, we have compared cytokine mRNA expression, cell phenotype, and cytotoxic activity in biopsies from 8 EBV-carrying and 6 EBV-HD patients. Neither the pattern of lymphokine production nor the cell phenotype of the in vivo-activated interleukin-2-responding populations provided a clear discrimination between EBV+ and EBV- cases. HLA class I-restricted EBV-specific cytotoxicity was shown in interleukin-2-dependent cultures from 3 of 3 EBV- tumors, whereas cultures from 6 of 6 EBV+ tumors were either noncytotoxic or exerted LAK-type cytotoxicity. EBV-specific cytotoxic T lymphocyte precursors were present in the blood of 1 patient carrying an EBV+ tumor. The results suggest that a tumor-associated suppression of EBV-specific T-cell responses may play an important role in the pathogenesis of EBV+ HD.
Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA A11 allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA A11-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14- or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA A11-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by A11-restricted CTL clones at concentrations as low as 5 x 10(-14) M.
Epstein-Barr virus (EBV)-specific CTL clones were isolated that recognized A-type EBV transformants but not B-type transformants. These A-type-specific CTL clones (HLA B8 restricted) were used to screen peptides derived from the EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 as potential CTL epitopes. Of the 76 peptides screened, one sequence from EBNA 3 (residues 329-353) was recognized by A-type-specific CTL clones after absorption onto target cells (either autologous B-type transformants or PHA blasts). This report is the first description of an EBV target epitope recognized by specific CTL clones.
In this report we describe a cytotoxic T-cell epitope in the Epstein-Barr virus nuclear antigen EBNA 6. This epitope is present on both A- and B-type transformants.
The potentially pathogenic effects of infection with Epstein-Barr virus (EBV), a B-lymphotropic agent with cell growth-transforming potential, are contained in healthy virus carriers by virus-specific cytotoxic T-lymphocyte (CTL) surveillance. The target antigens against which such CTL responses are directed are yet undefined, but the antigens probably derived from one or more of the EBV "latent" proteins constitutively expressed in virus-transformed B cells. We have analyzed target specificity of CTL responses from two EBV-immune donors that are preferentially reactive against autologous cells transformed with type A but not with type B virus isolates. Coding sequences for four EBV latent proteins with allelic polymorphism between A and B virus types--namely, the EBV nuclear antigens (EBNAs) EBNA 2, EBNA 3a, EBNA 3c, and EBNA leader protein--have been introduced into vaccinia virus vectors under control of vaccinia promoter P7.5 and used to express relevant EBNA proteins in appropriate target cells. Thus the CTL response from one donor has been mapped to type A EBNA 2 protein and from a second donor to type A EBNA 3a protein. Thereafter, a series of recombinant vaccinia viruses were constructed that carried specific internal deletions within the EBNA 2 type A coding sequence; by using these vectors, the above EBNA 2 type A-specific CTL response was shown to be directed against an epitope within a 100-amino acid fragment near the N terminus of the protein. This work clearly shows human CTL recognition of virus-coded nuclear antigens in the EBV system; moreover, it establishes an experimental approach that can be extended to all EBV latent proteins and to the more common CTL responses that cross-react against type A and type B virus isolates.
Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.
The Epstein-Barr virus nuclear antigen 5 (EBNA-5) is encoded by highly spliced mRNA from the major IR1 (BamHI-W) repeat region of the virus genome. A mouse monoclonal antibody, JF186, has been raised against a synthetic 18-amino-acid peptide deduced from the EBNA-5 message of B95-8 and Raji cells. The antibody showed characteristic coarse nuclear granules by indirect immunofluorescence and revealed multiple EBNA-5 species by immunoblotting and immunoprecipitation. The B95-8 line itself and all B95-8 virus-carrying cells, whether lymphoblastoid cell lines or in vitro-converted sublines of Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) lines, were EBNA-5 positive. Among 36 cell lines carrying different EBV strains, only 10 expressed the B95-8-Raji-prototype EBNA-5 recognized by JF186; this was probably due to genetic variation in the epitope recognized by JF186, as shown for P3HR-1. Human antibodies, affinity purified against EBNA-5-JF186 immunoprecipitates, detected EBNA-5 in the majority of EBV-positive BL lines and in all lymphoblastoid cell lines containing the BL-derived viruses. Thus, EBNA-5 can be expressed by all virus isolates examined, but is down-regulated, together with other latent gene products, in a minority of BL lines which have a particular cellular phenotype. EBNA-5 was detected as a ladder of protein species of 20 to 130 kilodaltons (kDa), with a regular spacing of 6 to 8 kDa, consistent with the coding capacity of the combined BamHI-W 66- and 132-base-pair exons, together with shifts of 2 to 4 kDa, consistent with the size of the separate 66- and 132-base-pair exons. Multiple EBNA-5 proteins can be expressed by the single cell as shown by cloning of newly infected cells.
Some EBV+ BL cell lines continue to grow as single cells on in vitro passage, show an unusually restricted expression of EBV-latent genes and retain a BL biopsy-like cell surface phenotype (group I/II lines); others change to growth in aggregates, show a broader pattern of virus latent gene expression, and develop a cell surface phenotype more characteristic of EBV-transformed LCL (group III lines). Here we show that the cell surface adhesion molecules LFA-1, ICAM-1, and LFA-3 are expressed at very low levels, if at all, on group I/II lines and are coordinately upregulated as BL lines move towards group III. The change to growth in aggregates reflects the increasing availability of LFA-1 and ICAM-1, the two ligands whose mutual interaction underlies homotypic BL cell adhesion in vitro. The low levels of ICAM-1 and LFA-3 on group I/II BL cell lines are also associated with an impaired ability to interact with EBV-specific CTL in the antigen-independent phase of effector/target conjugation. mAb blocking studies show that the small number of conjugates that are formed with group I/II BL targets involve the LFA-1/ICAM-1 adhesion pathway but not the LFA-3 pathway; in contrast, both pathways contribute to the efficient conjugate formation shown by group III BL or LCL targets. Earlier work identified one group III line, WW1 BL, as unusual since is expressed the full spectrum of EBV-latent proteins yet remained insensitive to lysis by EBV-specific CTL. Here we show that this line has an anomalous pattern of adhesion molecule expression with high levels of LFA-1 and ICAM-1 in the absence of detectable LFA-3. The WW1 BL cells form conjugates with EBV-specific CTL through the LFA-1/ICAM-1 pathway, but in the absence of a target LFA-3/effector CD2 interaction these conjugates do not achieve target cell lysis. This may reflect an important role for target LFA-3 molecules in activating EBV-specific CTL function. From these in vitro studies, we postulate that downregulation of the adhesion molecules LFA-3 and ICAM-1 on EBV+ BL underlies the ability of the malignant clone to evade EBV-specific T cell surveillance in vivo.
We have developed monoclonal antibodies to a 63,000-molecular-weight protein (p63) which is the product of the most abundant messenger RNA in Epstein-Barr virus-transformed cells and shown that the protein is associated specifically with plasma membranes. It was also found to be associated with the other membrane fractions and was found in all Epstein-Barr virus-transformed cells tested. In addition, p63 was present in virions, resulting in transient, early appearance in newly infected cells. Newly synthesized p63 was detected at the time cells underwent blast transformation (48 to 72 h postinfection). The possible role of this protein in transformation and as a target for cell-mediated cytotoxicity is discussed.
Sets of Burkitt lymphoma lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) derived from the same individuals were compared for sensitivity to cytotoxic T-lymphocyte (CTL) clones. Major histocompatibility complex class I antigen-restricted CTL clones were generated by stimulating the lymphocytes of an EBV-seropositive individual with the autologous LCL. One clone (BK-20) lysed the autologous and allogeneic HLA-A11-expressing LCLs but not mitogen-induced B lymphoblasts. Thus the clone was selectively cytotoxic for LCLs. Allospecific CTL clones directed against the HLA-A11 antigen were generated from an EBV-seronegative individual. One clone (WP-36) was selectively cytotoxic for the appropriate allospecific LCL, whereas another clone (WP-21) lysed also T and B lymphoblasts. None of the four Burkitt lymphoma lines established in parallel with the CTL-sensitive LCLs were lysed. Two of the Burkitt lymphoma lines were EBV-negative, and EBV-positive sublines were derived from these by in vitro infection. One but not the other of the two convertants became sensitive to all three types of CTL clones. The CTL-sensitive converted line had also acquired some LCL characteristics: increased cell size, aggregation, and a shift in several of the B-cell-specific surface markers. The CTL-resistant convertant expressed EBV antigens but showed no phenotypic change. These findings suggest that the cellular phenotype plays a decisive role in the sensitivity of B-cell-derived lines to the lytic effect of LCL-selective autologous and allogeneic CTLs.
Regulation of the expression of influenza A/PR/8/34 hemagglutinin (HA) by the vaccinia virus promoters PF (early), P7.5 (early and late) and PL11 (late) has been demonstrated using HA-vaccinia recombinant viruses VV-PR8-HA3, VV-PR8-HA6 and VV-PR8-HA, respectively. Levels of HA on the surface of VV-PR8-HA3 (PF)-infected cells were lower than with either VV-PR8-HA6 (P7.5) or VV-PR8-HA8 (PL11). Expression of HA under the control of the late promoter PL11 was inhibited in the absence of DNA replication. All three recombinant viruses stimulated a specific antibody response in mice which was dependent on the presence of infectious virus. Recognition of HA by cytotoxic T lymphocytes (CTL) was assessed by the ability of the viruses to stimulate naive precursors in vivo, to restimulate primed CTL in vitro and by target cell recognition. HA expressed under the control of either of the promoters with early function (PF or P7.5) was recognized by CTL when VV-PR8-HA3 or VV-PR8-HA6 were used to prime or restimulate splenocytes or to infect target cells. On the other hand, HA expressed by VV-PR8-HA8 (PL11) failed to prime for a CTL response in naive CBA/H mice, was ineffective at restimulation of primed splenocytes and failed to produce target cells for recognition by specific CTL. However, in BALB/c mice VV-PR8-HA8 did prime for a specific CTL response. These studies show that HA synthesized early in infection was recognized by both B and T cells while HA expressed after DNA replication was not generally recognized by T cells. The implications of the observations with the late promoter with respect to the use of late promoters in potential vaccinia virus-based vaccines are considered.
The EBV-encoded membrane protein (LMP) and the nuclear antigen EBNA-1 were compared for their capacity to induce rejection of transfected murine mammary carcinoma cells in syngeneic hosts. The tumorigenic potential of stable LMP and EBNA-1 expressing sublines of the ACA (H-2f)-derived mammary carcinoma line S6C was tested in pre-immunized syngeneic and semi-syngeneic animals. LMP expressing S6C cells elicited a strong rejection response as demonstrated by the lower tumor take and slower growth in immunized vs. control mice. In contrast, EBNA-1-expressing cells were non-immunogenic in syngeneic hosts and in one semi-syngeneic F1-hybrid. Rejection in 2 additional F1-hybrids did not appear to be due to EBNA-1-specific immune responses. Our findings support the hypothesis that the escape of EBV carrying tumor cells from EBV-specific immune surveillance may be facilitated by the fact that viral gene expression is limited to EBNA-1.
Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6-specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia.
The expression of Epstein-Barr virus (EBV)-encoded, growth-transformation-associated proteins was studied in lymphoproliferations of 9 allogeneic bone-marrow transplant (BMT) recipients. Immunoblots of cell lysates were probed with polyspecific and monospecific antisera directed against EBNA 1, 2, 3 and 6, and the membrane protein LMP. All tumors expressed EBNA 1 and LMP. EBNA 2 was detected in the tumors of 8 patients, and EBNA 3 and 6 in the tumors of 5 patients. The LMP regulatory sequences, 5' of the LMP gene, were totally unmethylated in all 7 cases, while the coding sequences of LMP and EBNA 2 were more methylated in CpG dinucleotides. EBV-transformed lymphoblastoid cell lines (LCL) express EBNA 1 to 6 and LMP; in contrast, Burkitt lymphomas express only EBNA 1. In vitro experiments have shown that EBNA 2, 3 and LMP can generate targets for cytotoxic T cells (CTL). These combined observations are consistent with the hypothesis that the EBV-associated lymphoproliferative disease of the BMT recipients escapes CTL-mediated rejection due to the failure of host immunosurveillance rather than to the down-regulation of immunogenic EBV-encoded antigens.
HLA class-I expression has been investigated by biochemical methods in 14 Burkitt lymphoma (BL) cell lines and the corresponding Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the same individuals. Selective down-regulation of one or more HLA class-I specificities was demonstrated in 9 out of 14 BL lines. The defect was restricted to a single HLA-A allele in 3 of the lines (BL29, BL72, WW-I-BL). Four lines (BL28, BL37, BL41 and Jijoye M13) showed down-regulation of both HLA-A and -C alleles, and one (BL36) failed to express one HLA-C allele. Only one BL line (WW-2-BL) had lost one HLA-A and one HLA-B allele. The allele-specific defects were mainly detected in cell lines that had maintained the phenotypic characteristics of the original tumor. Expression of B-cell activation markers and the EBV-encoded nuclear antigen (EBNA)-2 correlated with up-regulation of the Cw4 allele in the P79 subline of the BL line Jijoye. Treatment with gamma-interferon (IFN) resulted in full or partial reversion of the HLA class-I defects in some of the cases but had no significant effect in others. This was not due to a cell-line-related unresponsiveness to IFN, nor did it reflect an allele-specific mode of regulation because the same allele could respond differently in different cell lines. The data suggest that defective expression of HLA class-I antigens, which appears to be more prevalent for alleles within the HLA-A and -C loci, is a common feature of BL cell lines. Different regulatory mechanisms appear to be involved.
A pool of endogenous peptides bound to the human class I MHC molecule, HLA-B27, has been isolated. Microsequence analysis of the pool and of 11 HPLC-purified peptides provides information on the binding specificity of the HLA-B27 molecule. The peptides all seem to be nonamers, seven of which match to protein sequences in a database search. These self peptides derive from abundant cytosolic or nuclear proteins, such as histone, ribosomal proteins, and members of the 90K heat-shock protein family.
MHC class I antigens are highly polymorphic trans-membrane glycoproteins that play a key role in the regulation of immune responses. Lysis of virus infected or neoplastic cells by cytotoxic T-lymphocytes (CTL) depends on the cognate recognition of processed antigens and MHC class I molecules (Zinkernagel and Doherty 1979). Cell lines derived from Epstein-Barr virus (EBV) carrying Burkitt’s lymphoma (BL) are often resistant to lysis by HLA class I antigen restricted EBV-specific CTLs generated by stimulation of lymphocytes from EBV seropositive individuals with autologous virus infected cells (Rooney et al. 1985b, Tosteindottir et al. 1986). Since lymphocytes from BL patients are able to control the ^ proliferation of autologous EBV infected cells in in vitro regression assays (Rooney et al. 1985a), the CTL resistance of the tumor cells was interpreted to result from in vivo immunoselection. The finding that six of the seven EBV encoded antigens that may provide the target for the immune rejection of EBV transformed normal cells are not expressed in the tumors and are up-regulated after in vitro propagation (Rowe DT et al. 1986, Rowe M et al. 1987) was taken as a further evidence for the presence of immuno-logical control mechanisms.
Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) biopsy cells and early passage BL cell lines have been reported as showing an unusual type of virus-cell interaction; at least two EBV latent proteins appear not to be expressed. Serial passage of such lines is often accompanied by a broadening of virus latent gene expression and a corresponding change in the cell surface/growth phenotype towards that shown by in vitro transformed lymphoblastoid cell lines (LCLs). The sequence of events, both viral and cellular, involved in this transition needs to be defined properly. In the present work, phenotypically distinct cell clones have been derived from early passage cultures of a BL cell line in phenotypic transition, thereby giving access to relatively stable cell populations through which the different EBV-B cell interactions within the parental line can be studied. Clones retaining the original BL biopsy cell phenotype (CD10/CD77-positive, activation antigen/adhesion molecule-negative) expressed the virus-encoded nuclear antigen EBNA 1 but not any of the other known latent proteins, EBNAs 2, 3a, 3b, 3c, -LP and latent membrane protein (LMP). Other clones which had developed an LCL-like phenotype (CD10/CD77-negative, activation antigen/adhesion molecule-positive) now expressed all the above latent proteins and also contained significant numbers of cells in lytic cycle. Phenotypic change occurring within the parental BL cell line itself was initiated in a small subpopulation of cells in which the virus-encoded proteins EBNA 2 and LMP were transiently induced to an unusually high level of expression; this was accompanied by the first detectable changes in cell surface phenotype, namely the increase of cellular adhesion molecules. Some control over EBNA 2/LMP expression then appeared to be re-imposed since the presumed clonal descendents of these cells stably expressed EBNA 2 and LMP at much reduced levels typical of those seen in conventional LCLs.
Epstein-Barr virus (EBV) has been associated with serious or fatal lymphoproliferative disease in immunocompromised patients. EBV nuclear protein 2 and latent membrane protein are characteristically expressed in B lymphocytes proliferating in vitro in response to growth transformation by EBV. These two proteins are thought to be effectors of lymphocyte growth since they increase the expression of B-lymphocyte activation (CD23) and cell-adhesion (LFA 3 and ICAM 1) molecules in vitro. Using monoclonal antibody-immune microscopy, we have demonstrated that these two EBV proteins and their associated B-lymphocyte activation or adhesion molecules are expressed in the infiltrating B lymphocytes in immunocompromised patients with EBV lymphoproliferative disease. These monoclonal antibodies should be useful in the early diagnosis of EBV lymphoproliferative disease and in distinguishing it from other B-lymphocyte cancers associated with EBV, such as Burkitt's lymphoma. The finding of EBV nuclear protein 2 and latent membrane protein and their associated activation or adhesion molecules provides a further pathophysiologic link between EBV and the proliferation of B lymphocytes in immunocompromised patients.
Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis and is associated with Burkitt's lymphoma and nasopharyngeal carcinoma. The virus is harboured for life in all previously infected individuals and is apparently controlled by a population of EBV-specific memory T lymphocytes, specifically activated to recognize the functionally defined lymphocyte-detected membrane antigen. Two types (A and B) of EBV have been identified that show DNA sequence divergence within the BamH1 WYH region of the genome encoding the transformation-associated antigen, Epstein-Barr nuclear antigen 2 (EBNA 2) (ref. 4). To define the function of EBNA 2 in T-cell recognition, we have compared the ability of EBV-specific cytotoxic T-cell clones to distinguish between autologous B lymphocytes transformed by A- or B-type virus. We have now isolated both CD4 and CD8 cytotoxic T-cell clones that recognize autologous A-type but not B-type transformed lymphoblastoid cell lines, thus providing the first evidence that EBV-specific T-cell recognition can be mediated by EBNA 2. As this antigen is not expressed in Burkitt's lymphoma, this finding explains the failure of EBV-specific T-cell surveillance to eliminate the tumour.
Paired Epstein-Barr virus (EBV)-carrying cell lines have been established from Burkitt's lymphoma (BL) patients, one of each pair being the BL cell line derived from the malignant cells of the tumour, the other, the lymphoblastoid cell line (LCL) derived from the patient's normal B cells by experimental infection with the virus. Comparative studies have shown the following: (1) All the lines were to some extent sensitive to in-vitro activated natural-killer cells, individual pairs differing as to whether BL or LCL cells were more susceptible. (2) For six of the seven pairs tested, the BL cell line was clearly sensitive to allo-specific (anti-class 1 HLA) effector T cells, although levels of lysis were slightly below those observed for the corresponding LCL; only one BL cell line showed evidence of a dramatic reduction of HLA antigen expression, and this line was insensitive to allo-specific cytolysis. (3) For two of the three pairs tested to date, EBV-specific cytotoxic T-cell preparations from HLA antigen-matched donors lysed the LCL but not the BL cell line, despite the latter's apparent expression of the relevant restricting antigens. In both of these cases, it was known that the tumour arose in vivo in the face of prevailing EBV-specific T-cell surveillance. An escape of the malignant cells from such surveillance may therefore be important in the overall pathogenesis of EBV genome-positive BL.
Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.
A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant
vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant
vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression
of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis
began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication
was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system,
Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins
were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant
vaccinia virus expression system.
Post-transplant lymphomas or other lymphoproliferative lesions, which were usually associated with Epstein-Barr virus infections, developed in 8, 4, 3, and 2 recipients, respectively, of cadaveric kidney, liver, heart, and heart-lung homografts. Reduction or discontinuance of immunosuppression caused regression of the lesions, often without subsequent rejection of the grafts. Chemotherapy and irradiation were not valuable. The findings may influence policies about treating other kinds of post-transplantation neoplasms.
Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co-cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB-virus-transformed B-cell line. The fresh cells present in co-cultures from EB virus antibody-negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co-cultures from seropositive donors. Larger-scale co-cultures, set up at a ratio of 80-100 fresh UM cells to one autologous virus-transformed B cell, were harvested after 8 to 12 days and the T-cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co-cultures and were strongly active against the autologous virus-transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome-negative target cell lines chosen as sensitive indicators of non-specific cytotoxicity. Recognition of allogeneic EB-virus-transformed cells was restricted to those whose HLA-A and/or B and/or B and/or C antigen expression matched that of the effector cells themselves;; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB-virus-specific HLA-restricted memory T cells, present in the blood of previously-infected individuals, can be reactivated in vitro using the established autologous virus-transformed cell line as a stimulus. THe reactivated cytotoxic cells appear to recognize a virus-induced lymphocyte-detected membrane antigen, LYD-MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious monoucleosis.
Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original definition of the EBV-determined nuclear antigen (EBNA) [Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499-520]. Eight EBNA and EBV DNA-carrying lines showed nuclear staining with the antipeptide antibody, whereas five EBV DNA negative lines failed to stain. The staining pattern was more discretely punctate than the finely dispersed diffuse EBNA staining obtained with human antisera. Human EBV antibody-positive but not EBV-negative sera reacted with the synthetic peptide in an ELISA test. The peptide-specific antibodies were purified from the sera of healthy EBV-seropositive persons by affinity chromatography with the peptide. They gave an EBV-specific, brilliant punctate nuclear ACIF staining similar to that of the rabbit antipeptide antibodies. It was concluded that the glycine-alanine structure encoded by the IR3 region contains a native determinant of EBNA, detected by the ACIF test. Immunoblotting with the rabbit and human peptide-specific antibodies identified poly-peptides that varied between 70 and 92 kilodaltons in size in different EBV-positive cell lines, corresponding closely to a previously identified variation pattern in the size of EBNA. In addition, rabbit antipeptide antibodies identified two cellular polypeptides, 44 and 49 kilodaltons in size.
EBV-specific cytotoxic T cells can be generated in vitro in a secondary response. Several previous studies with bulk cultures provided evidence that cytotoxicity was restricted by HLA-A,B-related antigens. In the present family study, the EBV-specific cytotoxic T-cell response of a normal EBV-seropositive donor was analysed in detail by T-cell colony formation. Peripheral blood mononuclear cells were stimulated by the gamma-irradiated autologous lymphoblastoid cell line (LCL) and 3 days later seeded into agarose. Colonies were harvested, amplified by addition of interleukin-2 (IL-2), and analysed for T-cell markers and specificity in 51Cr-release assays. Twenty-two colonies were studied: all colonies were OKT3+, five were predominantly OKT4+, 9 were OKT8+ and 8 were mixtures. As expected from previous work, the OKT8+ colonies were cytotoxic for the autologous LCL target and cytotoxicity was blocked by monoclonal antibody (W6/32) to the nonpolymorphic determinants of HLA-A,B,C antigens. Significantly, the OKT4+ colonies tested also showed specific cytotoxicity, but lysis of the autologous LCL was blocked by the monoclonal antibody (OKlal) to the non-polymorphic determinants of HLA-DR antigens. Two interesting patterns of specificity were seen in cytotoxicity tests on sibling LCL targets. In one pattern, targets bearing the A11, B5, DR7 haplotype were lysed, while those bearing the A1, B8, DR3 were not, indicating haplotype preference. In the other pattern, there was lysis of the autologous cell line but not of the sibling targets. These results including HLA-DR-associated restriction, haplotype preference and strict self-preference, further illustrate the complexity of the EBV-cytotoxic T-cell response.
The crystal structures of major histocompatibility complex (MHC) molecules contain a groove occupied by heterogeneous material thought to represent peptides central to immune recognition, although until now relatively little characterization of the peptides has been possible. Exact information about the contents of MHC grooves is now provided. Moreover, each MHC class I allele has its individual rules to which peptides presented in the groove adhere.
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