EBV-specific T-cell Research Articles

Semiquantitative Epstein-Barr Virus (EBV) Polymerase Chain Reaction for the Determination of Patients at Risk for EBV-Induced Lymphoproliferative Disease After Stem Cell Transplantation

AbstractEpstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Semiquantitative Epstein-Barr Virus (EBV) Polymerase Chain Reaction for the Determination of Patients at Risk for EBV-Induced Lymphoproliferative Disease After Stem Cell Transplantation

Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Analysis of the T-cell micro-environment in Epstein-Barr virus-related post-transplantation B lymphoproliferative disease.

Epstein-Barr virus (EBV) post-transplantation B lymphoproliferative disease (BLPD) may undergo regression after immunosuppression withdrawal and restoration of EBV-specific cytotoxic T-cell (CTL) activity in the immunocompromised allografted host. The presence of morphologically normal T cells in the BLPD micro-environment may influence tumour behaviour in vivo. In this immunopathological study, the phenotype and the number of T cells and other immunoregulatory cells have been investigated in seven primary and four recurrent BLPD biopsies from nine solid organ transplant recipients. BLPD with either viral lymphadenopathic or polymorphic lymphoma appearances was found to contain sizeable T-cell populations, mainly of memory/helper (TCR alpha/beta +, CD3+, CD4+, CD45RO+) type. Cytotoxic (TCR alpha/beta +, CD3, CD8+, Tia-1+) T cells were strikingly low in all samples. Low CD28 and CD25 expression suggested that secondary signals for functional and sustained T-cell activation may be deficient in these tumours. No close correlation was found between the degree of T-cell infiltration and clinical outcome, although appreciably higher number of CD8+ T cells were detected in three BLPD tumours showing prolonged clinical remission after treatment. While some level of EBV-specific T-cell function may be present in untreated BLPD, the overall findings of this study suggest that the nature of T-cell infiltrates may reflect a response to immunosuppressive therapy rather than to EBV infection per se. The possibility that a local EBV-specific T-cell response is generated in BLPD undergoing regression after treatment needs to be investigated.

Biology and adoptive cell therapy of Epstein-Barr virus-associated lymphoproliferative disorders in recipients of marrow allografts.

Epstein-Barr virus (EBV) is an ubiquitous herpesvirus which is carried as a latent infection of B lymphocytes and salivary gland epithelial cells in over 90% of normal adults. Latently infected EBV-transformed B cells circulate at low frequency in the blood for the life of the host. These transformed B cells stimulate a heterogeneous and complex host cell response, ultimately leading to the development and maintenance of high frequencies of HLA-restricted T cells specific for the EBV-encoded nuclear antigens EBNA2-EBNA6 and the latency membrane proteins LMP-1 and LMP-2. Responses to latent EBV-encoded proteins are hierarchical with responses to certain epitopes predominating, dependent upon the HLA genotype of the host. Profound suppression of T-cell immunity may permit the emergence of polyclonal, oligoclonal or monoclonal EBV antigen-expressing lymphoproliferative disorders or malignant B-cell lymphomas expressing these latent EBV antigens. Adoptive transfer of small numbers of peripheral blood mononuclear cells or HLA-partially matched T cells from in vitro expanded EBV-specific T-cell lines derived from a seropositive marrow donor has induced durable regressions of bulky, widely metastatic monoclonal EBV lymphomas in a high proportion of cases. This review describes the current state of knowledge and hypothesis regarding the biology and immunology of EBV infection in the normal host, the features of donor, host and virus which contribute to the development of EBV-associated lymphoproliferative diseases and the mechanisms whereby they are controlled by adoptive transfer of immune T cells.

EBV-specific cytotoxic T-cells (CTL) recognize the immediate early transactivators Zta (BZLF1) and Rta (BRLF1) of EBV

EBV-specific cytotoxic T-cells (CTL) recognize the immediate early transactivators Zta (BZLF1) and Rta (BRLF1) of EBV

EBV-specific cytotoxic T-cells (CTL) recognize the immediate early transactivators Zta (BZLF1) and Rta (BRLF1) of EBV

EBV-specific cytotoxic T-cells (CTL) recognize the immediate early transactivators Zta (BZLF1) and Rta (BRLF1) of EBV

Frequency of multiple Epstein-Barr virus infections in T-cell-immunocompromised individuals.

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.

The Development of Cellular Immunity to Epstein-Barr Virus After Allogeneic Bone Marrow Transplantation

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a potentially lethal complication during the first 6 months after allogeneic bone marrow transplantation (BMT). To determine whether deficiencies of EBV-specific cellular immunity contribute to EBV-LPD susceptibility and distinguish patients at risk, we performed limiting dilution analysis to quantify anti-EBV cytotoxic T-lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or T-cell-depleted (TCD) grafts from EBV-seropositive donors. At 3 months post-BMT (n = 26), only five patients had EBV CTLp frequencies in the range of seropositive normal controls, irrespective of the type of transplant administered. By 6 months post-BMT, 9 of 13 patients tested had EBV CTLp frequencies within the normal range. The time period in which these patients had deficient cellular immunity to EBV corresponds to the period in which we have observed EBV-LPD in most prior patients. One patient with a low EBV CTLp frequency at 4 months post-BMT developed an EBV-LPD. Within 2 weeks of receiving an infusion of donor peripheral blood mononuclear cells (PBMC) providing less than 1,200 EBV-specific cytotoxic T-cell precursors, populations of EBV-specific CTL in the circulation were restored to levels detected in normal seropositive adults. Concurrently, the patient achieved a regression of the EBV-LPD, which has been sustained without further therapy. These studies indicate that recipients of both unmodified and TCD marrow grafts have profound deficiencies of EBV-specific T cell-mediated immunity early posttransplant, and that the period of risk for EBV-LPD closely corresponds to this interval of severe deficiency. Treatment of one patient with EBV-LPD with marrow donor-derived PBMC induced a rapid expansion of EBV-specific cytotoxic T-cell populations that occurred contemporaneously with the clinical regression of disease.

Local Suppression of Epstein-Barr Virus (EBV)-Specific Cytotoxicity in Biopsies of EBV-Positive Hodgkin's Disease

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.

Transfer of the HSV-tk Gene into Donor Peripheral Blood Lymphocytes for In Vivo Modulation of Donor Anti-Tumor Immunity after Allogeneic Bone Marrow Transplantation. The San Raffaele Hospital, Milan, Italy

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation

Summary Reactivation of Epstein-Barr virus (EBV) after bone-marrow transplantation leads in many cases to lymphoproliferative disease that responds poorly to standard therapy and is usually fatal. To prevent or control this complication, we prepared EBV-specific cytotoxic T-lymphocyte (CTL) lines from donor leucocytes and infused them into ten allograft recipients. Three of the patients had shown signs of EBV reactivation, with or without overt lymphoproliferation, and the others received CTL infusions as prophylaxis. No patient developed any complication that could be attributed to the CTL infusions. In the three patients with EBV reactivation, EBV DNA concentrations (measured by semiquantitative polymerase chain reaction [PCR]), which had increased 1000-fold or more, returned to the control range within 3-4 weeks of immunotherapy. The most striking consequence was the resolution of immunoblastic lymphoma in a 17-year-old patient who received four CTL infusions (two 1×10 7/m 2 and two 5×10 7/m 2). Because the CTL had been genetically marked before infusion, we were able to show by PCR analysis that they persisted for 10 weeks after administration. EBV-specific donor-type T-cell lines seem to offer safe and effective therapy for control of EBV-associated lymphoproliferation.

T-Cell Receptor Heterogeneity among Epstein-Barr Virus-Stimulated T-Cell Populations

The mechanism by which Epstein-Barr Virus (EBV) escapes T-cell activity during latency in immunocompetent individuals has long been debated. In order to identify potential weaknesses in the EBV-specific immune response, a study of T-cell receptors (TCR) within virus stimulated T-cell populations was performed. Membrane staining techniques and the polymerase chain reaction were used to address two questions: (1) Does EBV behave as a superantigen, thus stimulating, and possibly eliminating, T-cell subsets based on TCR Vβ expression?, and (2) Are T-cells dependent on a predominant TCR Vβ segment for effective cytotoxic function towards EBV? In search for superantigen effects, Vβ repertoires among PBL from seropositive and seronegative individuals were compared both before and after short term in vitro exposure to EBV. In order to characterize functional TCR, the Vβ usage among CD8 + EBV-specific cytotoxic T-cell clones was determined. Taken together, results illustrated the strengths rather than weaknesses of the EBV-specific T-cell immune response. T-cells did not respond to EBV in a manner typifying potent superantigen activity, nor did T-cells rely on the expression of a single Vβ gene segment for efficient, EBV-specific cytotoxic function.

Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders

Epstein-Barr virus (EBV) is associated with the development of two human B-cell malignancies, Burkitt's lymphoma and lymphomas that occur in the immunosuppressed host. The latter category of disease has become important recently as it is seen primarily in organ transplant recipients and individuals with acquired immunodeficiency syndrome. One possible mechanism for lymphoma development involves a reduction in or lack of EBV-specific cytotoxic T-cell recognition. In support of this model are previous observations that the expression of EBV nuclear antigen 2 (EBNA2) and latent membrane protein, two viral antigens associated with major histocompatibility complex class I-restricted T- cell killing, are downregulated in Burkitt's lymphoma and in early passage lymphoblastoid cell lines (LCL) derived from the malignant lesions. To determine whether a similar mechanism could occur in the development of posttransplant lymphoproliferative disorders (PTLD), we compared EBV gene expression among 23 PTLD tumor lesions obtained from 11 solid organ transplant recipients and among LCL derived from 3 of these lesions. In this report, we demonstrate, by Southern blot, Western blot, and immunofluorescence analysis, that (1) the tumor lesions exhibit varying patterns of restricted viral gene expression; (2) LCL derived from these lesions may represent the in vitro selection of cell subpopulations; and (3) immunosuppressed individuals have a markedly reduced antibody response to the latent cycle antigens, EBNA1, EBNA2, and EBNA-LP, but not to the lytic cycle viral capsid antigen when compared with normal immunocompetent controls.

Evidence for Restricted Epstein-Barr Virus Latent Gene Expression and Anti-EBNA Antibody Response in Solid Organ Transplant Recipients With Posttransplant Lymphoproliferative Disorders

Epstein-Barr virus (EBV) is associated with the development of two human B-cell malignancies, Burkitt's lymphoma and lymphomas that occur in the immunosuppressed host. The latter category of disease has become important recently as it is seen primarily in organ transplant recipients and individuals with acquired immunodeficiency syndrome. One possible mechanism for lymphoma development involves a reduction in or lack of EBV-specific cytotoxic T-cell recognition. In support of this model are previous observations that the expression of EBV nuclear antigen 2 (EBNA2) and latent membrane protein, two viral antigens associated with major histocompatibility complex class I-restricted T-cell killing, are downregulated in Burkitt's lymphoma and in early passage lymphoblastoid cell lines (LCL) derived from the malignant lesions. To determine whether a similar mechanism could occur in the development of posttransplant lymphoproliferative disorders (PTLD), we compared EBV gene expression among 23 PTLD tumor lesions obtained from 11 solid organ transplant recipients and among LCL derived from 3 of these lesions. In this report, we demonstrate, by Southern blot, Western blot, and immunofluorescence analysis, that (1) the tumor lesions exhibit varying patterns of restricted viral gene expression; (2) LCL derived from these lesions may represent the in vitro selection of cell subpopulations; and (3) immunosuppressed individuals have a markedly reduced antibody response to the latent cycle antigens, EBNA1, EBNA2, and EBNA-LP, but not to the lytic cycle viral capsid antigen when compared with normal immunocompetent controls.

Epstein‐barr virus‐infected b cells persist in the circulation of acyclovir‐treated virus carriers

In this study, infectious Epstein-Barr virus (EBV) shedding in the oropharynx and numbers of virus-infected B cells in the blood have been monitored in long-term virus carriers receiving acyclovir (ACV) therapy for herpes zoster. Eleven patients on oral ACV were followed prospectively before, during and for 2 weeks after treatment. As expected, the low levels of EBV shedding in these virus carriers (measured as cord-blood lymphocyte transforming activity in throat washings) were eliminated during the period of ACV treatment and returned at later times. Over the same period, however, the frequency of virus-infected B cells in the blood (measured by spontaneous transformation in limiting dilution assay) remained completely unchanged. Regression assays showed that these same patients had normal levels of EBV-specific cytotoxic T-cell immunity, so that the in vivo persistence of virus-infected B cells could not be ascribed to a defect in T-cell surveillance. We infer that the in vivo half-life of the virus-infected B-cell pool in long-term virus carriers is measured in months rather than days. We further suggest that such persistence requires a novel form of virus:B-cell interaction distinct from the type of "latent" infection displayed by in vitro-transformed cells.

Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP

Cytotoxic T cells from Epstein-Barr virus (EBV)-immune individuals specifically kill EBV-transformed B cells from HLA class I antigen-matched donors even though the latently infected cells express only a restricted set of virus genes. The virus-induced target antigens recognized by these immune T cells have not been identified. In our experiments, EBV DNA sequences encoding the virus latent gene products Epstein-Barr nuclear antigen (EBNA)1, EBNA 2, and EBNA-LP and the latent membrane protein (LMP) were individually expressed in a virus-negative human B-lymphoma cell line, Louckes. Transfected clones expressing LMP were killed by EBV-specific cytotoxic T-cell preparations from each of three virus-immune donors HLA matched with Louckes through HLA-A2, B44 antigens; control transfectants or clones expressing one of the EBNA proteins were not recognized. Expression of LMP in a second virus-negative B-cell line, BL41, sensitized these cells to EBV-specific cytolysis restricted through the HLA-A11 antigen. To distinguish between the viral protein and an induced human B-cell activation antigen as the target for T-cell recognition, LMP was then expressed in a murine mastocytoma cell line, P815-A11-restricted human T cells. The LMP-expressing P815-A11 transfectants were susceptible to lysis by EBV-specific cytotoxic T cells from three HLA-A11-positive individuals. Both Louckes and P815-A11 cells were also transfected with constructs capable of encoding a truncated form of LMP (Tr-LMP) which lacks the N-terminal 128 amino acids of the full-length protein. Tr-LMP-expressing transfectants were not recognized by the above T-cell preparations. The results suggest that LMP, and, in particular, epitopes derived from the N-terminal region of the protein, provides one of the target antigens for the EBV-induced human cytotoxic T-cell response.

Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains.

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.

Cytotoxic T-cell clones discriminate between A- and B-type Epstein-Barr virus transformants.

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.

Epstein Barr Virus-specific Immune Defects in Patients with Persistent Symptoms Following Infectious Mononucleosis

In a number of patients recovery from infectious mononucleosis (IM) following primary Epstein Barr virus (EBV) infection, is complicated by the persistence of symptoms for months or years. Normally recovery from infectious mononucleosis is associated with the development of EBV-specific antibodies and memory cytotoxic T-cells, which are present in the peripheral blood of all normal seropositive individuals. We studied four patients who had persistent symptoms for more than two years after infectious mononucleosis to determine if this abnormality was associated with a defect in EBV-specific or non-specific immune responses. All four patients had normal immunoglobulin concentrations, T- and B-cell numbers, T-cell proliferative responses and natural killer cell activity. However three of the four had reduced or absent antibodies to the EBV nuclear antigen (EBNA) although other EBV-specific antibody titres were normal. All four also had reduced EBV-specific cytotoxic T-cell activity as measured by the EBV regression assay. This defect was probably EBV-specific as alloreactive cytotoxic T-cell responses were normal. In addition, three of three patients tested had reduced in vitro antibody synthesis following pokeweed mitogen stimulation. These studies indicate that the syndrome of persistent symptoms following EBV mononucleosis may be associated with a defect in EBV-specific immunity, and thus suggest a possible immunological basis for the syndrome.

HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies

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.