Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas,chronic active EBV infection and hydroa vacciniforme-like eruptions

In this study, we describe the cytological and cytogenetic features of six Epstein-Barr virus (EBV)-infected natural killer (NK) cell clones. Three cell clones, SNK-1, -3 and -6, were derived from patients with nasal T/NK-cell lymphomas; two cell clones, SNK-5 and -10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK-11, was from a patient with hydroa vacciniforme (HV)-like eruptions. An analysis of the number of EBV-terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK-cell lymphomas but also those isolated from CAEBV and HV-like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T-cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV-positive NK cells, which proliferated in the diseased individuals. The results showed that EBV-positive NK cells in malignant and non-malignant lymphoproliferative diseases shared common cytological and cytogenetic features.     

A case of lymphomatoid gastropathy: a self-limited pseudomalignant natural killer (NK)-cell proliferative disease mimicking NK/T-cell lymphomas

Natural killer (NK)/T-cell lymphomas exhibit aggressive tumor behavior and have a poor prognosis. Recently, self-limited pseudomalignant NK-cell proliferative disorders of the stomach mimicking NK/T-cell lymphomas have been recognized. We report a rare case of lymphomatoid gastropathy in a 71-year-old female. The patient underwent esophagogastroduodenoscopy (EGD) because of slight epigastric discomfort which revealed a 10-mm, reddish, flat elevation with erosion on the posterior wall of the lower gastric body. Histological examination of biopsy specimens showed atypical NK/T cell infiltration with cytoplasmic CD3+, CD4?, CD5?, CD7+, CD8?, CD16?, CD20?, CD56+, CD68?, CD117?, MPO?, TIA1+, and granzyme B+. Epstein?Barr virus-encoded RNA in situ hybridization was negative. Three months later, repeated endoscopic examination surprisingly revealed spontaneous regression of the lesion without any treatment. It is important that endoscopists consider this rare entity in the differential diagnosis, and excessive treatment should be avoided.     

Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-deoxyphorbol ester. AIK-T8 and - T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.     

Uncommon cases of immature-type CD56+ natural killer (NK)-cell neoplasms, characterized by expression of myeloid antigen of blastic NK-cell lymphoma

Immature-type CD56⁺ natural killer (NK)-cell neoplasms are classified as either myeloid/NK-cell precursor acute leukemia or blastic NK-cell lymphoma. We identified two cases of immature-type CD56⁺ NK-cell neoplasms that were not categorizable as either of these entities. The first case involved a 74-year-old woman presenting with skin eruptions and pancytopenia due to bone marrow necrosis. Skin biopsy specimen revealed CD4⁺, CD7⁻, CD34⁻, CD43⁺, CD56⁺, CD68⁺, muramidase (lysozyme)⁺, and myeloperoxidase (MPO)⁻, and immunophenotyping of peripheral blood showed CD4⁺, CD7⁻, CD13⁺, CD33⁺, CD34⁻, CD43⁺, CD56⁺, cytoplasmic (cy)CD68⁺, CD123⁺, and HLA-DR⁺. The second case involved a 62-year-old man who had bilateral optic nerve tumor and presented with malignant cells in peripheral blood. Cell surface markers of malignant cells showed CD4⁺, CD7⁻, CD13⁺, CD33⁺, CD34⁻, CD43⁺, CD56⁺, cyCD68⁺, and HLA-DR⁺. The phenotypes of tumor cells in both cases were compatible with blastic NK-cell lymphoma, except for the expression of myeloid antigen. Clinical presentations of these cases showed characteristics of both blastic NK-cell lymphoma and myeloid/NK-cell precursor acute leukemia.     

Successful treatment of natural killer (NK) cell leukemia following a long-standing chronic active Epstein-Barr virus (CAEBV) infection with allogeneic bone marrow transplantation

The optimal treatment for natural killer (NK) cell leukemia after chronic active Epstein-Barr virus (CAEBV) infection has not been determined. A 15-year-old boy presented with NK cell leukemia following CAEBV infection for 5 years. The peripheral blood and BM had an increased number of CD3(-)CD56(+) large granular lymphocytes and a monoclonal integration of the EBV genome was detected. Chemotherapy was not sufficiently effective to control the disease. Allogeneic BMT from an HLA-identical sister was performed using a conditioning regimen consisting of total body irradiation, cyclophosphamide and thiotepa. The patient is disease-free with a perfect performance status 24 months after BMT. This is the first report to show that allogeneic BMT is potentially able to cure NK cell leukemia after CAEBV infection.     

Reduced natural killer (NK) function associated with high-risk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis with potential for progression to acute myeloid leukemia (AML). We compared natural killer (NK) cytolytic function in 48 MDS patients with 37 healthy donors and found reduced activity in the patient population (K562 cytolysis, 19% +/- 21% SD versus 40% +/- 17%) (P < .001). NK cytotoxicity in MDS patients was reduced against 3 disparate tumor targets with differential activating receptor requirement, suggesting global defects in NK function. Reduced NK function in MDS was significantly associated with higher International Prognostic Score (P = .01), abnormal karyotype (P = .05), the presence of excess blasts (P = .01), and age-adjusted bone marrow hypercellularity (P = .04). MDS patients had a display of the activating receptor NKp30, and NKG2D down-regulation closely correlated with impaired NK function (P = .001). NKG2D ligands (MICA and MICB) were expressed on CD34(+) cells from bone marrow of 30% of MDS patients and a leukemic cell line derived from an MDS patient (MDS1). Collectively, these findings suggest that impairment of NK cytolytic function derives in part from reduced activating NK receptors such as NKG2D in association with disease progression. Evasion of NK immunosurveillance may have importance for MDS disease progression.     

Clinical implications of plasma Epstein-Barr virus DNA in early-stage extranodal nasal-type NK/T-cell lymphoma patients receiving primary radiotherapy

The clinical value of plasma Epstein-Barr virus (EBV) DNA has not been evaluated in patients with early-stage extranodal nasal-type NK/T-cell lymphoma (NKTCL) receiving primary radiotherapy. Fifty-eight patients with stage I disease and 11 with stage II disease were recruited. High pretreatment EBV-DNA concentrations were associated with B-symptoms, elevated lactate dehydrogenase levels, and a high International Prognostic Index score. EBV-DNA levels significantly decreased after treatment. The 3-year overall survival (OS) rate was 82.6% for all patients. Stage I or II patients with a pretreatment EBV-DNA level of ≤ 500 copies/mL had 3-year OS and progression-free survival (PFS) rates of 97.1% and 79.0%, respectively, compared with 66.3% (P = .002) and 52.2% (P = .045) in patients with EBV-DNA levels of > 500 copies/mL. The 3-year OS and PFS rates for patients with undetectable EBV-DNA after treatment was significantly higher than patients with detectable EBV-DNA (OS, 92.0% vs 69.8%, P = .031; PFS, 77.5% vs 50.7%, P = .028). Similar results were observed in stage I patients. EBV-DNA levels correlate with tumor load and a poorer prognosis in early-stage NKTCL. The circulating EBV-DNA level could serve both as a valuable biomarker of tumor load for the accurate classification of early-stage NKTCL and as a prognostic factor.     

Characterization of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections

The pathogenic mechanisms involved in viral hepatitis are not completely understood. Evidence suggests that the pathology associated with hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are a result of the immune response in the liver to these viruses. The livers of patients with viral hepatitis have been shown to contain elevated numbers of T cells expressing the gamma/delta form of the T-cell receptor for antigen (TCRgammadelta). In this study, we investigated whether liver biopsy specimens obtained from individuals with viral (HCV and/or HBV) or nonviral hepatitis contained TCRgammadelta(+) T cells that could be expanded in vitro by cytokines. A high percentage of liver biopsy specimens obtained from HCV- and/or HBV-infected individuals contained high numbers of TCRgammadelta(+) T cells. In contrast, T-cell lines generated from liver biopsy tissues obtained from individuals with nonviral hepatitis or from normal controls had no preferential expansion of TCRgammadelta(+) T cells. Liver TCRgammadelta(+) T-cell lines from HCV-infected individuals had high levels of non-major histocompatibility complex (MHC)-restricted cytotoxic activity against different targets including primary hepatocytes and produced interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) following activation by anti-CD3. Surprisingly, none of these liver TCRgammadelta(+) T-cell lines could recognize any of the structural or nonstructural proteins of HCV and had no cytotoxic activity against cells infected with recombinant vaccinia viruses expressing different HCV proteins. However, the crosslinking of CD81, which has been shown to bind HCV particles and E2, resulted in significant levels of IFN-gamma and TNF-alpha production by liver TCRgammadelta(+) T cells. These results suggest that TCRgammadelta(+) T cells may play a role in the liver pathology of HCV infections.     

Chronic persistent Epstein-Barr virus infection of natural killer cells and B cells associated with granular lymphocytes expansion

B lymphocytes and epithelial cells are the only cell types known to be infected with Epstein-Barr virus (EBV) in normal individuals. Rarely, EBV also infects other cells, including natural killer (NK) cells, almost always in the context of fatal leukaemias or lymphoproliferative disorders. We report on a 6-year-old previously healthy girl who developed fevers and liver function abnormalities for 3 months. The peripheral blood revealed an abnormal expansion of large granular lymphocytes, comprising 24% of the white blood cells. Flow cytometric analysis of the peripheral blood mononuclear cells showed an abnormal increase of CD16-positive NK cells, 62% of which were EBV-infected by in situ EBER-1 hybridization. The circulating B cells were normal in number, but 18% were infected with EBV by in situ EBER-1 hybridization. Approximately 2 years after resolution of all symptoms and continued good health, 35% of the circulating mononuclear cells were EBV-infected, indicative of persistent expansion of EBV-infected cells. We conclude that abnormal expansions of EBV-infected NK and B cells can be associated with a chronic benign course.     

Quantification of circulating Epstein-Barr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBV-positive lymphomas in immunocompetent patients

In Epstein-Barr-virus (EBV)-positive lymphomas in immunocompetent patients, release of EBV DNA from tumor cells into the plasma might be useful for disease monitoring and prognostication. To test this hypothesis, we quantified serially plasma EBV DNA by quantitative polymerase chain reaction in 39 cases of EBV-positive (natural killer [NK] cell, n = 23; T cell, n = 8; B cell, n = 4; Hodgkin, n = 4) lymphomas. As control, EBV DNA was undetectable in 34 cases of EBV-negative lymphomas at diagnosis and during chemotherapy. In all cases of EBV-positive lymphomas, EBV DNA was detectable (10(5)-10(10) copies/mL) at diagnosis. It paralleled the clinical course, with EBV DNA becoming undetectable at remission and remaining elevated in refractory disease. On multivariate analysis, high-presentation EBV DNA (> 7.3 x 10(7) copies/mL) was significantly associated with an inferior overall survival (OS). Subgroup analysis of NK cell lymphomas, the largest cohort in this study, showed that presentation EBV DNA was correlated with disease stage and lactate dehydrogenase. On multivariate analysis, high-presentation EBV DNA (> 6.1 x 10(7) copies/mL) was significantly associated with an inferior disease-free survival. During treatment, patients with EBV DNA that showed further increases or failed to become undetectable had significantly inferior OS. In EBV-positive lymphomas, plasma EBV DNA is valuable as a tumor biomarker and for prognostication.     

Significance of circulating Epstein-Barr virus DNA monitoring after remission in patients with extranodal natural killer T cell lymphoma

Circulating Epstein-Barr virus (EBV)-DNA has been established as a useful parameter for diagnosis and predicting prognosis in patients with extranodal natural killer T cell lymphoma (ENKTL); however, the role of monitoring of circulating EBV-DNA after complete remission (CR) is not well established. From January 2008 to August 2016, 328 ENKTL patents were enrolled in 2 lymphoma cohorts. Of 171 patients achieved a CR, 81 had available monitoring data for circulating EBV-DNA with negative post-treatment EBV-DNA. Measurement of circulating EBV-DNA was performed from unfractionated whole blood and calculated according to WHO international standards. Median duration of follow-up was 40.4 months. In 31 of the 81 patients (38.8%), circulating EBV-DNA was detected at least once during follow-up, and 16 of these patients (51.6%) experienced relapse. In contrast, only 7 out of 50 (14.0%) patients with consistently undetectable circulating EBV-DNA experienced relapse (p?<?0.001). In multivariate analysis, positive conversion of circulating EBV-DNA was the only independent prognostic factor for occurrence of relapse (HR?=?6.552, p?<?0.001), progression-free survival (HR?=?4.549, p?=?0.01), and overall survival (HR?=?8.726, p?<?0.001). Patients with a higher level of circulating EBV-DNA than 3310 IU/mL (3.52 log₁₀ IU/mL) showed a strong tendency to relapse (73.3 vs. 31.3%, p?=?0.019). In conclusion, positive conversion of circulating EBV-DNA was a valuable indicator of relapse and inferior survival, especially if the level was higher than 3310 IU/mL in ENKTL patients had achieved CR. Close follow-up is necessary for patients developed detectable circulating EBV-DNA after remission.     

Nasal natural killer (NK) cell lymphoma: report of a case with activated NK cells containing Epstein-Barr virus and expressing CD21 antigen, and comparative studies of their phenotype and cytotoxicity with normal NK cells

Summary. Malignant lymphomas arising from the nasal cavity have been considered to be derived from T cells, but recent surface marker studies suggest that more than half of the lymphomas are derived from natural killer (NK) cells. Here we describe a case of nasal lymphoma whose lymphoma cells were identified as NK cells by morphological, phenotypic, immunogenotypic, and functional studies. We believe this is the first study with functional evidence of NK activity. When compared with normal freshly isolated NK cells or activated NK cells, the surface phenotypes and NK activity of the patient tumour cells were those of the activated, but not resting, NK cells. Also, Epstein-Barr virus (EBV) was detected in the tumour cells and the lymphoma cells were found to be monoclonally expanded. The patient's lymphoma cells also expressed EBV receptor CD21 (CR2) and CD30 (Ki-1) that have not been described on normal NK cells. We therefore examined highly enriched NK cells of normal donors, and found that some resting and/or activated NK cells express these antigens.     

The role of natural killer (NK) cells in resistance to herpes simplex virus I (HSV-I) infection

Summary Pretreatment of mice with cyclophosphamide (CY) increases susceptibility of adult mice to herpes simplex type I (HSV-I) infection. In searching for a CY sensitive mechanism, our attention has been focused on natural killer (NK) cells. Activity of these cells was increased in spleens of adult mice after inoculation with HSV-I and depressed after CY administration. The timing of recovery of NK cell activity after the administration of CY, was consistent with a role for these cells in collaboration with immune T lymphocytes when transferred to pre-treated, virus infected animals. It is postulated that sensitized T lymphocytes reacting with virus antigens release products that activate macrophages which in turn produce interferon. Interferon increases NK cell activity and NK cells inhibit viral replication. A correlation between resistance to HSV-I infection and NK cell activity of CBA or C57BL/10 mice was found. It is suggested that NK cells play a role in defence, not only against tumour cells, but also against a wide range of infective agents.     

The prognostic significance of lymphopenia in peripheral T-cell and natural killer/T-cell lymphomas: a study of 826 cases from the International Peripheral T-cell Lymphoma Project

Lymphopenia is a marker of inferior survival in patients with various malignancies. However, the prognostic significance of lymphopenia in peripheral T-cell lymphoma (PTCL) is unclear. We analyzed the prognostic significance of lymphopenia in 826 patients with different types of PTCL and natural killer/T-cell lymphoma (NKTCL) from the International Peripheral T-cell Lymphoma Project. Lymphopenia was defined as an absolute lymphocyte count of less than 1,000 cells per microliter. The overall frequency of lymphopenia was 35.3%, ranging from 21.1% in ALK(+) anaplastic large cell lymphoma (ALCL) to 47.5% in angioimmunoblastic T-cell lymphoma (AITL). Lymphopenia was independently associated with an inferior overall survival (OS) in patients with the lymphoma type of adult T-cell leukemia/lymphoma (ATLL), with a 2-year OS of 15% versus 40% for those without lymphopenia (P < 0.001). Lymphopenia was also an adverse predictor of survival in PTCL, not otherwise specified, but was associated with other unfavorable prognostic factors. A trend toward inferior survival for lymphopenic patients was also observed in AITL, ALK(-) ALCL and extranasal NKTCL lymphoma, whereas no difference in survival was found in nasal NKTCL, ALK(+) ALCL, or enteropathy-associated T-cell lymphoma. In this study, lymphopenia was identified as a new adverse prognostic factor in the lymphoma type of ATLL.     

Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood

The activity of natural killer (NK) cells in human umbilical cord blood (CB) has been reported to be low, compared with that in adult peripheral blood (PB) in vitro. To examine the cause of this, after dividing the CD56+/CD3- cells in CB and PB into CD56bright and CD56dim NK cells, the NK cell activities and the expression of various surface antigens were assayed for each fraction. The NK cell activity of CD56dim NK cells in CB was significantly lower than that in PB (P = 0.0003), whereas, there was no significant difference between the NK cell activity of CD56bright NK cells in PB and CB. The expression levels of adhesion molecules (CD2, CD11a, CD18, DNAX accessory molecule-1), CD16, and CD57 for CD56dim NK cells in CB were significantly lower than those in PB, and approximately one-third of CB CD56dim NK cells were capable of forming conjugates with K562 cells, compared with PB CD56dim NK cells. Furthermore, the inhibition of both the NK cell activities and binding of CD56dim NK cells in PB and CB by monoclonal antibody against each of these adhesion molecules suggests that they play an important role in NK cell activity. These findings show that the low NK cell activity in CB is caused by the low NK cell activity of CD56dim NK cells and that the low expression level of adhesion molecules on CB CD56dim NK cells may contribute to this low NK cell activity.     

Characterization of CD56-/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals

Natural killer (NK) cells are an important component of the innate immune response against viral infections. NK cell-mediated cytolytic activity is defective in HIV-infected individuals with high levels of viral replication. In the present study, we examined the phenotypic and functional characteristics of an unusual CD56(-)/CD16(+) (CD56(-)) NK subset that is greatly expanded in HIV-viremic individuals. The higher level of expression of inhibitory NK receptors and the lower level of expression of natural cytotoxicity receptors observed in the CD56(-) NK fraction compared with that of CD56(+) NK cells was associated with extremely poor in vitro cytotoxic function of this subset. In addition, the secretion of certain cytokines known to be important in initiating antiviral immune responses was markedly reduced in the CD56(-), as compared with the CD56(+) NK cell subset. These data suggest that the expansion of this highly dysfunctional CD56(-) NK cell subset in HIV-viremic individuals largely accounts for the impaired function of the total NK cell population.