Mosquito salivary gland extracts induce EBV-infected NK cell oncogenesis via CD4 T cells in patients with hypersensitivity to mosquito bites.

Severe hypersensitivity to mosquito bites (HMB) is characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. Patients with HMB often have natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection. Here we investigated whether mosquito bites have any influence on the oncogenesis of EBV-infected NK cells. We examined six HMB patients with EBV-infected NK cell lymphocytosis. We first demonstrated that CD4+ T cells, but not NK cells, proliferated well in response to mosquito salivary gland extracts (SGE), especially to SGE of Aedes albopictus. When NK cells were cocultured with autologous CD4+ T cells stimulated by mosquito SGE, the expression of viral oncogene latent membrane protein 1 (LMP1) was remarkably enhanced. Next, we stimulated mononuclear cells of the patients with mosquito SGE, and NK cell counts were monitored for 28 d. The counts changed little from initial levels in the culture with mosquito SGE, whereas they decreased steadily in the culture without the extracts. Furthermore, we detected LMP1 mRNA in the skin lesion induced by mosquito SGE. These results suggest that mosquito bites can induce expression of the viral oncogene LMP1 in NK cells via mosquito antigen-specific CD4+ T cells, which is involved in the oncogenesis of NK cells in vivo.

Enhanced T-cell response to mosquito extracts by NK cells in hypersensitivity to mosquito bites associated with EBV infection and NK cell lymphocytosis.

Hypersensitivity to mosquito bites is characterized by severe systemic as well as local symptoms, and associated with chronic active EBV infection and NK cell lymphocytosis. In this HEN disease, we investigated the response of PBMC to MSG extracts. PBMC were taken from three defined cases of HEN disease, three borderline cases, five individuals with simple exaggerated reactions to mosquito bites without systemic symptoms (simple responders), and eight healthy donors. PBMC, or purified CD4+, CD8+ or CD56+ cells, were cultured with MSG extracts prepared from each of five mosquito species to examine their proliferation and cytokine secretion. The patients with HEN disease had high stimulation indices with variations in responses to the extracts from Aedes albopictus, Aedes aegypti, Anopheles sinensis and Culex pipiens pallens. However, a non-Japan-habitant species Anopheles stephensi did not stimulate the patients' PBMC. Some borderline or simple responders showed moderate proliferation, and healthy donors had no reactive PBMC. In HEN disease, both CD56+ NK cells (producing IFN-gamma) and CD4+ Th0 cells (producing IL-4 and IFN-gamma) were increased in the blood. CD4+ cells, but not CD56+ NK cells or CD8+ cells, propagated in response to MSG extracts. However, this response of CD4+ cells and their IL-4 production were strongly enhanced by coexisting CD56+ cells. We suggest that the CD4+ T cell serving as the primary responder to MSG antigen and the NK cell functioning as the enhancer are both pathogenic in the development of HMB.

Clinicopathological states of Epstein-Barr virus-associated T/NK-cell lymphoproliferative disorders (severe chronic active EBV infection) of children and young adults.

Epstein-Barr virus (EBV)-associated T/NK-cell lymphoproliferative disorders (LPD) of children and young adults are sometimes termed as severe chronic active EBV infection (CAEBV), and are associated with an aggressive clinical course. However, these clinicopathological states and the role of EBV have not been clarified. A retrospective study was performed on 43 children and adult patients, who manifested EBV-associated T/NK-cell lymphoproliferative disorders (EBV-T/NK-LPD) and most of whom had experienced general illness with CAEBV for several months or years. Clinicopathologically, 43 patients were classified into four groups: group A (smoldering state) (n=7), morphological non-neoplastic LPD with chronic clinical course (several years); group B (chronic state) (n=10), non-neoplastic LPD with clonal EBV-infected cells and a chronic course; group C (leukemia/lymphoma state) (n=22), neoplastic LPD with a subacute course (years to months); group D (fulminant state) (n=4), neoplastic LPD with a fulminant course (weeks to days). The 43 patients comprised 21 males and 22 females. The median age of group A was 14 years, group B 12 years, group C 17 years, and group D 1 year. Four of 7 patients in group A, 3 of 10 in group B, 12 of 22 in group C, and all 4 in group D have died. Causes of death included hemophagocytic syndrome and/or tumor death. Genotypically and phenotypically, group C was composed of peripheral T-cell lymphoma (PTCL), and NK-cell leukemia/lymphoma (NKLL), and group D comprised cases of PTCL. Groups A and B exhibited increased NK- or T-cells (CD8>CD4), and rare B-cells. Serologic titers of EBV were only modestly elevated or not elevated in almost all cases. EBV early RNA-1 (EBER-1)-expressing EBV-infected cells were frequently encountered in each group, but the number of infected cells varied between the cases. The EBV genotype did not differ between the groups. Our findings support an important pathogenic role for EBV-infected T/NK-cell infection, rather than the EBV state, in CAEBV and consequent EBV-associated NK/T-neoplasia.

A case of a long-time survivor with chronic active Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is associated with hypersensitivity to mosquito bites (HMB) and fatal EBV-associated hemophagocytic syndrome (HPS). The prognosis of patients with chronic active EBV infection (CAEBV) is very poor. We report a rare case of an adult woman patient with a 28-yr history of HMB, who developed EBV-HPS. EBV genome was detected in the serum and peripheral blood lymphocytes. Clonal proliferation of EBV was demonstrated by Southern blot analysis using an EBV genome terminal-repeat probe. This is a very rare case of a long-term survivor with CAEBV. The patient was initially treated with immunochemotherapy and achieved complete remission. However, the patient immediately relapsed and underwent allogeneic bone marrow transplantation (BMT) from her HLA-matched brother. Peripheral blood cell recovered well, and EBV genome disappeared from the peripheral blood. Allogeneic BMT may be effective in eradicating EBV-HPS. Unfortunately, the patient died of graft vs. host disease on the 92nd day after BMT.

CD4+ T-lymphocyte-induced Epstein-Barr virus reactivation in a patient with severe hypersensitivity to mosquito bites and Epstein-Barr virus-infected NK cell lymphocytosis.

BACKGROUND: Natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection often shows severe hypersensitivity to mosquito bites (HMB) characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. However, the induction mechanism of HMB is still unclear. OBSERVATIONS: We investigated a typical case of HMB with EBV-positive NK cell lymphocytosis. CD4+ T cells dominantly infiltrated the site of the mosquito bite, while EBV-positive cells were few in comparison. CD4+ T cells, but not CD8+ T cells or NK cells, responded to the mosquito salivary gland extracts. Interestingly, coculturing of the NK cells and CD4+ T cells activated by mosquito extracts induced expression of EBV lytic-cycle proteins in the NK cells. Furthermore, the expression of BZLF1, a viral lytic-cycle transactivator, was detectable at the skin lesion induced by scratch patch testing with mosquito extract. The EBV DNA copy number levels in the plasma were elevated in systemic HMB symptoms compared with the normal condition. CONCLUSIONS: CD4+ T cells are important for the primary skin reaction to mosquito bites and might play a key role in reactivation of latent EBV infection in NK cells. This viral reactivation contributed to the pathogenesis of the infectious mononucleosis-like systemic symptoms of HMB in our present case.

Analysis of chromosome 6q deletion in EBV-associated NK cell leukaemia/lymphoma.

Deletions involving chromosome 6q have been reported in a number of human cancers such as ovarian and breast tumours as well as haematopoietic malignancies. It seems that this region might contain tumour-suppressor genes. Putative natural killer cell lymphomas/leukaemias (NKLL) represent a group of recently characterized haematolymphoid malignancies sharing an immunophenotype of CD3/Leu4- CD3epsilon+ CD56+, a genotype of germline T-cell receptor genes, and have a close association with Epstein-Barr virus (EBV). Deletion at 6q21-q25 was demonstrated in three recently reported cases of NKLL. Here we investigated the possible involvement of 6q deletions in the pathogenesis, and especially the tumorigenesis of NKLL. The regions of D6S1574 (6p25), DS276 (6p12), D6S257 (6q11), D6S434 (6q14), D6S287 (6q15), D6S292 (6q21), D6S308 (6q22), D6S264 (6q25), and D6S446 (6q26) were analysed by PCR in 25 cases of NKLL, including seven cases with chronic NK leukaemia, six with acute NK leukaemia and 12 with NK lymphoma. 6q deletions, especially 6q15-25, were frequently detected, but 6p deletions were not detected in any cases. Analysis of 6q21 showed possible deletion in two of seven cases (29%) with chronic NK leukaemia, three of six (50%) with acute leukaemia, and 12 of 12 (100%) with NK lymphoma. The frequency of deletion increased in clinical phases. In three cases with lymphoma, fluorescence in situ hybridisation was performed, which confirmed 6q21 deletion in two cases, although 6q telomeric and centromeric regions were preserved. The other case failed to show deletion. Our results suggest that 6q deletion, especially 6q21-25, might be involved in NKLL tumorigenesis, and support the presence of the tumour suppressor genes associated with the development of NKLL.