Abstract
Abstract 3672
Poster Board III-608
Introduction
Epstein Barr virus (EBV) is predominantly B lymphotrophic both in-vitro and in vivo, where in immunocompetent individuals the virus persists asymptomatically in the B lymphoid compartment under host T cell control. EBV's association with B cell malignancies, such as Hodgkin and Burkitt lymphoma, can be viewed as rare accidents of the virus' lifelong interaction with the B cell system. By contrast, EBV infection of NK and T cells is considered a rare event but is nonetheless strongly associated with a spectrum of rare lymphoproliferations: EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV), aggressive NK leukaemia and NK/T lymphoma all characterised pathogenically by the presence of monoclonal EBV in the T and/or NK cells. The mechanism of viral entry and its contribution to lymphoproliferation in these cell lineages remains to be clearly defined. The majority of reported cases of EBV-HLH occur in the context of primary EBV infection in children or adolescents, some of whom have a defined inherited immune defect. Adult cases of EBV-HLH occur extremely rarely and appear to be more frequent in individuals of East Asian origin. Furthermore, the vast majority of analysed cases of EBV-HLH have identified CD8+ lymphocytes as the predominant virus-bearing cell. To-date, EBV infection of (CD3-CD56+) circulating NK cells has not been reported and the pattern of viral gene expression remains unclear.
Methods/Patients
We analysed peripheral blood from three consecutive cases of EBV-HLH, referred to our laboratory between 2007-2009, to identify the predominant virus-harbouring cell. All three cases occurred in adults (mean age 44yrs), with no history of inherited immunodeficiency, who presented with clinical and laboratory features consistent with a diagnosis of HLH; fever, hepatosplenomegaly, pancytopenia, markedly elevated serum ferritin and lactate dehydrogenase and EBV copy number of 105-106 per millilitre of whole blood. Haemophagocytosis was unequivocally present on tissue biopsy from two patients. Mononuclear cells were separated using the MoFlo™ cell sorter into pure populations. Patient 1 and 2: CD19+CD3 −CD56−, CD3+CD19−CD56−, and CD56+CD3−CD19−. Patient 3: CD19+CD3 –CD16−, CD3+CD19−CD16−, CD16+CD3−CD19− and CD3−CD19−CD16−. DNA was subsequently extracted from each population and assayed by quantitative PCR, expressed as genome copies per million cells.
Results
In all three cases we found the predominant EBV load within the non-B, non-T lymphocyte populations; definitively shown to be the CD56+CD3− cell fraction in 2 cases and for case 3 within CD3−CD19−CD16− lymphocytes likely to represent CD56+CD16- NK cells (a minority population in normal peripheral blood). A representative figure is shown:
We then quantitatively examined latent and lytic viral gene transcripts by real-time PCR and, in contrast to previously published data, we found a tightly restricted pattern of EBV gene expression with extremely high levels of EBER (EBV-encoded RNA) transcripts present. Lymphocytes derived from tonsillar tissue and peripheral blood, from both healthy and immunosuppressed individuals, served as control samples and demonstrated the predominant EBV genome load in the CD19+ B-cells but not the T or NK fractions.
Conclusion
This novel finding of high EBV genome copy numbers and a restricted pattern of viral gene expression, within circulating natural killer cells in the context of adult EBV-HLH, is both pathogenically intriguing and importantly, has relevance for the investigation of targeted therapies for this aggressive disease.
Disclosures:
No relevant conflicts of interest to declare.
Differential Methylation of Epstein-Barr Virus Latency Promoters Facilitates Viral Persistence in Healthy Seropositive Individuals
