Epstein-Barr Virus Infection Of Cells Research Articles

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

Caebv Disease Originates from Infected Hematopoietic Stem Cells

Background Chronic active Epstein-Barr Virus (EBV) disease (CAEBV) is lethal syndrome due to persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Methods Bone marrow and peripheral cells derived from five CAEBV patients, one EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) patient, and two healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative PCR (qPCR), PrimeFlow and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between CAEBV patients and controls, and between infected and uninfected cells. One CAEBV patient was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again six month after HSCT. Results EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, so as hematopoietic stem cells (HSCs) in the CAEBV patients. EBV-infected HSCs exhibited a higher differentiation rate towards downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV infected cells were thoroughly removed from the hematopoietic system after HSCT. Conclusions Token together,multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected hematopoietic stem cells. Figure 1. EBV-infected HSCs were detected in CAEBV patients with multiple assays.

Mechanism and therapeutic implications of pomalidomide-induced immune surface marker upregulation in EBV-positive lymphomas

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/β in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom’s effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

The Role of NK Cells in EBV Infection and Related Diseases: Current Understanding and Hints for Novel Therapies

The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus most often transmitted during infancy and infecting the vast majority of human beings. Usually, EBV infection is nearly asymptomatic and results in life-long persistency of the virus in a latent state under the control of the host immune system. Yet EBV can cause an acute infectious mononucleosis (IM), particularly in adolescents, and is associated with several malignancies and severe diseases that pose a serious threat to individuals with specific inborn error of immunity (IEI). While there is a general consensus on the requirement for functional CD8 T cells to control EBV infection, the role of the natural killer (NK) cells of the innate arm of immunity is more enigmatic. Here we provide an overview of the interaction between EBV and NK cells in the immunocompetent host as well as in the context of primary and secondary immunodeficiencies. Moreover, we report in vitro data on the mechanisms that regulate the capacity of NK cells to recognize and kill EBV-infected cell targets and discuss the potential of recently optimized NK cell-based immunotherapies for the treatment of EBV-associated diseases.

APX‑115A, a pan‑NADPH oxidase inhibitor, reduces the degree and incidence rate of dry eye in the STZ‑induced diabetic rat model.

Dye eye disease (DED) is a common ocular disorder in patients with diabetes. It has been reported that APX-115A, a pan-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitor, has an apoptosis-inducing effect on Epstein-Barr virus-infected retinal epithelial cells, but its effects in DED are poorly understood. Therefore, a rat model of diabetes was used in the present study to investigate whether APX-115A has an impact on DED in diabetic rats. A diabetic model was established in male Sprague Dawley rats via the intraperitoneal injection of streptozotocin. The eyeballs of the rats were treated with a solution containing APX-115A or a saline control. Tear secretion was measured with the phenol red thread tear test, and the morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, localization of NAPDH oxidase 2 (NOX2) in the eyeball and lacrimal gland tissues was detected by immunohistochemistry. The APX-115A treatment had no effect on body weight, blood glucose level or the size of the lacrimal glands. However, morphological changes, namely intracellular vacuoles and acinar atrophy, were observed in the lacrimal glands of the diabetic rats, and APX-115A treatment attenuated these changes. Immunohistochemistry revealed that NOX2 expression was decreased in the lacrimal glands of the diabetic rats, and APX-115A treatment did not attenuate the reduction in NOX2. The corneas of the diabetic rats treated with APX-115A exhibited no change in thickness but had lower NOX2 expression levels compared with those of the control diabetic rats. APX-115A also increased tear secretion and ameliorated the histological changes associated with diabetes. Furthermore, the NOX2 expression levels in the corneas of the diabetic rats treated with APX-115A were restored to the levels observed in normal rats. These findings suggest that APX-115A has potential as a therapeutic agent for DED.

Modulation of peroxisomal compartment by Epstein-Barr virus.

Viruses utilize clever strategies of interacting with various cellular factors, to remodel an organelle function, for the establishment of successful infection. In recent decades, numerous studies revealed the exploitation of the peroxisomal compartment by viruses. Epstein-Barr virus (EBV) is a ubiquitous virus linked with various cancers and neurological disorders. Till now, there is no report regarding the impacts of EBV infection on peroxisomal compartment. Therefore, we investigate the modulation of peroxisomal proteins in EBV transformed cell lines and during acute EBV infection. EBV positive Burkitt lymphoma cells of different origins as EBV transformed cells along with EBV negative Burkitt lymphoma cells as a control were used in this study. For acute EBV infection experiments, we infected peripheral blood mononuclear cells with EBV for three days. Thereafter, analyzed the gene expression patterns of peroxisomal proteins using qPCR. In addition, quantification of lipid content was performed by using fluorescence microscopy and biochemical assay. Our results revealed that, the peroxisomal proteins were distinctly regulated in EBV transformed cells and during acute EBV infection. Interestingly, PEX19 was significantly upregulated in EBV infected cells. Further, in correlation with the altered expression of peroxisomes proteins involved in lipid metabolism, the EBV transformed cells showed lower lipid abundance. Conversely, the lipid levels were increased during acute EBV infection. Our study highlights the importance of investigating the manipulation of the peroxisomal compartment by putting forward various differentially expressed proteins upon EBV infection. This study provides a base for further investigation to delve deeper into EBV and peroxisomal interactions. The future research in this direction could provide involvement of novel signaling pathways to understand molecular changes during EBV mediated pathologies.

Transcriptome reprogramming of Epstein-Barr virus infected epithelial and B cells reveals distinct host-virus interaction profiles

Epstein-Barr virus (EBV) is an opportunistic pathogen that can manifest itself as a potential contributor to human diseases years after primary infection, specifically in lymphoid and epithelial cell malignancies in immune-competent and immune-compromised hosts. The virus shuttles between B cells and epithelial cells during its infection cycle, facilitating its persistence and transmission in humans. While EBV efficiently infects and transforms B-lymphocytes, epithelial cells are not as susceptible to transformation in vitro. We utilized a 3D platform for culturing normal oral keratinocyte cells (NOKs) using Matrigel for greater insights into the molecular interactions between EBV and infected cells. We determined the transcriptome of EBV infected NOKs and peripheral blood mononuclear cells (PBMCs) for 7 and 15 days. LMPs (−1, −2A, and −2B) and EBNAs (−1, −2, −3A, −3B and −3C) were detected in all samples, and lytic gene expression was significantly higher in NOKs than PBMCs. We identified over 2000 cellular genes that were differentially expressed (P-value<0.05). Gene ontology (GO) and pathway analyses significantly identified pathways related to collagen-activation, chemokine signaling, immune response, metabolism, and antiviral responses. We also identified significant changes in metalloproteases and genes encoding chemotactic ligands and cell surface molecules. C-X-C chemokine receptor type 4 (CXCR4) was dramatically downregulated in PBMCs and upregulated in NOKs. However, MMP1 was significantly downregulated in NOKs and upregulated in PBMCs. Therefore, multiple pathways contribute to distinct pathologies associated with EBV infection in epithelial and B cells, and MMP1 and CXCR4 are critical molecules involved in regulation of latent and lytic states linked to viral associated diseases.

Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection

To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample. Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully. A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

Highly multiplexed, single-cell spatial phenotyping of Epstein-Barr virus infected tissues.

Abstract The Epstein-Barr virus (EBV), a group 1 carcinogen, is a causative factor in nine different cancers that cause ~165,000 deaths globally each year. Almost all adults carry EBV asymptomatically; the virus persisting primarily in rare memory B cells for the lifetime of the host. However, in some infected individuals, for reasons that are unknown, EBV may contribute to the development of cancer. The immune microenvironment in which infected cells reside may influence or trigger cancer development, but we know very little about how this occurs. We have begun to address these critical questions by deploying a spatial biology assay for comprehensive study of the immune microenvironment of EBV-infected tissues. Using AKOYA’s immunofluorescence based Phenocycler assay, we developed an antibody panel that can detect 40 or more different biomarkers in situ at single cell resolution. The panel contains antibodies directed at EBV infected cells at various stages of viral infection, as well as biomarkers for different immune cell lineages, immune cell activation states and tissue structures. This design allows simultaneous identification of infected immune cells within their microenvironment, while, crucially, maintaining spatial relationships between them. We have now set out to produce unbiased and comprehensive spatial phenotypic maps of normal and EBV infected human FFPE tissues. Our data will provide an initial understanding of how the immune microenvironment of virus-infected cells influences tissue pathology and, ultimately, the development of cancer.

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection.

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

The Role of NK Cells in EBV Infection and EBV-Associated NPC.

A vast majority of the population worldwide are asymptomatic carriers of Epstein-Barr Virus (EBV). However, some infected individuals eventually develop EBV-related cancers, including Nasopharyngeal Carcinoma (NPC). NPC is one of the most common EBV-associated epithelial cancers, and is highly prevalent in Southern China and Southeast Asia. While NPC is highly sensitive to radiotherapy and chemotherapy, there is a lack of effective and durable treatment among the 15%–30% of patients who subsequently develop recurrent disease. Natural Killer (NK) cells are natural immune lymphocytes that are innately primed against virus-infected cells and nascent aberrant transformed cells. As EBV is found in both virally infected and cancer cells, it is of interest to examine the NK cells’ role in both EBV infection and EBV-associated NPC. Herein, we review the current understanding of how EBV-infected cells are cleared by NK cells, and how EBV can evade NK cell-mediated elimination in the context of type II latency in NPC. Next, we summarize the current literature about NPC and NK cell biology. Finally, we discuss the translational potential of NK cells in NPC. This information will deepen our understanding of host immune interactions with EBV-associated NPC and facilitate development of more effective NK-mediated therapies for NPC treatment.

Patients with autoimmune diseases have an altered activity of the PD-1 pathway and proportions of Epstein-Barr virus infected cells in benign lymphadenopathies

Patients with autoimmune diseases (AD) have an increased risk to develop benign lymphadenopathies compared to patients without AD. The aim with this study was to determine the role of the PD-1 pathway and the number of cells harboring Epstein-Barr virus (EBV) in benign lymphadenopathies in patients with AD (cases) compared to patients without AD (controls). Pathology registries were screened to identify patients with biopsies diagnosed as benign lymphadenopathy and medical journals were reviewed for information on AD. Immunohistochemical stainings (PD-1 and PD-L1) and EBER in situ hybridization for EBV were applied on lymph node biopsies in patients with AD (n = 22) and patients without AD (n = 57). The case group was compared with the control group with Wilcoxon-signed rank, chi-square and Fischeŕs exact test. There was a statistically significantly higher proportion of PD-1+ cells and a tendency for a lower prevalence of PD-L1+ and EBV+ cells in cases compared to controls. Apparently, patients with AD have an altered immune response as revealed in benign lymphadenopathies compared to patients without AD. If this association might be a piece of the puzzle for the increased risk of development of lymphomas in patients with AD remains to be determined.

Upregulation of GLS1 Isoforms KGA and GAC Facilitates Mitochondrial Metabolism and Cell Proliferation in Epstein-Barr Virus Infected Cells.

Epstein–Barr virus or human herpesvirus 4 (EBV/HHV-4) is a ubiquitous human virus associated with a wide range of malignant neoplasms. The interaction between EBV latent proteins and host cellular molecules often leads to oncogenic transformation, promoting the development of EBV-associated cancers. The present study identifies a functional role of GLS1 isoforms KGA and GAC in regulating mitochondrial energy metabolism to promote EBV-infected cell proliferation. Our data demonstrate increased expression of GLS1 isoforms KGA and GAC with mitochondrial localization in latently EBV-infected cells and de novo EBV-infected PBMCs. c-Myc upregulates KGA and GAC protein levels, which in turn elevate the levels of intracellular glutamate. Further analysis demonstrated upregulated expression of mitochondrial GLUD1 and GLUD2, with a subsequent increase in alpha-ketoglutarate levels that may mark the activation of glutaminolysis. Cell proliferation and viability of latently EBV-infected cells were notably inhibited by KGA/GAC, as well as GLUD1 inhibitors. Taken together, our results suggest that c-Myc-dependent regulation of KGA and GAC enhances mitochondrial functions to support the rapid proliferation of the EBV-infected cells, and these metabolic processes could be therapeutically exploited by targeting KGA/GAC and GLUD1 to prevent EBV-associated cancers.

Dasatinib exacerbates splenomegaly of mice inoculated with Epstein-Barr virus-infected lymphoblastoid cell lines

Latent infection of Epstein-Barr virus (EBV) is associated with a poor prognosis in patients with B cell malignancy. We examined whether dasatinib, a multi kinase inhibitor, which is broadly used for chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia is effective on EBV-positive B cell malignancies, using lymphoblastoid cell lines (LCLs) in vitro and in vivo. As a result, in vitro experiments showed that dasatinib induced cell death of the EBV-LCLs which was not accompanied with a lytic reactivation of EBVs. To evaluate the effectiveness in EBV latency type III represented by immunodeficiency lymphoma, LCL-inoculated immunodeficient NOD/shi-scid/Il2rgnul (NOG) mice were treated with dasatinib. However, in vivo experiments revealed that dasatinib treatment exacerbated tumor cell infiltration into the spleen of LCL-inoculated NOG mice, whereas tumor size at the inoculated site was not affected by the treatment. These results suggest that dasatinib exacerbates the pathogenesis at least in some situations although the drug is effective in vitro. Hence, we should carefully examine a possibility of dasatinib repositioning for EBV+ B cell malignancies.

Immunopathogenetic role of plasmocytoid dendritic cells in Epstein-Barr virus infection

Plasmocytoid dendritic cells (pDCs) play a key role in immunity against most viruses, given their unparalleled ability to produce large amounts of IFN I type. Studies aimed at studying pDCs in Epstein-Barr virus infection (EBV) are still few. The outcome of EBV largely depends on the ability of the patient’s immune system to form an adequate immune defense, that provides not only a rapid recovery, but also prevents the prolonged course of the disease, leading to the development of immune deficiency and other complications. The development of effective immunorehabilitation of EBV, able to prevent the chronization of this disease, remains one of the most important areas of scientific and practical activities.

Epstein-Barr virus infection in the development of neurological disorders

Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus that contributes to the etiology of diverse human cancers and auto-immune diseases. EBV establishes a relatively benign, long-term latent infection in over 90 percent of the adult population. Yet, it also increases risk for certain cancers and auto-immune disorders depending on complex viral, host, and environmental factors that are only partly understood. EBV latent infection is found predominantly in memory B-cells, but the natural infection cycle and pathological aberrations enable EBV to infect numerous other cell types, including oral, nasopharyngeal, and gastric epithelia, B-, T-, and NK-lymphoid cells, myocytes, adipocytes, astrocytes, and neurons. EBV infected cells, free virus, and gene products can also be found in the CNS. In addition to the direct effects of EBV on infected cells and tissue, the effect of chronic EBV infection on the immune system is also thought to contribute to pathogenesis, especially auto-immune disease. Here, we review properties of EBV infection that may shed light on its potential pathogenic role in neurological disorders.

Alterations in cellular expression in EBV infected epithelial cell lines and tumors.

The Epstein Barr virus (EBV) is linked to the development of two major epithelial malignancies, gastric carcinoma and nasopharyngeal carcinoma. This study evaluates the effects of EBV on cellular expression in a gastric epithelial cell line infected with or without EBV and a nasopharyngeal carcinoma cell line containing EBV. The cells were grown in vitro and as tumors in vivo. The effects on cellular expression were determined using both 2D DIGE proteomics and high throughput RNA sequencing. The data identify multiple pathways that were uniquely activated in vitro. RNA sequences mapping to the mouse genome were identified in both the EBV positive and negative tumor samples in vivo, although, differences between the EBV positive and negative cells were not apparent. However, the tumors appeared to be grossly distinct. The majority of the identified canonical pathways based on two fold changes in expression had decreased activity within the tumors in vivo. Identification of the predicted upstream regulating factors revealed that in vitro the regulating factors were primarily protein transcriptional regulators. In contrast, in vivo the predicted regulators were frequently noncoding RNAs. Hierarchical clustering distinguished the cell lines and tumors, the EBV positive tumors from the EBV negative tumors, and the NPC tumors from the gastric tumors and cell lines. The delineating genes were changed greater than 4 fold and were frequently regulated by protein transcription factors. These data suggest that EBV distinctly affects cellular expression in gastric tumors and NPC and that growth in vivo requires activation of fewer cellular signaling pathways. It is likely that the broad changes in cellular expression that occur at low levels are controlled by regulatory viral and cellular RNAs while major changes are affected by induced protein regulators.

Elevated numbers of PD-L1 expressing B cells are associated with the development of AIDS-NHL

The risk for non-Hodgkin lymphoma (NHL) is markedly increased in persons living with human immunodeficiency virus (HIV) infection, and remains elevated in those on anti-retroviral therapy (cART). Both the loss of immunoregulation of Epstein-Barr virus (EBV) infected cells, as well as chronic B-cell activation, are believed to contribute to the genesis of AIDS-related NHL (AIDS-NHL). However, the mechanisms that lead to AIDS-NHL have not been completely defined. A subset of B cells that is characterized by the secretion of IL10, as well as the expression of the programmed cell death ligand-1 (PD-L1/CD274), was recently described. These PD-L1+ B cells can exert regulatory function, including the dampening of T-cell activation, by interacting with the program cell death protein (PD1) on target cells. The role of PD-L1+ B cells in the development of AIDS-NHL has not been explored. We assessed B cell PD-L1 expression on B cells preceding AIDS-NHL diagnosis in a nested case-control study of HIV+ subjects who went on to develop AIDS-NHL, as well as HIV+ subjects who did not, using multi-color flow cytometry. Archival frozen viable PBMC were obtained from the UCLA Multicenter AIDS Cohort Study (MACS). It was seen that the number of CD19+CD24++CD38++and CD19+PD-L1+cells was significantly elevated in cases 1–4 years prior to AIDS-NHL diagnosis, compared to controls, raising the possibility that these cells may play a role in the etiology of AIDS-NHL. Interestingly, most PD-L1+ expression on CD19+ cells was seen on CD19+CD24++CD38++ cells. In addition, we showed that HIV can directly induce PD-L1 expression on B cells through interaction of virion-associated CD40L with CD40 on B cells.

PB2052 PHENOTYPIC IDENTIFICATION OF EBV‐INFECTED LYMPHOCYTES FOR THE DIFFERENTIAL DIAGNOSIS OF HLH, CAEBV AND PTLD; A CLINICALLY APPLICABLE, FLOW‐CYTOMETRY‐ BASED PERIPHERAL BLOOD ASSAY

Background:Epstein–Barr virus (EBV) is a ubiquitous lymphotropic virus which preferentially infects B‐cells1,2, but can also infect T‐cells and NK‐cells. EBV can cause B‐cell or T/NK lymphoproliferative (LPD) disorders and EBV‐related haemophagocytic lymphohistiocytosis (HLH)2. These rare but distinct diseases share clinical features of lymphadenopathy, fever, malaise, organomegaly and night sweats1. High levels of EBV DNA, as assessed by qPCR on whole blood or plasma are typical but, per se, have limited clinical value. EBV copy number in individual lymphocyte subsets offers much greater clinical value but is technically challenging and hence rarely investigated.Aims:We have applied a dual in‐situ hybridisation‐flow cytometry technique (EBER‐FlowRNA) allowing the detection and phenotypic characterisation of specific lymphocyte subsets infected by EBV, impacting the clinical management of patients with EBV‐associated lymphoproliferative disease.Methods:Following informed consent, blood samples from patients with suspected EBV‐related LPD were obtained at diagnosis. All patients were part of the observational ‘Studies of EBV‐associated NK/T‐cell diseases and formation of a registry’ (07/H1208/62). All analyses were performed at the Institute for Immunology and Immunotherapy (Birmingham University, UK). Clinical information for the cases was retrospectively obtained from clinical records.Peripheral blood mononuclear cells (PBMC) were isolated from fresh patient blood and subjected to multicolour cell surface staining using up to 10 individual fluorophores. The cells were then fixed/permeabilised and subjected to EBER in‐situ hybridisation (PrimeFlowRNA) using pairs of 20‐mer specific probes, previously amplified and loaded with up to 400 fluorescent molecules; Beta‐2‐microglobulin probe pairs were used as positive control. Samples were processed by multicolour flow cytometry (BD Fortessa) and analysed with FlowJo software.Results:We present 4 representative clinical cases. Clinical characteristics described in Table 1. Flow cytometry of the samples was performed within 48 h of sample collection. Case 1 reflected a CD3+/CD4+/EBER+ population compatible with relapsed CAEBV, excluding the alternative diagnosis of EBV‐related B‐cell PTLD. Case 2 was an unusual CAEBV with gastric involvement and a CD3lo/CD4+/EBER+ population in peripheral blood, consistent with EBER positive malignant clone in the gastric biopsy. Case 3 presented with florid HLH and a CD19+/EBER+ clone consistent with primary EBV infection in the context of immunosuppression, treated successfully with Rituximab. Case 4 presented with a systemic illness and hepatic impairment, with identification of a CD56+/CD8+/EBER+ clone representing aggressive NK cell leukaemia. In cases 1, 2 and 4, EBER‐flowRNA prevented inappropriate use of empirical rituximab for raising EBV DNA‐aemia, whereas in case 3, EBER‐flowRNA excluded a T/NK LPD and supported rituximab use.Summary/Conclusion:In line with previously reported methodology4,5, our customised flow‐cytometry approach successfully identified the EBV‐infected lymphocyte subsets with a short turnaround time (48 hours sample‐to‐result). Additionally, successful EBER fluorescent labelling allowed for cell sorting and subsequent genomic and transcriptomic studies of EBV infected cells. Importantly, flow cytometry findings directed appropriate therapeutic approaches. Application of this technique in a larger cohort is warranted. We propose EBER‐FlowRNA as a relevant, rapid and feasible differential diagnostic tool for EBV‐related LPD.image

Determination of Epstein-Barr Virus-Infected Lymphocyte Cell Types in Peripheral Blood Mononuclear Cells as a Valuable Diagnostic Tool in Hematological Diseases.

BackgroundHigh loads of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells (PBMCs) can be indicative of a broad spectrum of diseases, ranging from asymptomatic infection to fatal cancers.MethodsWe retrospectively investigated the EBV-infected cell types in PBMCs among 291 patients. Based on EBV-infected cell types, the clinical features and prognoses of 93 patients with EBV-associated (EBV+) T/natural killer (NK)–cell lymphoproliferative diseases (LPDs) T/NK-LPDs) were investigated over a 5-year period.ResultsAlthough B-cell-type infection was found in immunocompromised patients and patients with asymptomatic high EBV carriage, infectious mononucleosis, EBV+ B-cell LPDs and B-cell lymphomas, T-cell, NK-cell or multiple-cell-type infection in immunocompetent hosts were highly suggestive of EBV+ T/NK-LPDs, EBV+ T/NK-cell lymphomas, and aggressive NK-cell leukemia. Patients with non–B-cell infection had a poorer prognosis than those with B-cell-type infection. In our cohort, 79.6% of patients with EBV+ T/NK-LPDs were >18 years old, and NK cells were identified as EBV-infected cell type in 54.8%. Nearly half of patients with EBV+ T/NK-LPDs had genetic defects associated with immunodeficiency. However, hemophagocytic lymphohistiocytosis, and not genetic defects, was the only parameter correlated with poor prognosis of EBV+ T/NK-LPDs.ConclusionsDetermination of EBV-infected cell types among PBMCs is a valuable tool for the differential diagnosis of EBV+ hematological diseases.In this study, determination of Epstein-Barr virus-infected cell types in peripheral blood mononuclear cells of 291 patients with high Epstein-Barr virus loads were retrospectively investigated, which indicate it is a valuable tool for Epstein-Barr virus-associated hematological diseases.