Epstein-Barr Virus-associated Hemophagocytic Lymphohistiocytosis Research Articles

Emapalumab as a therapeutic intervention for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis: A case series.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is characterized by a severe cytokine storm, heightened inflammatory response, and immune-mediated damage to tissues and organs. Standard treatment protocols for hemophagocytic lymphohistiocytosis often fall short in effectively controlling EBV-HLH, leading to a need for novel therapeutic options. Emapalumab, a monoclonal antibody targeting interferon-gamma, has shown promise due to its targeted cytokine modulation capabilities and favorable safety profile. This study aimed to evaluate the efficacy and safety of emapalumab in pediatric patients with EBV-HLH. The case series involved 4 pediatric patients diagnosed with EBV-HLH who did not achieve disease control despite receiving comprehensive treatment. All 4 pediatric patients were diagnosed with EBV-HLH. Emapalumab was introduced as an adjunctive therapeutic intervention alongside the HLH-94 or L-DEP regimens for these patients. Among the 4 patients, 1 experienced severe multiorgan dysfunction and opted to discontinue therapy. The remaining 3 patients showed controlled disease progression with significant clinical improvements following emapalumab administration. These improvements included reduced levels of inflammatory markers, normalization of blood counts and liver function, and decreased Epstein-Barr virus viral load. The findings suggest that emapalumab may be an effective and safe treatment option for pediatric EBV-HLH. However, further research is necessary to confirm these outcomes, especially in critically ill patients.

Treatment of pediatric refractory or relapsed Epstein-Barr virus-associated hemophagocytic syndrome with PD-1 inhibitors.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a type of pediatric HLH that occurs frequently in Asia. Although immunochemotherapy based on etoposide and hormone has improved survival rates, there are still about 30% of HLH patients that do not respond. The objective of the article is to examine the efficacy and safety of programmed cell death protein 1 (PD-1) inhibitors for children with relapsed/refractory (r/r) EBV-HLH. A retrospective case note review of four pediatric patients with r/r EBV-HLH who were treated with PD-1 inhibitors at Sun Yat-sen Memorial Hospital, Sun Yat-sen University. All four patients responded to PD-1 inhibitors and achieved partial response after their first infusion. Plasma EBV DNA copy number and HLH-related monitoring indicators decreased in all of these patients. All patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), and two were still alive at the last follow-up on December 30, 2022. Two patients died because of transplantation-related complications. Serious side effects included increased liver enzymes and edema in two patients. PD-1 inhibitors are an effective salvage therapy and can provide a bridge to allo-HSCT for pediatric patients with r/r EBV-HLH. However, side effects should be monitered.

Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Enhancing diagnostic precision in EBV-related HLH: a multifaceted approach using 18F-FDG PET/CT and nomogram integration

BackgroundThe hyperinflammatory condition and lymphoproliferation due to Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) affect the detection of lymphomas by 18F-FDG PET/CT. We aimed to improve the diagnostic capabilities of 18F-FDG PET/CT by combining laboratory parameters.MethodsThis retrospective study involved 46 patients diagnosed with EBV-positive HLH, who underwent 18F-FDG PET/CT before beginning chemotherapy within a 4-year timeframe. These patients were categorized into two groups: EBV-associated HLH (EBV-HLH) (n = 31) and EBV-positive lymphoma-associated HLH (EBV + LA-HLH) (n = 15). We employed multivariable logistic regression and regression tree analysis to develop diagnostic models and assessed their efficacy in diagnosis and prognosis.ResultsA nomogram combining the SUVmax ratio, copies of plasma EBV-DNA, and IFN-γ reached 100% sensitivity and 81.8% specificity, with an AUC of 0.926 (95%CI, 0.779–0.988). Importantly, this nomogram also demonstrated predictive power for mortality in EBV-HLH patients, with a hazard ratio of 4.2 (95%CI, 1.1–16.5). The high-risk EBV-HLH patients identified by the nomogram had a similarly unfavorable prognosis as patients with lymphoma.ConclusionsThe study found that while 18F-FDG PET/CT alone has limitations in differentiating between lymphoma and EBV-HLH in patients with active EBV infection, the integration of a nomogram significantly improves the diagnostic accuracy and also exhibits a strong association with prognostic outcomes.

Clinical Characteristics Analysis of EBV-associated Hemophagocytic Lymphohistiocytosis Patients with Acute Kidney Injury

To analyze the clinical characteristics of patients with Epstein-Barr virus(EBV)-associated hemophagocytic lymphohistiocytosis(HLH) with acute kidney injury(AKI). EBV-HLH patients who were hospitalized in our hospital from January 2014 to December 2020 were collected, and their clinical characteristics, treatment, concurrent acute kidney injury and prognosis were retrospectively analyzed. In this study, the incidence of AKI complicated by EBV-HLH was 65.5%, and the 28-day mortality rate was 15.3%. Compared with non-AKI group, patients in the AKI group had higher levels of bilirubin, lactate dehydrogenase, creatinine, urea nitrogen, and β2-microglobulin（β2-MG）, poorer coagulation, and lower soluble IL-2 receptor （sCD25）. Patients in the AKI group had a higher proportion of chemotherapy, transplantation, mechanical ventilation, and the application of vasoactive medications, and were hospitalized for longer periods of time, with higher in-hospital mortality rates and 28-day mortality rates. Patients in the AKI group were analyzed in subgroups according to the Kidney Disease Improving Global Outcomes （KDIGO）classification, and the levels of leukocytes, bilirubin, albumin, creatinine, urea nitrogen, β2-MG, activated partial thromboplastin time （APTT）, and prothrombin time activity （PTA）were more responsive to the severity of the patient's condition. KDIGO grade 2 and 3 had higher proportions of receiving transplants, diuretics, organ support (mechanical ventilation, application of vasoactive medications, and renal replacement therapy), and admissions to the intensive care unit （ICU）, and with higher in-hospital mortality rates and 28-day mortality rates. Regression analysis found that creatinine, β2-MG, APTT, transplantation, and chemotherapy were independent risk factors for the development of AKI; the application of vasoactive drugs was both an independent risk factor for the development of AKI and for death at 28 days; and chemotherapy, length of hospitalization, and HGB and fibrinogen levels were protective factors for death at 28 days. AKI in EBV-HLH has high incidence and high rate of progression to severe disease and death, early attention should be given and strengthened in order to carry out early treatment and improve the prognosis of patients.

Clinical utility of plasma Epstein-Barr virus DNA monitoring in pediatric Epstein‐Barr virus-associated hemophagocytic lymphohistiocytosis: a Chinese retrospective observational study

BackgroundEpstein-Barr virus DNA (EBV-DNA) is closely related to the pathogenesis and prognosis of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The quantitative measurement of blood EBV-DNA is widely used in EBV-HLH, but there remains a lack of evidence to guide clinicians.MethodsA retrospective analysis was conducted on clinical manifestations, laboratory tests, 310 blood EBV-DNA loads, and prognosis of 51 pediatric patients diagnosed with EBV-HLH. Receiver operating characteristic (ROC) curves were utilized to determine the optimal cutoff values of EBV-DNA for predicting mortality and evaluating the active status of EBV-HLH.ResultsEBV-positive- lymphoma-HLH had higher initial plasma EBV-DNA load(1.10 × 106copies/ml) compared to the EBV-HLH group (1.98 × 104 copies/ml) (P = 0.006), and experienced recurrently elevated plasma EBV-DNA levels during treatment. The optimal cut-off value of initial plasma EBV-DNA load in predicting mortality was 2.68 × 105 copies/ml, with a sensitivity of 88.57% and a specificity of 56.25%. For determining the active status of HLH, the optimal cutoff value of PBMC EBV-DNA load during treatment was 2.95 × 105 copies/ml, with a sensitivity of 69.14% and a specificity of 64.71%. The cut-off value of plasma EBV-DNA for determining active status was 1.32 × 103 copies/ml, with a sensitivity of 84.34%, and a specificity of 87.67%. Patients with higher PBMC and plasma EBV-DNA at initial and those with repeated elevated plasma EBV-DNA during treatment had worse prognoses (P < 0.05).ConclusionDynamic monitoring of EBV-DNA is a valuable tool for assessing disease status and predicting the prognosis of EBV-HLH, with plasma EBV-DNA being more effective than PBMC EBV-DNA. Patients with high levels of PBMC and plasma EBV-DNA at initial and those with repeated elevated plasma EBV-DNA during treatment had worse prognoses.

Rituximab as a Therapeutic Strategy in Hemophagocytic Lymphohistiocytosis: Efficacy, Outcomes, and Survival-Insights From a Systematic Review.

Hemophagocytic lymphohistiocytosis (HLH) is a severe immunologic disorder that can be fatal if left untreated. The condition is characterized by excessive immune system activation and is often triggered by infections such as Epstein-Barr virus (EBV). Rituximab, an anti-CD20 monoclonal antibody, has been suggested as a treatment, particularly for EBV-associated HLH. A systematic review was conducted using PRISMA guidelines, with a literature search spanning PubMed, Scopus, Web of Science, and the Cochrane Library. The inclusion criteria focused on studies that assessed rituximab's efficacy in treating HLH. Quality assessment was performed using the Joanna Briggs Institute Critical Appraisal Checklist for Case Reports. Of 783 identified records, 24 studies were included in the final analysis. Rituximab was typically administered at 375mg/m2, with varying doses and treatment frequency. Clinical response, often seen within 1 month, was assessed by improvements in clinical symptoms and laboratory findings. Survival rates posttreatment displayed a wide range, with instances of complete remission and disease-free periods, as well as reports of relapse and mortality. Rituximab demonstrates the potential for significant clinical benefit in treating HLH, particularly when associated with EBV, showing promise in reducing disease activity and contributing to remission. These findings encourage further research and clinical trials to refine the therapeutic protocols and better understand the long-term effects of rituximab in HLH management.

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.

Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds,one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

Secondary hemophagocytic lymphohistiocytosis in pediatric patients with visceral leishmaniasis and Epstein-Barr virus infection.

Visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH) is indistinguishable from those of HLH of other etiologies due to the overlap symptoms, posing a serious threat to life. In this study, we aimed to provide insights for early diagnosis and improve outcomes in pediatric patients with VL-HLH. We retrospectively analyzed the clinical and laboratory data of 10 pediatric patients with VL-HLH and 58 pediatric patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The median time from symptom onset to cytopenia in patients with VL-HLH and EBV-HLH was 11 days (interquartile range, 7-15 days) and five days (interquartile range, 3.75-9.25 days) (P = 0.005). Both groups showed liver injury and increased lactate dehydrogenase levels; however the levels of aspartate aminotransferase, alanine aminotransferase, direct bilirubin, and lactate dehydrogenase in patients with VL-HLH were significantly lower than those in patients with EBV-HLH (P < 0.05). The fibrinogen and triglyceride levels were almost normal in VL-HLH patients but were significantly altered in EBV-HLH cases ( P < 0.05). The positive rate of first bone marrow microscopy examination, anti-rK39IgG detection, and blood metagenomic next-generation sequencing was 50%, 100%, and 100%, respectively. After VL diagnosis, eight patients were treated with sodium stibogluconate and two were treated with liposomal amphotericin B. All the patients with VL-HLH recovered. Our study demonstrates that regular triglyceride and fibrinogen levels in pediatric patients with VL-HLH may help in differential diagnosis from EBV-HLH. VL-HLH is milder than EBV-HLH, with less severe liver injury and inflammatory responses, and timely treatment with antileishmanial agents is essential to improve the outcomes of pediatric patients with VL-HLH.

Novel plasma microRNA expression features in diagnostic use for Epstein-Barr virus-associated febrile diseases

BackgroundEpstein-Barr virus (EBV) is widely infected in humans and causes various diseases. Among them, microRNAs of EBV play a key role in the progression of EBV-associated febrile diseases. There're few specific indicators for rapid differential diagnosis of various febrile diseases associated with EBV, and the lack of more reliable screening methods with high diagnostic utility has led to spaces for improvement in the accurate diagnosis and efficient treatment of relevant patients, making EBV infection a complicated clinical problem. With recent advances in plasma microRNA testing, the apparent presence of EBV microRNAs in plasma can help screen for EBV infection. The gene networks targeted by these microRNAs can also indicate potential biomarkers of EBV-associated febrile diseases. This study aimed to identify some novel miRNAs as potential biomarkers for early diagnosis of respectively EBV-associated febrile diseases. Materials and methodsA total of 110 participants were recruited for this task. First, we performed high-throughput sequencing and preliminary PCR validation of differentially expressed miRNAs in 15 participants with EBV-associated fever (divided into 5 groups, common EBV carriers, infectious mononucleosis (IM) and chronic active EBV infection (CAEBV), EBV-associated Hemophagocytic Lymphohistiocytosis (EBV-HLH), and 3 healthy individuals). After a comprehensive analysis, 10 miRNAs with abnormal expression were screened, and then qRT-PCR was performed in the rest of 95 participants to detect the validation of miRNAs expression in plasma samples. Thereafter, we further investigated their potential for clinical application in EBV-related febrile diseases by using a combination of Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and Protein-protein interaction network analysis. ResultsThrough identification and detailed analysis of the obtained data, we found significant differences in the expression of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p in blood samples from patients with different EBV-related febrile diseases. We found that the expression levels of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p in plasma are indicative of determining different disease types of EBV-related febrile diseases, while EBV-miR-BART22 and EBV-miR-BART2-3p may be potential therapeutic targets. ConclusionThe expression levels of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p suggest that they may be used as transcriptional features for early differential diagnosis of EBV-related febrile diseases, and EBV-miR-BART22 and EBV-miR-BART2-3p may be potential therapeutic targets.

Comparison of plasma proteomic profiles of patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis and infectious mononucleosis.

Primary Epstein-Barr virus (EBV) infection occasionally causes EBV-infectious mononucleosis (EBV-IM) and EBV-hemophagocytic lymphohistiocytosis (EBV-HLH). Although EBV-IM is mostly mild and self-limiting, EBV-HLH is a life-threatening disease characterized by excessive immune activation. However, the pathogenesis of EBV-HLH is yet to be fully elucidated. A diagnostic biomarker for EBV-HLH is desirable because early diagnosis and treatment are critical for the effective management of patients. In this study, the proteomic profiling of plasma was performed using liquid chromatography-mass spectrometry to identify proteins specific to EBV-IM and EBV-HLH. Furthermore, pathway analysis was performed for the proteins upregulated in patients with EBV-IM and EBV-HLH. Compared to healthy controls, 63 and 18 proteins were upregulated in patients with EBV-IM and EBV-HLH, respectively. Pathway and process enrichment analyses revealed that the complement system was the most enriched category of upregulated proteins in EBV-IM, whereas proteins related to immune effector processes were the most enriched in EBV-HLH. Among the 18 proteins upregulated in EBV-HLH, seven were exclusive to EBV-HLH. These specific proteins were associated with three pathways, and apolipoprotein E was commonly found in all the pathways. Proteomic analysis may provide new insights into the host response to EBV infection and the pathogenesis of EBV-related diseases.

Characteristics of Virology and Immune Inflammation of Epstein-Barr Virus Infection Related Non-Neoplastic Diseases in Children.

The goal was to study the difference of virological, immunologic, and inflammatory indicators between Epstein-Barr associated infectious mononucleosis (EBV-IM) and EBV associated hemophagocytic lymphohistiocytosis (EBV-HLH) and to explore the evaluation indicators for monitoring the therapeutic efficacy of EBV-HLH. Twenty children with EBV-IM (IM group) and 10 children with EBV-HLH (HLH group) were selected. Virology indicators were detected; the absolute count of lymphocyte, and lymphocyte subsets were detected; the levels of immunoglobulin and ferritin were assayed. Compared to the IM group, the HLH group showed a decrease in EBV-specific VCA-IgM antibody levels (U = 29.0, p = 0.006) and an increase in EBV-specific NA-IgG antibody levels (U = 17.0, p = 0.001), while there was no significant difference in EB-DNA loads (t = 0.417, p = 0.680). The counts of lymphocytes, and various lymphocyte subsets in the HLH group were lower than those in the IM group. Inflammatory markers in the HLH group were significantly higher than those in IM group. Dynamic monitoring of virological, immunological, and inflammatory indicators in HLH patients during treatment showed that EBV DNA gradually decreased in patients with good prognosis. Inflammatory indicators significantly decreased and returned to normal, lymphocyte count significantly increased and returned to normal during treatment. However, patients with poor prognosis showed rebound increase in EBV DNA and inflammatory indicators in the later stage of treatment, while lymphocyte count further decreased with the recurrence of the disease. Exhausted and damaged immune function in host by persistent stimulation of EB viral antigen is one of the main pathogeneses of EB-HLH. Lymphocyte count and serum ferritin level are effective indicators to monitor the therapeutic efficacy during the treatment to HLH.

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.

Single-Cell RNA Sequencing Reveals Contradictory Immune Microenvironment in Hemophagocytic Syndrome

Hemophagocytic lymphohistiocytosis (HLH), a formidable and rare clinical syndrome impacting multiple organs, manifests as a perilous condition. However, the intricate pathophysiological mechanisms underlying HLH remain yet to be fully elucidated. In this study, we obtained peripheral blood samples from 9 HLH patients spanning the years 2018 to 2022. Employing single-cell RNA sequencing (scRNA-seq), we conducted a comprehensive analysis of the alterations in the immune microenvironment, delving into the role of EBV infection in HLH and its influence on pathogenesis. By comparing with health donor peripheral blood , we identified a total of 11 significant immune cell populations among 72,779 single-cell transcriptomes, comprising 20,337 cells from healthy controls and 52,442 cells from HLH patients. Notably, we found that the proportion of T cells increased significantly, and other immune cells decreased in HLH patients. In terms of function, we found that the overall T cell killing ability was weakened, and the factor secretion ability was enhanced. Concerning myeloid cells, the impact of CD16+ monocytes on T cells exhibited a pronounced increase, along with an enhanced ability to secrete IL-18 factors, potentially serving as the main source of cells promoting T cell function. Different subpopulations of T cells have significant changes in the pathogenesis of HLH. Remarkably, both CD4+ T cells and CD8+ T cells exhibited HLA-DR+GZMK+ cell subgroups with upregulated IFN-γ production and response, suggesting their association with the hyperinflammatory state in HLH. A correlation analysis between the proportion of HLA-DRA+ GZMK+ T cells and the severity score revealed a significant correlation (p&amp;lt;0.05), with the ROC curve further affirming their predictability in assessing high inflammatory activity. Among CD4+ T cells, the proportion of Treg cells and the secretion of inhibitory factors such as TGFb1 decreased. Additionally, we noted the presence of a functionally impaired subset of CBLB+CD8+ T cells, indicating the considerable variability in T cell subset functionality in HLH. Next, a comprehensive analysis of the alterations in myeloid cells, B cells, and NK cells in HLH patients was conducted. We observed a substantial reduction in the number of DC cells and immune-related functions within various myeloid cell subsets. Regarding B cells, an increase in anergy B cells was observed, whereas the population of plasma cells, an effective B cell type, exhibited a decrease. In the context of NK cells, there was a significant increase in the proportion of NK cell subsets associated with IFN-γ secretion. Moreover, EBV-infected T cells in EBV-associated HLH were identified as the principal source of IFN-γ. Through single-cell EBV-CALL analysis, we found that IFN-γ secretion was primarily attributed to EBV-infected T cells, subsequently influencing CD16+ monocytes. Additionally, IL-16 production was predominantly observed in EBV-infected CD16+ monocytes, thereby impacting lymphocytes. Moreover, Monocytes were stimulated by EBV-infected CD4+ T cells and Atypical T cells, leading to the secretion of a pro-inflammatory factors such as MIF. Furthermore, our investigation unveiled that EBNA2 could promote MYC into the nucleus and enhance the proliferation and IFN-γ secretion of T cells. In conclusion, our comprehensive analysis of the immune microenvironment changes following the onset of HLH has yielded valuable insights and novel perspectives for understanding the underlying mechanisms and potential clinical treatments of this condition.

Clinical and Pathological Characteristics of Severe EBV Infection in Japanese Children; a Disease Spectrum from Acute Liver Failure to Hemophagocytic Lymphohistiocytosis

Abstract Background Epstein-Barr virus (EBV) is a ubiquitous herpesvirus known as a cause of infectious mononucleosis (IM). While most EBV infection is mild and self-limited, severe EBV diseases such as severe IM, EBV-associated hemophagocytic lymphohistiocytosis (HLH) and EBV-associated acute liver failure (ALF) may occur on rare occasions. Interestingly, the severe EBV disease with ALF may occasionally present with overlapping features of HLH; however, it remains unknown if the presence or absence of HLH reflects a distinctive underlying pathophysiology of EBV infection. To investigate the clinical and pathological characteristics of EBV-associated ALF among children, we conducted a single-center, retrospective study at the largest tertiary children’s hospital in Japan. Methods We enrolled children under 18 years old who required medical management in the intensive care unit due to primary EBV infection between January 2010 and April 2021. Patients with underlying immunodeficiencies and other medical conditions were excluded. Clinical data, type of EBV-infected cell lineage, and pathological exam were reviewed. Fourteen patients with EBV-associated ALF were identified in the initial screening, and two were excluded (due to underlying medical conditions). Thus, the details of 12 patients were further analyzed. Five patients fulfilled the criteria for HLH, while seven presented as isolated ALF only. Results The most common symptoms of these patients include fever (92%), vomiting (58%), and consciousness disturbance (100%). EBV loads were higher when HLH co-existed (median 2,700 copies/μg of DNA vs. 130,000 copies/μg of DNA, p = 0.01). Identification of EBV-infected cells revealed that the most common cell type was CD8+T cells (8/12, 80%), followed by CD3+T cells (1/12, 10%), and CD19+ cells (2/12, 20%). EBV infected in both CD8+T and CD19+ cells in one case and EBV-infected cells were not identified in two cases. In addition, an increase of activated CD8+ T cells (CD3+CD8+HLA-DR+) was observed in both HLH and ALF cases. Among 12 patients with ALF, seven patients underwent life-saving liver transplantation (LT) due to ALF. Of five patients with ALF+HLH, all patients received corticosteroid, four received cyclosporin or etoposide, and one underwent hematopoietic stem cell transplantation (HSCT). Overall mortality is 17% (2/12 patients), and both patients were complicated with HLH. Conclusion This study suggests that HLH and ALF can be an indistinguishable spectrum of severe EBV disease, and the underlying pathogenesis may be associated with the unusual type of EBV-infected cells. Hence, the identification of EBV-infected cells should be considered to guide the diagnosis and treatment. Our study also revealed that LT and cytotoxic anti-inflammatory therapy effectively treated patients with EBV-associated ALF with or without HLH, given the high mortality (&amp;gt;70%) of this disease spectrum. Further studies investigating EBV-associated ALF pathology are warranted to determine optimal management.

Comparative study of biomarkers for the early identification of Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis in infectious mononucleosis

Background and aimEpstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and infectious mononucleosis (EBV-IM) share mimic symptoms in the early stages of childhood development. We aimed to examine the clinical features and laboratory indices of these two diseases in children and uncover unique indicators to assist pediatricians in identifying these diseases early.MethodsWe collected clinical data from 791 pediatric patients diagnosed with EBV-IM or EBV-HLH, compared the clinical traits and laboratory biomarkers presented in the two groups, and constructed predictive models based on them.ResultsPatients with EBV-IM had greater ratios of cervical lymphadenopathy, eyelid edema, and tonsillitis, whereas individuals with EBV-HLH were more likely to have hepatomegaly and splenomegaly. When using the criteria of interleukin (IL)-10 > 89.6 pg/mL, interferon (IFN)-γ > 45.6 pg/mL, ferritin > 429 μg/L, D-dimer > 3.15 mg/L and triglycerides > 2.1 mmol/L, the sensitivity was 87.9%, 90.7%, 98.1%, 91.1% and 81.5% to predict EBV-HLH, while the specificity was 98.4%, 96.3%, 96.5%, 94.1% and 80.6%, respectively. A logistic regression model based on four parameters (IL-10, ferritin, D-dimer, and triglycerides) was established to distinguish EBV-HLH patients from EBV-IM patients, with a sensitivity of 98.0% and a specificity of 98.2%.ConclusionsIL-10, IFN-γ, ferritin and D-dimer levels are significantly different between EBV-HLH and EBV-IM. Predictive models based on clinical signs and laboratory findings provide simple tools to distinguish the two situations.

Chronic active Epstein-Barr virus disease originates from infected hematopoietic stem cells

AbstractChronic active Epstein-Barr virus (EBV) disease (CAEBV) is a lethal syndrome because of persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Bone marrow and peripheral cells derived from 5 patients with CAEBV, 1 patient with EBV-associated hemophagocytic lymphohistiocytosis, and 2 healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative polymerase chain reaction, PrimeFlow, and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between patients with CAEBV and controls, and between infected and uninfected cells. One patient with CAEBV was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again 6 months after HSCT. EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, as well as the hematopoietic stem cells (HSCs) of the patients with CAEBV. EBV-infected HSCs exhibited a higher differentiation rate toward 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. Taken together, multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected HSCs, which might potentially lead to innovative therapy strategies for CAEBV.

An analysis of sintilimab combined with ruxolitinib as compassionate therapy for 12 adults with EBV-associated hemophagocytic lymphohistiocytosis.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a severe hyperinflammatory illness that affects adults and is caused by an EBV infection. Without allogeneic hematopoietic stem cell transplantation (allo-HSCT), the overall survival of adult patients with EBV-HLH is unsatisfactory, necessitating the development of innovative therapeutic approaches. The clinical records of twelve EBV-HLH patients who received sintilimab therapy combined with ruxolitinib on a compassionate basis at the First Affiliated Hospital of Soochow University were retrospectively examined in this investigation. All the patients responded without fever, but three patients relapsed within a week. Among the nine patients achieving complete response (CR), 55.6% (5/9) maintained CR for >4.5 months, and 33.3% (3/9) relapsed following CR. Neither patients with no response (NR) nor relapsed patients were fit for allo-HSCT, and all died soon after discharge. Six patients had clinical CR with a median follow-up of 5 (4.4-14.7) months. There were no documented severe negative effects. Additional information on this innovative treatment for adult EBV-HLH is provided in our report.

Sintilimab treatment for chronic active Epstein–Barr virus infection and Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis in children

BackgroundChronic active Epstein–Barr virus infection (CAEBV) and Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) are rare but life-threatening progressive diseases triggered by EBV infection. Glucocorticoid/immunosuppressants treatment is temporarily effective; however, most patients relapse and/or progress. Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy; however, there are risks of transplantation-associated complications. Currently there is no standard treatment for CAEBV and EBV-HLH. Programmed death protein 1 (PD-1) inhibitors have achieved a high response in many EBV-related diseases. Sintilimab (a recombinant human IgG4 monoclonal antibody against PD-1) disrupts the interaction between PD-1 and its ligand, leading to T cell reinvigoration.MethodsA retrospective analysis was performed on three children with CAEBV or EBV-HLH in the Children’s Hospital of Soochow University between 12 December 2020 and 28 November 2022. The efficacy of sintilimab was evaluated.ResultsThree patients, including two males and one female, were analyzed. Among them, two children were diagnosed with CAEBV with intermittent fever for more than four years, and one child was diagnosed with EBV-HLH. After sintilimab treatment and a mean follow-up of 17.1 months (range 10.0–23.3 months), patients 1 and 3 achieved a complete clinical response and patient 2 achieved a partial clinical response. All three children showed a > 50% decrease in EBV-DNA load in both blood and plasma. EBV-DNA copies in sorted T, B, and NK cells were also markedly decreased after sintilimab treatment.ConclusionOur data supported the efficacy of PD-1 targeted therapy in certain patients with CAEBV and EBV-HLH, and suggested that sintilimab could provide a cure for these diseases, without HSCT. More prospective studies and longer follow-up are needed to confirm these conclusions.