CAEBV infection is characterized by clonal proliferation of EBV-infected T cells or NK cells in East Asian countries. It is categorized as an EBV-positive T/ NK cell tumor in the 2016 revision of the WHO classification of hematopoietic and lymphoid tissue tumors, generating widespread interest. The pathogenesis of CAEBV infection is still unclear, and it has been suggested that two factors may be present: the characteristics of the virus and the immune deficiency of the host.15 Although EBV can contribute to the development of CAEBV infection by expressing various proteins that interfere with the secretion of cytokines and inhibit apoptosis of infected cells, the prevalence of EBV infection in the population is high. In contrast, the incidence of CAEBV infection is low. Thus, a specific EBV strain may cause CAEBV infection with high virulence or a unique immune escape mechanism. Studies have identified EBV strains with high lytic replication rates.16,17 Yet, no CAEBV infection outbreaks or household transmission have been detected to date, suggesting that host immune factors rather than viral factors may play a key role in CAEBV infection.
CAEBVH is an essential clinical phenotype of CAEBV infection. Clinical observation found that patients of CAEBVH had more apparent symptoms, faster progression, and poorer prognosis than patients without liver damage.18 Therefore, this study analyzed the clinicopathological characteristics and investigated the mechanism of CAEBVH.
In our study, clinical features mainly included abnormal liver function (9/10), hepatomegaly (9/10), fever (7/10), splenomegaly (10/10), and elevated EBV-DNA viral load. This was mainly following T cell type, characterized by high fever, enlarged liver or spleen or lymph nodes, and EBV antibody-specific change. As we know, CAEBV infection can understand inflammatory diseases that lead to HLH, and ferritin is an indicator. Ferritin is closely related to the progression and regression of disease conditions. Some patients had elevated ferritin (5/7) and progressed to HLH (4/7) in our study. In our study, 4 patients who had HLH with high ferritin died or recurred.
There are three main features in the liver histopathological examination. One is that inflammatory changes with steatosis in patients with CAEBVH. Secondly, the inflammatory cells, mainly lymphocytes, infiltrated the confluent area and hepatic sinusoids. Thirdly, EBER-ISH was positive in these lymphocytes. It was shown not EBV but lymphocytes caused liver damage in CAEBVH, unlike viral hepatitis, B. And consistent with clinical features, T cells are the target cells of EBV infection.
Immune cell phenotyping showed decreased CD8+T lymphocytes, suggesting that CD8+ T lymphocytes may play an essential role in CAEBV infection. To explore the definite mechanism of CAEBVH, we detected mutations in host immune genes using whole-exome sequencing. Our study showed mutant genes were primarily enriched in 'T cell activation'. We further found more CD8+T lymphocytes in the CAEBV infection group than in the control group. However, no difference between CD3+T, CD4+T, CD20+B lymphocytes, and CD56+NK cells. In the present research, reduced number and impaired function of EBV-specific CD8+T lymphocytes were also found in CAEBV hosts. 19,20
CD8+T lymphocytes are essential immune cells in the body and play a critical role in fighting virus infection in the host.21 It was reported that the perforin/granzyme B and the Fas/FasL apoptosis played a vital role in CD8+T lymphocytes.22–25 The TUNEL assay showed distinct apoptosis in the CAEBVH group than controls in our study. Perforin and granzyme B induced target cell death via non-caspase-dependent apoptotic pathway.26 And it was found perforin gene mutations in a patient with CAEBV infection.27 But our study found the expression of granzyme B and perforin had no significant difference between the two groups, and there were no perforin gene mutations. Hence, perforin and granzyme pathways need to be furth confirmed.
We further investigated the role of the caspase-dependent apoptotic pathway in CAEBVH. It was showed that Fas, FasL and caspase-8 were highly expressed in the CAEBVH group. Nomura et al.28 found caspase-3, critical apoptosis pathway proteins, highly expressed in CAEBV patients. When Fas of lymphocytes combined with FasL of tissue cells, activating caspase-8 of tissue cells to conduct caspase-dependent apoptosis pathway.29,30 In our study, high expression of caspase-8 in lymphocytes suggested lymphocytes were undergone excessive apoptosis due to abnormal proliferation.31,32 Activated caspase-8 also promoted the secretion of the inflammatory cytokine to cause further tissue damage.33 Keiko Nomura et al.34 had identified three Japanese patients with CAEBVH who had original Fas pathway-associated mutations. Thus, gene mutations associated with the Fas/FasL pathway may lead to CAEBVH; however, whether the mutations were primary or secondary need further studies.
KEGG enrichment suggested the complement activation may be a possible mechanism of CAEBVH. C3 can activate CD8+T cells and promote proliferation.35 C1q was activated when it was bound to the immune complex and subsequently initiated the classical complement pathway. The classical pathway and lectin pathway activated C4d. And C3d was engaged in the three pathways, including classical, lectin and alternative pathways.36 Our study found the expression of C1q in LSECs and GC was higher in the CAEBVH group than in the controls. C3d in the LSECs was higher in the CAEBVH group. No C4d expression was observed in either group, considering that the alternative pathway, not the lectin and classical pathway, might be an essential pathogenic mechanism of CAEBV infection. Owing to C4d is a marker of B-lymphocyte immune response, such as immune complexes and antibodies. It was reflected the T-lymphocyte rather than the B-lymphocyte involved in the mechanism of CAEBVH. The imbalance of C3-derived fragments was the key to autoimmune and neoplastic diseases.37,38 Therefore, we inferred that the complement pathway could be involved in CAEBVH, which might help drug development efforts.
In summary, we hypothesize that EBV infection of lymphocytes leads to Fas/FasL and complement activation in CAEBH, which causes a storm of inflammatory cytokine and neoplastic diseases. Therefore, CAEBV infection treatment requires control of inflammation and immune cell proliferation. Standard therapies are antiviral and immunosuppression. Although patients (5/10) received the antiviral and immunosuppressive treatment, they still died with a median survival time of 3.0 (1.5, 4.0) months in our study. Some reports showed that allogeneic hematopoietic stem cell transplantation (allo-HSCT) could cure CAEBVH 39. In other words, immune replacement and reconstitution played a vital role in CAEBVH. It is implied gene mutations in host may be the essential reason. Therefore, targeted gene therapy may have implications for clinical treatment.