In recent years, tumor microenvironment immune cells have been analyzed in various types of malignancies, and the characteristics of the pathological conditions related to tumor progression and treatment resistance have been clarified.20 EBV-T/NK-LPD is an extremely rare disease, and few reports have been published regarding tumor environmental immune cells. Therefore, we investigated the differences in gene expression among the three groups of EBV-T/NK-LPDs. As a result, EBV-related gene expression levels were similar in the comparative analysis of the three groups, supporting the previous report.10 However, examination of infiltrating immune cells suggested that systemic CAEBV disease might have a unique pattern compared with other related diseases. These results suggest that the presence of host immune abnormalities has been assumed.21,22
In our analysis, the differences in Tfh cell- and B-cell-related gene expressions were observed in the systemic CAEBV disease group, suggesting a relationship between the two cell types. A statistically significant increase in the number of CD20-positive cells was also observed in the group with high B-cell and Tfh cell-related gene expression levels, suggesting that B-cell gene expression levels are clinically significant in predicting the prognosis of systemic CAEBV disease. There have been several study reports on the relationship between Tfh and B cells.23 Tfh cells have a number of functions necessary to support the work of B cells within the germinal center.24 The proliferation and differentiation of B cells induced by Tfh cells producing cytokines, such as IL-21, and CD40-mediated differentiation and class switching of B cells by Tfh cells expressing CD40L, which is a member of the TNF family, have been reported.25 Considering these reports, abnormal Tfh cell-related gene expression may explain abnormal immunoglobulin productions and autoimmune disease-like symptoms during the exacerbation of clinical symptoms in systemic CAEBV disease. Furthermore, the group with increased CD20-positive cells had higher EBV-related gene expression than the group without increased CD20-positive cells. Survival analysis demonstrated a significantly poorer prognosis in the group with a higher number of CD20-positive cells than in the group with a lower number of CD20-positive cells. In addition, patients with a higher number of CD20-positive cells had increased number of EBV-positive B cells. As biopsies are often performed at the time of exacerbation of clinical symptoms, these results suggest that the expansion of EBV-positive B cells might be observed during the exacerbation period, resulting in poor prognosis. A previous study reported a decrease in the number of EBV-specific CTLs in systemic CAEBV disease26, suggesting that EBV cannot be eliminated owing to some reasons at the time of exacerbation. Details of the subtypes of Tfh cells have recently been reported. Follicular regulatory T helper cells are involved in B-cell differentiation, and follicular cytotoxic T helper cells are involved in viral clearance.27,28 These Tfh subtype dysfunctions for eliminating EBV may exist in systemic CAEBV disease.
In addition, our study showed that the LMP1 gene expression level was associated with CD20-positive B-cell count in systemic CAEBV disease, in contrast to the EBNA1 gene expression level. LMP1 in EBV-infected cells increases IL-6, granulocyte-macrophage colony-stimulating factor (CSF), and IL-1β expression levels through increased glycolysis, and these cytokines induce expansion of MDSC that suppresses T, NK, and other myeloid cells in peripheral blood mononuclear cell samples.29,30 However, our result showed that there was no correlation between LMP1 gene expression levels and MDSC-related gene expression levels. The immune cell environment between peripheral blood and tissue lesions may differ; however, further experiments on the roles of MDSC within lesions in systemic CAEBV disease are necessary. Tfh-cell/B-cell gene and LMP-1 expression levels may be related to the mechanisms of EBV-infected B-cell expansion by inhibiting normal immune reactions, which cause unique clinical symptoms and worse prognoses of systemic CAEBV disease. However, further studies are required to confirm these findings.
Gene expression levels in systemic CAEBV disease and EBV-associated T-/NK-cell lymphoma (ENKTL and EBV-nodal T- and NK-cell lymphoma) were also compared in this study. Most of these diseases are currently difficult to diagnose only by histopathological evaluation without information on clinical courses or situations.31 We performed a comparative analysis of immune cell-related genes among the three groups and extracted statistically significant genes in our cohort. In the systemic CAEBV disease cohort, T-cell acute leukemia protein 1 (TAL1), cluster of differentiation 36 (CD36), interleukin-34 (IL-34), cathepsin G (CTSG), and complement factor D (CFD) were the most variable genes, none of which was reported to be expressed in the microenvironment analyses of EBV-T/NK-LPD. TAL1 has been reported to be overexpressed in 30–40% of pediatric T-cell acute lymphoblastic leukemia.32 CAEBV is frequently observed in children; thus, it may have some relationship with pathogenic mechanisms. The mechanism by which CD36 overexpression in CTL induces a decrease in cytotoxic cytokines is known and may be involved in the decreased function of EBV-specific CTL.33 In addition, IL-34 is a cytokine involved in monocyte activation and binds to CSF-1, nuclear factor kappa B (NF-kB), phosphatidylinositol 3kinase/protein kinase B, p38 mitogen-activated protein kinase, extracellular signal-regulated protein kinases 1 and 2, c-Jun N-terminal kinase, Janus kinase (JAK), signal transducer, and activator of transcription 3 (STAT3).34–38 As NF-kB signaling and JAK-STAT signaling activation have been reported in CAEBV cell-line experiments, our results may suggest that these signaling pathways may be related with the development of systemic CAEBV disease.39, 40 CTSG is involved in systemic inflammation and autoimmune diseases and is possibly involved in the clinical symptoms of systemic CAEBV disease.41 As described above, top fluctuate genes may explain the unique symptoms. It will be necessary to investigate in detail whether these genetic abnormalities are actually involved in systemic CAEBV disease and whether they can serve as differential markers for EBV-T/NK-LPDs.
In contrast, our results did not show any trend toward differences in EBV-associated gene expression among EBV-T/NK-LPDs. Host-side genetic abnormalities, including mainly DDX3X, and EBV-side genetic abnormalities, especially BERT gene deletion, are commonly observed in EBV-associated malignant lymphomas with high frequencies.10 Furthermore, abnormalities such as EBV-related cytokine BALF are also involved in tumor progression.10 However, EBV-related gene expression levels are not useful for differentiating EBV-related malignant lymphoma based on our finding that there is no difference in EBV-related gene expression.
In this study, we performed a comparative analysis of microenvironmental immune cell- and EBV-related genes in significantly rare diseases, including systemic CAEBV disease and EBV-associated T-/NK-cell lymphoma (ENKTL and EBV-nodal T- and NK-cell lymphoma). Although there are few reports on the analysis of tumor-infiltrating cell-related genes in EBV-T/NK-LPD, such as systemic CAEBV disease, ENKTL, and EBV-positive nodal T- and NK-cell lymphoma, our study was able to clarify the abnormalities of gene expression and the differences in gene expression of EBV-positive T/NK cell malignant lymphoma. Owing to the extremely rare nature of these diseases, the analysis results could not be fully validated. To establish better diagnostic and therapeutic methods, it is necessary to accumulate data and specimens to clarify the mechanisms of systemic CAEBV disease and EBV-positive T-/NK-cell lymphoma.