The Potential of Plasma CXCL10 As a Biomarker That Reflects Pathogenesis and Prognosis in Systemic Chronic Active EBV Disease

Background and Purpose:

Systemic Chronic Active Epstein-Barr Virus disease (sCAEBV) is a refractory disease characterized by the activation and clonal proliferation of EBV-infected T or NK cells, leading to a progressive and fatal course. The primary clinical feature is persistent systemic inflammation. Its only curative treatment is hematopoietic stem cell transplantation (Blood Adv. 2020;4:2918), but following disease activities of inflammatory symptoms are poor prognostic factors after the treatment: fever, elevated ALT, vasculitis, uveitis, and progressive skin lesions. Elucidating the mechanisms behind the onset and the control of these inflammatory symptoms is essential to establish effective treatments. The aim of this study is to identify cytokines that contribute to the pathogenesis of sCAEBV and serve as biomarkers.

Materials and Methods:

The diagnosis of sCAEBV was made based on the diagnostic criteria in accordance to the WHO classification. Cytokine multiplex analysis was performed using the Human Cytokine/Chemokine Panel 38 Plex (EMD Millipore) following the manufacturer’s instructions. Single-cell RNA sequencing of peripheral blood mononuclear cells was performed using a DNBSEQ-G400 sequencer. Microarray analyses were used to examine gene expression variations in patient cells’ pre- and post-CXCL10 stimulation with 3D-Gene Human Oligo chip 25k. The probabilities of overall survival (OS) were visualized by the Kaplan-Meier method, and the comparisons were made using a log-rank test.

Results:

We comprehensively analyzed the concentrations of 38 inflammatory cytokines in the plasma of 19 patients with sCAEBV and 47 healthy individuals. Compared to healthy individuals, CXCL10 was identified as a cytokine with significantly higher concentrations in sCAEBV patients. The peripheral blood mononuclear cells (PBMCs) of sCAEBV patients contain EBV-infected T- or NK-cells. To identify the cells producing CXCL10, we performed single-cell RNA sequencing analysis on the PBMCs of five sCAEBV patients whom we could obtain sufficient samples from. CXCL10mRNA was strongly expressed in monocytes. Meanwhile, its receptor, CXCR3, was highly expressed in the fraction of EBV-infected T- or NK-cells of patients. We also confirmed the expression of CXCR3 in EBV-infected cells from patients by flow cytometry. To investigate the effect of CXCL10 on EBV-infected cells, we stimulated sCAEBV-derived cell lines (Br J Hem. 2003:21;805) with CXCL10 and observed the changes. Stimulating at the concentrations corresponding to those in the patient's blood did not affect the proliferation or cell death of the sCAEBV cell lines. However, microarray analysis before and after the stimulation showed significant changes in genes related to cell adhesion. Finally, to verify the significance of CXCL10 as a biomarker, we measured its concentrations before and after chemotherapy. In patients whose disease activity decreased after treatment, CXCL10 levels significantly decreased. Moreover, patients with high levels of CXCL10 at diagnosis resulted in significantly low overall survival rates. Additionally, cases with high levels of plasma CXCL10 (> median) at diagnosis resulted in poorer OS compared to those with low levels (p < 0.05).

Conclusion:

CXCL10 has a potential to be a biomarker that reflects treatment efficacy and prognosis of sCAEBV. We need further validation in a larger number of cases and a proof of its role in the pathogenesis to confirm its potentiality.