Unstable EBV latency drives inflammation in multiple sclerosis patient derived spontaneous B cells

Abstract

Epidemiological studies have demonstrated that Epstein-Barr virus (EBV) is a known etiologic risk factor, and perhaps prerequisite, for the development of MS. EBV establishes life-long latent infection in a subpopulation of memory B cells. Although the role of memory B cells in the pathobiology of MS is well established, studies characterizing EBV-associated mechanisms of B cell inflammation and disease pathogenesis in EBV (+) B cells from MS patients are limited. Accordingly, we analyzed spontaneous lymphoblastoid cell lines (SLCLs) from multiple sclerosis patients and healthy controls to study host-virus interactions in B cells, in the context of an individual's endogenous EBV. We identify differences in EBV gene expression and regulation of both viral and cellular genes in SLCLs. Our data suggest that EBV latency is dysregulated in MS SLCLs with increased lytic gene expression observed in MS patient B cells, especially those generated from samples obtained during "active" disease. Moreover, we show increased inflammatory gene expression and cytokine production in MS patient SLCLs and demonstrate that tenofovir alafenamide, an antiviral that targets EBV replication, decreases EBV viral loads, EBV lytic gene expression, and EBV-mediated inflammation in both SLCLs and in a mixed lymphocyte assay. Collectively, these data suggest that dysregulation of EBV latency in MS drives a pro-inflammatory, pathogenic phenotype in memory B cells and that this response can be attenuated by suppressing EBV lytic activation. This study provides further support for the development of antiviral agents that target EBV-infection for use in MS.

Keywords: Epstein-Barr Virus; Multiple Sclerosis; latency; lymphoblastoid cell lines (LCLs); lytic activation.

Figure 1. Generation and growth of SLCLs from the PBMC of MS patients and healthy controls (HC).

(A) SLCLs were generated from PBMCs isolated from MS patients and healthy controls by extended culture of PBMCs without addition of exogenous, lab strain EBV. Three weeks after ex vivo culture, cyclosporin A was added to eliminate residual T -cells. (B) cell viability in SLCLs, LCLs (B95.8), and EBV (−) BJAB cells measured by Cell Titer-Glo^® Cell luminescent Cell Viability Assay (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA followed by Tukey’s multiple comparison test). (C) Proliferation index in SLCLs, LCLs (B95.8), and EBV (−) BJAB cells measured by CFSE (*p<0.05, **p<0.001; one-way ANOVA followed by Tukey’s multiple comparison test). (D) Viability of long-term culture of AMS SLCLs compared to HC and SMS SLCLs, LCLs (B95.8), and EBV (−) BJAB cells (**P=0.0023, Log-rank Mantel-Cox test).

Figure 2. Evidence of increased lytic activity in SLCLs from AMS patients.

(A) EA-D and Zta expression in AMS and SMS by flow cytometry (B) quantitation of EAD and Zta expression by flow cytometry (*p<0.05; one-way ANOVA followed by Tukey’s multiple comparison test) (C) Western blot of EBV latent (EBNA1, EBNA2, LMP1, EBNA3C) and lytic (Zta and EA-D) genes relative to β-actin. (D) Expression of EBV lytic (LC3, Zta) and latent (EBNA1, LMP1) genes by RT-qPCR.

Figure 3. Whole genome NGS sequencing of endogenous EBV of SLCLs from AMS, SMS, and HC.

(A) Copies of EBV genome per 1 M reads. (B) heterogeneity of EBV sequences found within samples from AMS, SMS, and HC. (C) AMS, SMS, and HC endogenous EBV aligned to the wild-type EBV genome (NC_007605.1). (D) Protein coding variations in EBNA3A identified in MS patients.

Figure 4. Heterogeneity in oriP and impaired EBNA1 binding in SLCLs from MS patients.

(A) Heterogeneity in oriP visualized by the number of mutations compared to the reference. (B) Sequence alignment comparing variations in region 7780-7816 in oriP.(C) Phylogenetic analysis of OriPform SLCLs and other EBV associated diseases, including: infectious mononucleosis (IM), diffuse large B-cell lymphoma (DLBL), NK/T lymphoma, chronic active EBV (CAEBV), eBL (endemic Burkitt’s lymphoma), nasopharyngeal carcinoma (NPC), gastric cancer (EBVaGC), post-transplant lymphoproliferative disorder (PTDL), post-transplant B-lymphoma (PTBL). The oriP protein sequences for each category are shown in the upper panel. (D) ChIP assay for EBNA1 binding to the DS, Qp, and cellular locus CLIC1 in SLCLs. P values were determined for three biological replicates (***P < 0.001, ** P<0.01, *P<0.05; Two-way ANOVA). Immunoprecipitation was performed with IgG as a control (not shown).

Figure 5. Increased inflammation in SLCLs generated from AMS patients.

(A) RNA-seq showing top cellular genes that are upregulated (red) or downregulated in AMS, SMS, and HC SLCLs. (B) RT-qPCR for BIRC3, FOXP1, CD44, and HAVCR2. (C) Ingenuity pathway analysis showing top regulators of transcription and pathways that are upregulated or downregulated in AMS SLCLs compared to HC SLCLs. (D) RT-qPCR of IL-12β and LTA E) Intracellular cytokine staining for IL-6, GMCSF, LTA, and IL-10 in AMS, SMS, and HC SLCLS. (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA followed by Tukey’s multiple comparison test).

Figure 6. Tenofovir alafenamide (TAF) decreases EBV lytic activity and inflammation in SLCLs from MS patients.

TAF (5μM) was added to SLCLs and LCLs (B95.8) for 72 hours with medium changed daily. (A) Cell death (% live) in cells treated with 12.5 or 25 μg/ml GCV with medium changed twice per day for four days. (B) EBV viral load in LCLs and SLCLs treated with TAF. (D) IL-6 and LTA expression by intracellular cytokine staining. (C) Zta expression by RT-qPCR. (D) EA-D expression by RT-qPCR. (F-G) Cytokine production during a mixed lymphocyte reaction as measured by EliSPOT. (**p<0.01, ***p<0.001; one-way ANOVA followed by Tukey’s multiple comparison test)