High‐throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV‐reactive CD8⁺ T cells

Abstract

Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS).Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBVreactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8 + T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4 + T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8 + T cells in MS. Keywords:Cerebrospinal fluid r Epstein-Barr virus r High-throughput sequencing r MS r TCR Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Andreas Lossius e-mail: andreas.lossius@rr-research.no * These authors contributed equally to this work as senior authors. Eur. J. Immunol. 2014. 44: 3439-3452 Introduction T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS) [1]. The T cells access the central nervous system (CNS) via the cerebrospinal fluid (CSF) or cross the blood-brain barrier through the perivascular spaces, which communicate with the CSF [2]. The CSF is also contiguous with the extracellular fluid of the brain and is believed to reflect inflammation within the CNS better than blood [3]. Although moderately increased in MS [4], the low numbers of T cells in CSF have complicated the characterization of these cells. Thus, the clonal composition of T cells in the CSF and their relationship to T cells in the blood is poorly understood. Cloning and sequencing of individual T-cell receptors (TCRs) [5], spectratyping [6,7], or flow cytometric staining for TCR variable (V) β-chain families [8] has previously permitted mapping of only a narrow part of the intrathecal TCR repertoire. These studies have yielded partly conflicting results with respect to clonal diversity and TCRV β-chain usage, and the overlap between the T-cell populations in CSF and blood has not been quantified. Epstein-Barr virus (EBV) has consistently been associated with MS in epidemiological studies [9], and MS patients display a perturbed immune respo...

EBV and Autoimmunity

“…There is thus a convergence of epidemiological, clinical, and immunological evidence that anti-EBNA1 antibodies and CD4+ positive EBNA1-specific T cells cross-reacting with myelin antigens contribute to the pathological process in MS. CD4+ T cells recognizing other EBV antigens and myelin epitopes have also been reported in blood (Lang et al 2002) and the cerebrospinal fluid (Holmoy and Vartdal 2004). These findings do not exclude an important role for EBV-specific CD8+ T cells, although comparisons of their frequency and function in blood and cerebrospinal fluid (CSF) from individuals with MS and matched healthy controls have given somewhat mixed results, which could be in part attributable to methodological differences (Hollsberg et al 2003;Gronen et al 2006;Jilek et al 2008Jilek et al , 2012Jaquiery et al 2010;Angelini et al 2013;Pender et al 2014a;Lossius et al 2014). B cells must also play an important role, either through their antigen-presentation activity or other mechanisms.…”

Exploring the origins of grey matter damage in multiple sclerosis

“…Several epidemiological studies have suggested that Epstein-Barr virus (EBV) is one of the strongest candidates for this infectious agent [56][57][58] and EBV proteins and RNA have been detected in B-cells in the meninges and perivascular spaces of MS cases with extensive meningeal infiltrates and cortical demyelination [59][60][61] . It has been proposed that failure to control latent EBV infection in an immune privileged site, such as the subarachnoid space, could lead to recurrent intrathecal reactivation of EBV and tissue damage in the nearby grey matter 62,63 . However, a number of other studies have been unable to detect EBV in the brain or lesions of MS patients 64,65 and this remains a highly debated controversy 66 .…”

Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire

“…There have been several reports of CDR3 motifs associated with pathogens such as Clostridium tetani (toxoid), Candida albicans, Mycobacterium tuberculosis, HSV, CMV, EBV, and influenza A (32)(33)(34)(35)(36)(37)(38)(39)(40). We investigated the overlap of the signature CDR3 motifs with these pathogen-associated motifs for TCRβ (Dataset S3).…”