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Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS). While T cells dominate the immune response, both B and T cells contribute to the demyelination and neuronal injury that underlies clinical disease manifestation. Although MS is widely regarded as an autoimmune disease, the exact disease aetiology remains unclear and both genetic and environmental factors contribute to disease susceptibility (1). The traditional view of MS pathogenesis is that an environmental factor triggers the peripheral activation of T cells cross-reactive for an antigen in the CNS in genetically susceptible individuals. These T cells then migrate to the CNS where they promote neuronal injury by the release of cytotoxic or inflammatory mediators (2). B cells in turn, may contribute to disease pathogenesis via the production of soluble mediators and/or by endorsing the activation of T cells (3). The local inflammatory milieu in the CNS also promotes the
accumulation of bystander lymphocytes, which may contribute to neuronal injury (4, 5). Since both the diseaseinitiating steps and the target auto-antigens are unclear in MS, discerning between disease-relevant and bystander immune cells is necessary as to shed light on disease-initiating triggers, to identify prognostic factors of disease progression and to monitor the efficacy of immunotherapies.
Historically, one of two measures has been employed to identify immunological signatures in MS, immune phenotyping and clonal analysis. The first approach relies on describing the immune phenotype of cells based on known pro-inflammatory markers, such as cytokines. These studies have revealed that lymphocytes with an
antigen-experienced, memory phenotype accumulate in the CNS and peripheral compartments in MS, and exhibit a higher propensity to secrete inflammatory mediators when restimulated ex vivo compared to healthy controls (6). The second approach relies on the ability to track clonally related lymphocytes based on Antigen receptor sequencing. A clonotype refers to a unique antigen receptor sequence that relates lymphocytes that are the progeny of a single cell that was activated in an antigen-specific manner and expanded in the periphery.
Antigen receptor sequencing analyses of both the B and T cell repertoires have revealed that clonally expanded lymphocytes can be found in both the CNS and peripheral compartments in MS (7-10), confirmatory of the traditional view that antigen-specific lymphocytes activated and expanded in the periphery are the dominant disease drivers in MS. Unfortunately, current methodologies are limited to unveiling either the immune phenotype or the antigen receptor sequence and thus fail to reveal whether clonally expanded lymphocytes account for the inflammatory immune cell repertoire in MS (11). Therefore, coupled information about the immune phenotype and clonotype would help to discern potentially disease-relevant, antigen-specific immune cells in MS.
Transcriptomic analysis of immune phenotypes and clonotypes by single-cell RNA sequencing Studying immune processes in MS is challenging due to the limited access to bio-materials and low-throughput methodologies. Recent advances in RNA sequencing promise a deeper understanding of immune processes in MS by permitting the high-throughput sequencing of thousands of cells at a single-cell resolution (11). This technology dramatically surpasses current methodologies of generating transcriptional libraries based on bulk tissue extracts or pooled cells for several reasons, including: (1) it avoids amplification bias that can skew measures of clonal expansion; (2) reveals coupled information about a cell’s immune phenotype in addition to ist antigen receptor sequence, and (3) permits a deeper sequencing of the antigen receptor, thus lending the possibility to predict three-dimension conformation of the antigen binding groove and antigen specificity (12, 13).
Therefore, single-cell RNA sequencing can jointly elucidate the immune phenotype and clonotype of immune cells in MS.