Comprehensive Analysis of TCR-\u03b2 Repertoire in Patients with Neurological Immune-mediated Disorders

Alessandra De Paula Alves Sousa,Kory R Johnson,Paolo A Muraro,Joan Ohayon,Jun Zhu,Steven Jacobson

doi:10.1038/s41598-018-36274-7

Abstract

In this study we characterized the TCR repertoire profiles in patients with chronic progressive inflammatory neurological disorders including HAM/TSP, associated with human T-cell lymphotropic virus type I (HTLV-I) infection, and multiple sclerosis (MS), an inflammatory, demyelinating disease of the CNS of unknown etiology. We hypothesized that a T-cell receptor (TCR) clonal repertoire ‘signature’ could distinguish HAM/TSP patients from healthy controls, as well as from patients with a more heterogeneous CNS-reactive inflammatory disease such as MS. In this study, we applied an unbiased molecular technique – unique molecular identifier (UMI) library-based strategy to investigate with high accuracy the TCR clonal repertoire by high throughput sequencing (HTS) technology. cDNA-TCR β-chain libraries were sequenced from 2 million peripheral mononuclear cells (PBMCs) in 14 HAM/TSP patients, 34 MS patients and 20 healthy controls (HC). While HAM/TSP patients showed a higher clonal T-cell expansion compared to MS and HC, increase of the TCR clonal expansion was inversely correlated with the diversity of TCR repertoire in all subjects. In addition, longitudinal analysis of TCR repertoires from HAM/TSP patients demonstrated a correlation of the TCR clonal expansion with HTLV-I proviral load. Surprisingly, MS patients showed a higher diversity of TCR repertoires than other groups. Despite higher TCR clonal expansions in HAM/TSP patients, no disease-specific TCRs were shared among patients. Only non-shared or “private” TCR repertoires was observed. While no clones that shared the same CDR3 amino acid sequences were seen in either HC or MS patients, there was a cluster of related CDR3 amino acid sequences observed for 18 out of 34 MS patients when evaluated by phylogenetic tree analysis. This suggests that a TCR-repertoire signature may be identified in a subset of patients with MS.

Highlights

human T-cell lymphotropic virus type I (HTLV-I) mediated HAM/TSP, the antigen(s) driving the inflammatory autoimmune-mediated response in multiple sclerosis (MS) patients are still unknown viruses have long been considered potential environmental ‘triggers’ in this disorder[7]
To interrogate the T-cell receptor repertoire (TCR) clonal expansion, we first modeled the coefficient of variation (CV) for each clonotype by the average number of unique unique molecular identifier (UMI) observed across technical triplicates (Fig. 1B)
From inspection of this model (Fig. 1B), exponential variability in number of unique UMIs can be seen for those clones at an inflection point on the curve of less than 8 unique UMIs (23 read count) that was associated with a CV~ 15.0% Based on this observation, we classified the TCR clones in each subject into one of 3 groups: (1) singletons or clones with 1 unique UMI; (2) clones with ≥2 < 8 unique UMIs and (3) expanded clones with ≥8 unique UMIs

Summary

Introduction

HTLV-I mediated HAM/TSP, the antigen(s) driving the inflammatory autoimmune-mediated response in MS patients are still unknown viruses have long been considered potential environmental ‘triggers’ in this disorder[7]. It is been hypothesized that in MS, environmental triggers (including viruses) in genetically susceptible individuals lead to immunopathogenic T cell responses that contribute to the development of disease[8,9]. We have reported that the TCR clonal repertoire in the peripheral blood of MS patients was different than what is observed from the T-cell profile within the CSF. The rationale for using this robust unbiased technical approach was to accurately compare the clonal expansion of TCR-β repertoires in circulating PBMCs of patients with neurological immune-mediated diseases including HAM/TSP, MS, and healthy control (HC) individuals. We wished to more comprehensively address whether a shared T-cell repertoire was present in HAM/TSP or MS patients in comparison with healthy controls, underlying a potential antigen-driven clonal expansion in these patients

Methods

Results

Conclusion