Quantitative and qualitative characterization of expanded CD4+ T cell clones in rheumatoid arthritis patients.

Kazuyoshi Ishigaki,Sakae Tanaka,Kazuhiko Yamamoto,Yuho Kadono,Tetsuro Yasui,Yuta Kochi,Keishi Fujio,Hirofumi Shoda

doi:10.1038/srep12937

Abstract

Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4+ T cell-mediated immunity. Although expanded CD4+ T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4+ T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.

Highlights

There have been some studies on error correction algorithms for NGS T cell receptor (TCR) repertoire analysis[18], careful applications are required because inaccurate algorithm might distort the results of TCR repertoire analysis
In two Rheumatoid arthritis (RA) patients with stable disease and fixed treatments, the TCR repertoire analysis of peripheral blood (PB) memory CD4+ T cells was repeatedly performed with 3-month intervals by a single-cell analysis, which is the gold standard method
We proposed an original and rational strategy for the detailed characterization of each expanded CD4+ T cell clones (ECs) quantitatively and qualitatively by combining a comprehensive TCR repertoire analysis and single-cell transcriptome analysis

Summary

Introduction

The accuracy and the validity of error correction algorithms for NGS TCR repertoire analysis have to be tested using reference data obtained by single-cell Sanger’s sequencing. Due to the lack of reliable markers of clonal expansion, single-cell transcriptome analysis is the only method that allows ECs to be characterized because TCR sequencing and gene expression analysis have to be investigated simultaneously and analyzed comprehensively in a single-cell resolution. Single-cell transcriptome analysis enables us to track the gene expression changes of the same clones in different sites. In this way, through in-depth characterization of ECs, we might be able to detect pathogenic clones and unveil the new aspects of pathophysiology of RA. We performed a single-cell transcriptome analysis of the most expanded CD4+ T cell clones (mECs) in PB and the synovium and tracked the shift in gene expression profiles between them

Methods

Results

Conclusion