Ultra-efficient sequencing of T Cell receptor repertoires reveals shared responses in muscle from patients with Myositis

Janelle M. Montagne, Xuwen Alice Zheng, Iago Pinal-Fernandez, Jose C. Milisenda, Lisa Christopher-Stine, Thomas E. Lloyd, Andrew L. Mammen, H. Benjamin Larman

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Myositis, or idiopathic inflammatory myopathy (IIM), is a group disorders of unknown etiology characterized by the inflammation of skeletal muscle. The role of T cells and their antigenic targets in IIM initiation and progression is poorly understood. T cell receptor (TCR) repertoire sequencing is a powerful approach for characterizing complex T cell responses. However, current TCR sequencing methodologies are complex, expensive, or both, greatly limiting the scale of feasible studies. Methods: Here we present Framework Region 3 AmplifiKation sequencing (“FR3AK-seq”), a simplified multiplex PCR-based approach for the ultra-efficient and quantitative analysis of TCR complementarity determining region 3 (CDR3) repertoires. By using minimal primer sets targeting a conserved region immediately upstream of CDR3, undistorted amplicons are analyzed via short read, single-end sequencing. We also introduce the novel algorithm Inferring Sequences via Efficiency Projection and Primer Incorporation (“ISEPPI”) for linking CDR3s to their associated variable genes. Findings: We find that FR3AK-seq is sensitive and quantitative, performing comparably to two different industry standards. FR3AK-seq and ISEPPI were used to efficiently and inexpensively characterize the T cell infiltrates of surgical muscle biopsies obtained from 145 patients with IIM and controls. A cluster of closely related TCRs was identified in samples from patients with sporadic inclusion body myositis (IBM). Interpretation: The ease and minimal cost of FR3AK-seq removes critical barriers to routine, large-scale TCR CDR3 repertoire analyses, thereby democratizing the quantitative assessment of human TCR repertoires in disease-relevant target tissues. Importantly, discovery of closely related TCRs in muscle from patients with IBM provides evidence for a shared antigen-driven T cell response in this disease of unknown pathogenesis. Funding: This work was supported by NIH grant U24AI118633 and a Prostate Cancer Foundation Young Investigator Award.

Original languageEnglish (US)
Article number102972
JournalEBioMedicine
Volume59
DOIs
StatePublished - Sep 2020

Keywords

  • Autoimmunity
  • Idiopathic Inflammatory myopathy
  • Myositis
  • TCR repertoire

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint

Dive into the research topics of 'Ultra-efficient sequencing of T Cell receptor repertoires reveals shared responses in muscle from patients with Myositis'. Together they form a unique fingerprint.

Cite this