TY - JOUR
T1 - Ultra-efficient sequencing of T Cell receptor repertoires reveals shared responses in muscle from patients with Myositis
AU - Montagne, Janelle M.
AU - Zheng, Xuwen Alice
AU - Pinal-Fernandez, Iago
AU - Milisenda, Jose C.
AU - Christopher-Stine, Lisa
AU - Lloyd, Thomas E.
AU - Mammen, Andrew L.
AU - Larman, H. Benjamin
N1 - Funding Information:
We would like to thank the Johns Hopkins Medical Institute Deep Sequencing and Microarray Core Facility and the Johns Hopkins Institute of Genetic Medicine Genetics Resources Core Facility.
Funding Information:
This work was supported by NIH grant U24AI118633 and a Prostate Cancer Foundation Young Investigator Award. The funders had no role in the design of this study, data collection, data analyses, data interpretation, or writing of this manuscript.
Publisher Copyright:
© 2020 The Authors
PY - 2020/9
Y1 - 2020/9
N2 - 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.
AB - 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.
KW - Autoimmunity
KW - Idiopathic Inflammatory myopathy
KW - Myositis
KW - TCR repertoire
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U2 - 10.1016/j.ebiom.2020.102972
DO - 10.1016/j.ebiom.2020.102972
M3 - Article
C2 - 32891935
AN - SCOPUS:85090160120
SN - 2352-3964
VL - 59
JO - EBioMedicine
JF - EBioMedicine
M1 - 102972
ER -