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A Simple Assay to Quantify Mycobacterial Lipid Antigen-specific T Cell Receptors in Human Tissues and Blood

Angela X. Zhou, Thomas J. Scriba, Cheryl L. Day, Deanna A. Hagge, Chetan Seshadri

Abstract
T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals.

Introduction
Mycobacterial diseases such as leprosy and tuberculosis (TB) are major causes of morbidity and mortality across the world. A major barrier to eradication of these diseases is the lack of timely and accurate diagnosis. In both diseases, T cells feature prominently in pathophysiology and diagnosis.
T cells express a heterodimeric T cell receptor (TCR) composed of an ɑ and β chain that is expressed at the cell surface and mediates recognition of specific antigens.

Methods:

Ethics statement
The study was approved by the Institutional Review Board of the University of Washington, the University of Cape Town, and the Nepal Health Research Council (NHRC). For the South Africa Tuberculosis Cohort, written informed consent was obtained from all adult participants, as well as from the parents and/or legal guardians of the adolescents who participated.

Quantitative PCR
Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) or T cell populations using RNeasy Mini or Micro Kits (Qiagen, Hilden, Germany)

Discussion
In summary, we developed a simple qPCR assay to measure the abundance of a mycobacterial lipid-specific TCR and used this assay in a cohort of M.tb-reactive donors in South Africa and leprosy patients in Nepal. To our knowledge, our results are the first to show that GEM T cells are a natural part of the skin T cell repertoire. In almost all samples tested, we were readily able to detect the sequence above the limit of detection, confirming their designation as ‘donor-unrestricted’ T cells (DURTs).

Acknowledgments: Dr. Thomas Hawn for facilitating access to archived cDNA from dermal biopsy specimens. Dr. Krystle Yu for cloning the GEM TCR-ɑ and GEM TCR-β plasmids.

Citation: Zhou AX, Scriba TJ, Day CL, Hagge DA, Seshadri C (2021) A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood. PLoS Negl Trop Dis 15(12): e0010018. https://doi.org/10.1371/journal.pntd.0010018

Editor: Katharina Röltgen, Stanford University, UNITED STATES

Received: August 16, 2021; Accepted: November 23, 2021; Published: December 16, 2021.

Copyright: © 2021 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: Funding was received from the National Institutes of Health (R01-AI125189 to CS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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