A multi-tetramer flow cytometry panel for studying the phenotypes of human donor-unrestricted T cells in clinical studies — ASN Events
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  3. A multi-tetramer flow cytometry panel for studying the phenotypes of human donor-unrestricted T cells in clinical studies

A multi-tetramer flow cytometry panel for studying the phenotypes of human donor-unrestricted T cells in clinical studies (#97)

Erik D Layton 1 , Malisa Smith 1 , Krystle K Quan 1 , Thomas J Scriba 2 , Chetan Seshadri 1
  1. Department of Medicine, University of Washington, Seattl, Washington, United States of America
  2. Department of Pathology, South African Tuberculosis Vaccine Initiative (SATVI) and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, University of Cape Town, Cape Town, South Africa

The non-polymorphic antigen presenting molecules CD1, MR1, BTN3A1 are used by antigen presenting cells to present non-peptide antigens to T cells.  These “donor-unrestricted T cells” (DURTs) have not been extensively evaluated in human clinical studies unlike T-cells specific for peptide antigens. To address this, we developed a multi-parameter flow cytometry panel aimed at characterizing and quantifying the phenotypes of DURTs in clinical specimens. The core of this panel consists of the T-cell lineage (CD3, CD4, and CD8), memory (CD45RA, and CCR7) markers, as well as markers for δ1 and δ2 expressing γδ T cells. 5-OP-RU loaded MR1 tetramer and α-galactosylceramide (α-GalCer) loaded CD1d tetramer identify canonical populations of mucosal associated invariant T (MAIT) cells and invariant NKT cells respectively. Anti-TRAV1-2 helps define MAIT cells and germline-encoded mycolyl reactive (GEM) T cells. GEM T cells are specific for the immunodominant antigen, glucose monomycolate (GMM), which is presented by CD1b and common to many mycobacteria. In contrast with the CD1b GMM tetramer, CD1b diacylated sulfoglycolipid (Ac2SGL) tetramer distinguishes T cells specific for virulent mycobacteria. Optimization of this panel was done through antibody titrations and fluorescence minus one (FMO) experiments using T-cell lines specific for each of the tetramer reagents.  Tetramers were qualified with respect to linearity, range, limit of detection, limit of quantification, reproducibility, repeatability, intermediate precision, and accuracy.  Finally, we used this panel to perform a cross-sectional study of a well characterized cohort of South African adolescents with and without latent tuberculosis infection (LTBI). We used t-Distributed Stochastic Neighbor Embedding (t-SNE) to visualize the data, and we found qualitative differences among cells specific to CD1b-GMM and CD1b-Ac2SGL in the presence of LTBI. This panel is easily modified to include additional tetramers or phenotypic markers to characterize DURTs in studies of mycobacterial infection, disease, and vaccination.