These tetramers allowed engagement of more than one copy of the T cell receptor (TCR) on the surface of a T cell, resulting in increased avidity of the connection and allowing recognition of antigen-specific T cells by circulation cytometry, even those present at low frequencies. formally validated this assay, we performed a cross-sectional study of healthy U.S. settings and South African adolescents with and without latent tuberculosis illness (LTBI). We display that GMM-specific T cells are specifically recognized in South African subjects with LTBI and not in U.S. healthy settings. This assay can be expanded to include additional tetramers or phenotypic markers to characterize GMM-specific T cells in studies of mycobacterial illness, disease, or vaccination. (M.tb)-infected human beings (Gilleron RCBTB1 et al., 2004; Layre et al., 2009; Montamat-Sicotte et al., 2011; Moody et al., 2000; Seshadri et al., 2015). However, it is currently unfamiliar whether lipid-specific T-cells become triggered or increase as a result of mycobacterial vaccination. A major barrier to progress has been the lack of formally validated assays to quantify and characterize T-cell reactions to lipid antigens. Tetramers take advantage of multimerization to generate high avidity reagents that can bind to and track rare antigen-specific T cells within a larger mixed human population of T cells. Tetramers based on the major histocompatibility complex (MHC) Class I and Class II proteins have significantly advanced Lomeguatrib our understanding of T cell reactions to peptide antigens but are limited by the highly polymorphic nature of MHC (Altman et al., 1996). On the other hand, CD1 genes are structurally non-polymorphic, so a Lomeguatrib CD1 tetramer can in basic principle be used on everyone, therefore permitting a global analysis of antigen-specific T cell reactions for the first time. The development of soluble lipid-loaded CD1 tetramers changed the panorama for investigation of T-cell phenotypes and functions (Benlagha et al., 2000; Karadimitris et al., 2001; Matsuda et al., 2000). These tetramers allowed engagement of more than one copy of the T cell receptor (TCR) on the surface of a T cell, resulting in increased avidity of the connection and allowing recognition of antigen-specific T cells by circulation cytometry, actually those present at low frequencies. Initially developed for CD1d, tetramers have now been prolonged to CD1a, CD1b, and CD1c, including those loaded with mycobacterial lipid antigens to facilitate studies in individuals with latent and active tuberculosis (Wayne et al., 2018; Kasmar et al., 2013, 2011; Ly et al., 2013). However, these reagents have not yet found their way into validated end point assays that may be employed in medical Lomeguatrib settings. Here, we present the formal validation of an assay using CD1b Lomeguatrib tetramers loaded with glucose monomycolate (GMM), a major component of the mycobacterial cell wall (Brennan et al., 1970). GMM comprises up to 2% of total extractable lipid and is produced by many mycobacterial varieties, including and (Brennan et al., 1970; Moody et al., 2000; Moody, 1997; Silva, 1985). Because the glucose moiety is definitely host-derived, GMM in infected tissues signals the presence of pathogenic mycobacteria and provides an antigenic target for T cells (Moody et al., 2000). Therefore, GMM has been observed to be an immunodominant antigen in experimental illness of cattle and studies of humans with latent tuberculosis (Nguyen et al., 2009; Seshadri et al., 2015). We used GMM-specific T-cell lines to establish the operating characteristics in a circulation cytometry assay and to optimize and validate the tetramer assay according to the following guidelines: linearity, range, limit of detection, repeatability, reproducibility, intermediate precision, and accuracy. We used this assay to study a cohort of healthy subjects and find that GMM-CD1b tetramer positive cells are specifically recognized in South African adolescents at high risk for M.tb exposure but not in U.S. subjects at low risk for exposure. We expect that this assay will find energy in natural history studies of M. tb exposure and disease as well as investigations into the immunogenicity of novel whole cell mycobacterial vaccines. 2. METHODS 2.1. Tradition Media Press (R10) for washing peripheral blood mononuclear cells (PBMC) consisted of RPMI 1640 (Gibco, Waltham, MA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT). Our foundation T cell press (TCM) consisted of RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL Penicillin, 100 mg/mL Streptomycin, 55 mM 2-mercaptoethanol, 0.3X Essential Amino Acids, 60 mM Non-essential Amino Acids, 11 mM HEPES, and 800 mM L-Glutamine (Gibco, Waltham, MA) sterile-filtered. Our TCM comprising human being serum (TCM/HS) consisted of 10% human being serum (derived from healthy donors), 100 U/mL Penicillin, 100 mg/mL Streptomycin, and 400 mM L-Glutamine (Gibco, Waltham, MA). 2.2. Preparation and storage of GMM lipids Glucose monomycolate (C32-GMM) isolated from was generously provided by the laboratory of D. Branch Moody. Stock GMM was solvated Lomeguatrib in chloroform:methanol (2:1, v:v).