Microbiology & Immunology - Research Publications

Permanent URI for this collection

http://hdl.handle.net/11343/226

Filters

2012 - 2013 2
Reset filters

Settings
Statistics
Citations
Type: Journal Article × Subject: CD1d tetramers ×

Search Results

Now showing 1 - 2 of 2
  • Item
    Thumbnail Image
    Ex-vivo α-Galactosylceramide activation of NKT cells in humans and macaques
    Fernandez, CS ; Cameron, G ; Godfrey, DI ; Kent, SJ (ELSEVIER SCIENCE BV, 2012-08-31)
    NKT cells are key mediators of antiviral and anticancer immunity. Experiments in mice have demonstrated that activation of NKT cells in vivo induces the expression of multiple effector molecules critical to successful immunity. Human clinical trials have shown similar responses, although in vivo activation of NKT cells in humans or primate models are far more limited in number and scope. Measuring ex vivo activation of NKT cells by the CD1d-restricted glycolipid ligand α-Galactosylceramide (α-GalCer) through cytokine expression profiles is a useful marker of NKT cell function, but for reasons that are unclear, this approach does not appear to work as well in humans and non-human primate macaque models in comparison to mice. We performed a series of experiments on human and macaque (Macaca nemestrina) fresh whole blood samples to define optimal conditions to detect NKT cell cytokine (TNF, IFNγ, IL-2) and degranulation marker (CD107a) expression by flow cytometry. We found that conditions previously described for mouse splenocyte NKT cell activation were suboptimal on human or macaque blood NKT cells. In contrast, a 6h incubation with brefeldin A added for the last 4h, in a 96-well plate based assay, and using an α-GalCer concentration of 1 μg/ml were optimal methods to stimulate NKT cells in fresh blood from both humans and macaques. Unexpectedly, we noted that blood NKT cells from macaques infected with SIV were more readily activated by α-GalCer than NKT cells from uninfected macaques, suggesting that SIV infection may have primed the NKT cells. In conclusion, we describe optimized methods for the ex vivo antigen-specific activation of human and macaque blood NKT cells. These assays should be useful in monitoring NKT cells in disease and in immunotherapy studies.
  • Item
    Thumbnail Image
    Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells
    Reantragoon, Rangsima ; Corbett, Alexandra J. ; Sakala, Isaac G. ; Gherardin, Nicholas A. ; Furness, John B. ; CHEN, ZHENJUN ; Eckle, Sidonia B.G. ; Uldrich, Adam P. ; Birkinshaw, Richard W. ; Patel, Onisha ; KOSTENKO, LYUDMILA ; MEEHAN, BRONWYN ; KEDZIERSKA, KATHERINE ; Liu, Ligong ; Fairlie, David P. ; Hansen, Ted H. ; GODFREY, DALE I. ; ROSSJOHN, JAMIE ; MCCLUSKEY, JAMES ; KJER-NIELSEN, LARS (Rockefeller University Press, 2013)
    Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) alpha-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8 alpha(+)CD8 beta(-/lo), implying predominant expression of CD8 alpha alpha homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH2OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V. 19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-beta repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.