Cutting Edge: CD1a Tetramers and Dextramers Identify Human Lipopeptide–Specific T Cells Ex Vivo

Abstract

Human CD1a mediates foreign antigen recognition by a T cell clone, but the nature of possible T cell receptor interactions with CD1a-lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide antigen, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD ≈ 100µM). Detection of ternary CD1a-lipid-TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Further, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a-antigen-TCR. Further, these studies demonstrate the existence of lipopeptide specific T cells in humans ex vivo.

αβ T cell antigen receptor recognition of CD1a presenting self lipid ligands

“…3c-e), and we obtained a similar value for insect-derived CD1a-endo (data not shown). This value was comparable or moderately greater than reported CD1a-mediated TCR interactions involving individual antigenic ligands 10,18 . For these experiments, approximately 3,000 response units of CD1a-endo was coupled to the streptavidin surface plasmon resonance chip via the carboxy-terminal biotin tag on CD1a, and the maximal response achieved was 1,100 response units (40% of maximal predicted response units).…”

“…All surface plasmon resonance experiments were conducted at 25 °C in duplicate with a BIAcore 3000 instrument in 10 mM HEPES-HCl (pH 7.5) and 150 mM NaCl. The buffer was supplemented with 0.5% bovine serum albumin (Sigma) to prevent nonspecific interaction between the chip and the fluidics according to published protocols 10,18,33 ; this was in place of detergents that would strip lipids from CD1a complexes. Biotinylated, deglycosylated CD1a-jun-BirA-His 6 -β 2 m-Fos proteins were immobilized onto a Sensor Chip SA (GE Healthcare) with a surface density of approximately 3,000 response units.…”

“…1a). This was unexpected because tetramers of MHC, MR1 or CD1 do not typically bind to TCRs until defined antigens have been loaded within the antigen-binding cleft, which alters the surface of the antigen-presenting molecule to generate complexes whose avidity exceeds a threshold needed for distinct staining [15][16][17][18] . Control experiments indicated that staining by tetramers of CD1a presenting endogenous ligands (CD1a-endo) was not due to TCR overexpression or nonspecific interactions, as CD1a-endo tetramers did not bind to Jurkat cells expressing an irrelevant TCR derived from a human MR1-restricted mucosal-associated invariant T cell (MAIT cell) 19 (Fig.…”

Human autoreactive T cells recognize CD1b and phospholipids

“…For the generation of dextramers, CD1a and CD1b monomers produced in human cell line HEK 293T (NIH tetramer facility) were loaded with antigens and assembled into phycoerythrin-or allophycocyaninlabeled dextramers, as described (8,9). Dextramers were incubated with cells for 30 min at room temperature before adding monoclonal antibodies for an additional 30 min on ice.…”

“…Prior work using human T-cell clones (3)(4)(5)(6)(7) and the recently developed human CD1a, CD1b, and CD1c tetramers (8)(9)(10) has demonstrated the existence of mycobacteria-reactive T cells, including polyclonal T cells with stereotyped T-cell receptors (TCRs), known as germline-encoded mycolyl lipid-reactive T cells (11) and LDN5-like T cells (12). At this time, nearly all known foreign antigens presented by CD1b, including mycolic acid, glucose monomycolate, glycerol monomycolate, and sulfoglycolipids, are selectively synthesized by Mycobacterium tuberculosis and related mycobacterial species.…”