Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material

Human γδ T cells recognize CD1b by two distinct mechanisms

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids

Atypical sideways recognition of CD1a by autoreactive γδ T cell receptors

CD1a is a monomorphic antigen-presenting molecule on dendritic cells that presents lipids to αβ T cells. Whether CD1a represents a ligand for other immune receptors remains unknown. Here we use CD1a tetramers to show that CD1a is a ligand for Vδ1+ γδ T cells. Functional studies suggest that two γδ T cell receptors (TCRs) bound CD1a in a lipid-independent manner. The crystal structures of three Vγ4Vδ1 TCR-CD1a-lipid complexes reveal that the γδ TCR binds at the extreme far side and parallel to the long axis of the β-sheet floor of CD1a’s antigen-binding cleft. Here, the γδ TCR co-recognises the CD1a heavy chain and β2 microglobulin in a manner that is distinct from all other previously observed γδ TCR docking modalities. The ‘sideways’ and lipid antigen independent mode of autoreactive CD1a recognition induces TCR clustering on the cell surface and proximal T cell signalling as measured by CD3ζ phosphorylation. In contrast with the ‘end to end’ binding of αβ TCRs that typically contact carried antigens, autoreactive γδ TCRs support geometrically diverse approaches to CD1a, as well as antigen independent recognition

Human γδ T cells recognize CD1b by two distinct mechanisms

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids

CD1b Tetramers Broadly Detect T Cells That Correlate With Mycobacterial Exposure but Not Tuberculosis Disease State

The non-polymorphic nature of CD1 proteins creates a situation in which T cells with invariant T cell receptors (TCRs), like CD1d-specific NKT cells, are present in all humans. CD1b is an abundant protein on human dendritic cells that presents M. tuberculosis (Mtb) lipid antigens to T cells. Analysis of T cell clones suggested that semi-invariant TCRs exist in the CD1b system, but their prevalence in humans is not known. Here we used CD1b tetramers loaded with mycolic acid or glucose monomycolate to study polyclonal T cells from 150 Peruvian subjects. We found that CD1b tetramers loaded with mycolic acid or glucose monomycolate antigens stained TRAV1-2+ GEM T cells or TRBV4-1+ LDN5-like T cells in the majority of subjects tested, at rates ~10-fold lower than NKT cells. Thus, GEM T cells and LDN5-like T cells are a normal part of the human immune system. Unlike prior studies measuring MHC- or CD1b-mediated activation, this large-scale tetramer study found no significant differences in rates of CD1b tetramer-mycobacterial lipid staining of T cells among subjects with Mtb exposure, latent Mtb infection or active tuberculosis (TB) disease. In all subjects, including 'uninfected' subjects, CD1b tetramer+ T cells expressed memory markers at high levels. However, among controls with lower mycobacterial antigen exposure in Boston, we found significantly lower frequencies of T cells staining with CD1b tetramers loaded with mycobacterial lipids. These data link CD1b-specific T cell detection to mycobacterial exposure, but not TB disease status, which potentially explains differences in outcomes among CD1-based clinical studies, which used control subjects with low Mtb exposure

CD1b Tetramers Broadly Detect T Cells That Correlate With Mycobacterial Exposure but Not Tuberculosis Disease State

The non-polymorphic nature of CD1 proteins creates a situation in which T cells with invariant T cell receptors (TCRs), like CD1d-specific NKT cells, are present in all humans. CD1b is an abundant protein on human dendritic cells that presents M. tuberculosis (Mtb) lipid antigens to T cells. Analysis of T cell clones suggested that semi-invariant TCRs exist in the CD1b system, but their prevalence in humans is not known. Here we used CD1b tetramers loaded with mycolic acid or glucose monomycolate to study polyclonal T cells from 150 Peruvian subjects. We found that CD1b tetramers loaded with mycolic acid or glucose monomycolate antigens stained TRAV1-2+ GEM T cells or TRBV4-1+ LDN5-like T cells in the majority of subjects tested, at rates ~10-fold lower than NKT cells. Thus, GEM T cells and LDN5-like T cells are a normal part of the human immune system. Unlike prior studies measuring MHC- or CD1b-mediated activation, this large-scale tetramer study found no significant differences in rates of CD1b tetramer-mycobacterial lipid staining of T cells among subjects with Mtb exposure, latent Mtb infection or active tuberculosis (TB) disease. In all subjects, including 'uninfected' subjects, CD1b tetramer+ T cells expressed memory markers at high levels. However, among controls with lower mycobacterial antigen exposure in Boston, we found significantly lower frequencies of T cells staining with CD1b tetramers loaded with mycobacterial lipids. These data link CD1b-specific T cell detection to mycobacterial exposure, but not TB disease status, which potentially explains differences in outcomes among CD1-based clinical studies, which used control subjects with low Mtb exposure

Dual TCR-a Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRa-chain that uses T cell receptor a-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), we performed bulk TCRa- and TCRb-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRa-chains also coexpressed an invariant MAIT TCRa-chain. Transfection of each non-TRAV1-2 TCRa-chain with the TCRb-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRa-chain expression in human T cells and competition for the endogenous b-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity. The Journal of Immunology, 2022, 208: 1-7

Dual TCR-α expression on mucosal-associated invariant T cells as a potential confounder of TCR interpretation

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non–TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRβ-chain sequencing and single-cell–based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non–TRAV1-2 TCRα-chain with the TCRβ-chain from the same cell demonstrated that the non–TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous β-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non–TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity