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Human T cells are activated by both peptide and nonpeptide Ags produced by Mycobacterium tuberculosis. T cells recognize cell wall lipids bound to CD1 molecules, but effector functions of CD1-reactive T cells have not been systematically assessed in M. tuberculosis–infected humans. It is also not known how these features correlate with T cell responses to secreted protein Ags. We developed a flow cytometric assay to profile CD1-restricted T cells ex vivo and assessed T cell responses to five cell wall lipid Ags in a cross-sectional study of 19 M. tuberculosis–infected and 22 M. tuberculosis–uninfected South African adolescents. We analyzed six T cell functions using a recently developed computational approach for flow cytometry data in high dimensions. We compared these data with T cell responses to five protein Ags in the same cohort. We show that CD1b-restricted T cells producing antimycobacterial cytokines IFN-γ and TNF-α are detectable ex vivo in CD4+, CD8+, and CD4−CD8− T cell subsets. Glucose monomycolate was immunodominant among lipid Ags tested, and polyfunctional CD4 T cells specific for this lipid simultaneously expressed CD40L, IFN-γ, IL-2, and TNF-α. Lipid-reactive CD4+ T cells were detectable at frequencies of 0.001–0.01%, and this did not differ by M. tuberculosis infection status. Finally, CD4 T cell responses to lipids were poorly correlated with CD4 T cell responses to proteins (Spearman rank correlation −0.01; p = 0.95). These results highlight the functional diversity of CD1-restricted T cells circulating in peripheral blood as well as the complementary nature of T cell responses to mycobacterial lipids and proteins. Our approach enables further population-based studies of lipid-specific T cell responses during natural infection and vaccination.

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 μM). 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. Furthermore, 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/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

CD1 proteins evolved to present diverse lipid Ags to T cells. In comparison with MHC proteins, CD1 proteins exhibit minimal allelic diversity as a result of nonsynonymous single nucleotide polymorphisms (SNPs). However, it is unknown if common SNPs in gene regulatory regions affect CD1 expression and function. We report surprising diversity in patterns of inducible CD1a expression on human dendritic cells (DCs), spanning the full range from undetectable to high density, a finding not seen with other CD1 isoforms. CD1a-deficient DCs failed to present mycobacterial lipopeptide to T cells but had no defects in endocytosis, cytokine secretion, or expression of costimulatory molecules after LPS treatment. We identified an SNP in the 5′ untranslated region (rs366316) that was common and strongly associated with low CD1a surface expression and mRNA levels (p = 0.03 and p = 0.001, respectively). Using a CD1a promoter-luciferase system in combination with mutagenesis studies, we found that the polymorphic allele reduced luciferase expression by 44% compared with the wild-type variant (p < 0.001). Genetic regulation of lipid Ag presentation by varying expression on human DCs provides a mechanism for achieving population level differences in immune responses despite limited structural variation in CD1a proteins.

Mycobacterium tuberculosis remains a major pathogen of worldwide importance, which releases lipid Ags that are presented to human T cells during the course of tuberculosis infections. Here we report that cellular infection with live M. tuberculosis or exposure to mycobacterial cell wall products converted CD1− myeloid precursors into competent APCs that expressed group 1 CD1 proteins (CD1a, CD1b, and CD1c). The appearance of group 1 CD1 proteins at the surface of infected or activated cells occurred via transcriptional regulation, and new CD1 protein synthesis and was accompanied by down-regulation of CD1d transcripts and protein. Isolation of CD1-inducing factors from M. tuberculosis using normal phase chromatography, as well as the use of purified natural and synthetic compounds, showed that this process involved polar lipids that signaled through TLR-2, and we found that TLR-2 was necessary for the up-regulation of CD1 protein expression. Thus, mycobacterial cell wall lipids provide two distinct signals for the activation of lipid-reactive T cells: lipid Ags that activate T cell receptors and lipid adjuvants that activate APCs through TLR-2. These dual activation signals may represent a system for selectively promoting the presentation of exogenous foreign lipids by those myeloid APCs, which come into direct contact with pathogens.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b · DAF and CD1c · DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c · DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b · DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c · DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b · DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.

Human CD1 proteins have differentially shaped grooves and present bacterial or self lipid antigens to T cells, but it is unknown whether the pool of endogenous lipids are differently sampled by each type of CD1 proteins. We used recently established lipidomic approach to analyze the molecules extracted from human CD1a, CD1b, CD1c, and CD1d proteins. In the study, we demonstrated that this highly sensitive and accurate lipidomic approach detected more than one thousand diverse molecules defined by accurate mass retention time values, which represented hundreds of lipid species associated with CD1 proteins. More than fifty percent of these molecules were shared by all four types of CD1 proteins and additional thirty percent shared by two or three CD1 isoforms. Further mass spectrometry analyses of collision-induced dissociation showed that the molecules eluted from all four types of CD1 proteins mainly are lipids from subcellular membranes, including phospholipids of conventional, ether-linked, and lyso forms and sphingolipids. Surprisingly, CD1 isoforms with bigger grooves preferred to lipids with shorter chains, which can be explained by small spacer or scaffold lipids that bind together with antigens. Whereas previous studies argued that one lipid molecule blocks CD1 grooves, analogous to MHC class II-associated CLIP peptide, we conclude that CD1 proteins broadly sample cellular lipids and two small lipids are able to be adapted into a big groove.