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The CD1 family of Ag-presenting molecules is able to display lipids to T cells by binding them within a hydrophobic groove connected to the protein surface. In particular, the CD1b isotype is capable of binding ligands with greatly varying alkyl chain lengths through a complex network of interconnected hydrophobic pockets. Interestingly, mycobacterial lipids such as glucose monomycolate exclusively bind to CD1b. We determined the crystal structure of one of the three expressed bovine CD1b proteins, CD1b3, in complex with endogenous ligands, identified by mass spectrometry as a mixture of phosphatidylcholine and phosphatidylethanolamine, and analyzed the ability of the protein to bind glycolipids in vitro. The structure reveals a complex binding groove architecture, similar to the human ortholog but with consequential differences. Intriguingly, in bovine CD1b3 only the A', C' and F' pockets are present, whereas the T' pocket previously described in human CD1b is closed. This different pocket conformation could affect the ability of boCD1b3 to recognize lipids with long acyl chains such as glucose monomycolate. However, even in the absence of a T' tunnel, bovine CD1b3 is able to bind mycolates from Rhodococcus ruber in vitro.

The CD1 family of proteins has evolved to bind a range of endogenous and foreign lipids and present these at the cell surface for antigen-specific recognition by T cells. The distinct intracellular trafficking pathways of CD 1 molecules indicate that collectively, they have the potential to survey the endocytic system widely for antigen, consistent with a role in the presentation of lipids derived from intracellular microbial pathogens. In keeping with this idea, CDla, CDlb, CDlc and CDld have now been shown to present foreign lipid antigens derived from mycobacteria, Gram-negative bacteria and also protozoan species to T cells. These antigens are extremely diverse chemically, and include naturally occurring lipopeptide, glycolipid and phospholipid structures that are distinct from mammalian lipids. CD1-restricted mycobacterial lipids defined to date derive from the highly complex microbial cell envelope. They play a variety of physiological roles for the microbe, including formation of the plasma membrane and protective cell wall and as metabolic intermediates in iron-scavenging pathways. In each case, alkyl chains of CD 1-restricted lipid antigens are accommodated within a deep hydrophobic groove in the membrane-distal alphal-alpha2 domains of the CD1 molecule, with hydrophilic elements solvent-exposed and accessible for recognition by the T cell receptor. Variation in the number, length and saturation of alkyl chains, and the precise chemistry and chirality of the lipid headgroup, clearly exert dominant influences on antigenicity, mediated by effects on CD1 binding and T cell receptor recognition. In the context of structural studies of CD1-lipid complexes, these data suggest that the CD1 isoforms have evolved binding specificities for different classes of foreign lipids, and strongly support a model for antigen recognition involving fine discrimination of lipid headgroup components by the alpha beta T cell receptor. In this review, we summarise our current knowledge of foreign lipid antigens bound by CD 1, focusing on the roles their distinct structural features play in presentation and T cell antigen recognition, and their likely function in antimicrobial T cell responses.

Members of the CD1 family of antigen-presenting molecules bind and present a variety of mammalian and microbial glycolipids for specific recognition by T cells. CD1 proteins accomplish their antigen-presenting function by binding the alkyl chains of the antigens within a deep, hydrophobic groove on the membrane distal surface of CD1, making the hydrophilic elements of the antigen available for contact with the variable regions of antigen-specific T-cell receptors. Most models of CD1-restricted T cells function in infectious, neoplastic, or autoimmune diseases and are based on the premise that CD1-restricted T-cell responses are initiated by alterations in cellular glycolipid content. Although a growing number of self, altered self and foreign glycolipid antigens have been identified, the cellular mechanisms that could lead to the generation of antigenic glycolipids within cells, or control the presentation of particular classes of altered self or microbial glycolipids in disease states have only recently come under investigation. Here we review the structures of known glycolipid antigens for T cells and discuss how the chemical nature of these antigens, which is quite different from that of peptides, influences their recognition by T cells.

T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

The CD1 family of proteins mediates a newly described pathway for presentation of lipids and glycolipids for specific recognition by T cells. All four of the known human CD1 proteins (CD1a, CD1b, CD1c and CD1d) as well as murine CD1d have now been shown to mediate T-cell recognition of lipid or glycolipid antigens. These antigens include naturally occurring foreign glycolipids from intracellular pathogens or synthetic glycolipids that are related in structure to mammalian glycolipids. The CD1b and CD1d-presented antigens differ in their fine structures but reveal a general motif in which a rigid hydrophilic cap is bound to two aliphatic hydrocarbon chains. Different T-cell populations recognize individual antigens without cross-reactivity to closely related antigen structures or CD1 isoforms, documenting the complexity and fine specificity of CD1-mediated T-cell responses. Mapping of the molecular determinants of recognition for CD1b and CD1d-presented antigens reveals that T cells discriminate the fine structure of the hydrophilic cap of the antigen, but both the length and structure of the lipid chains may be altered without loss of recognition. This pattern of lipid antigen recognition may be accounted for by a simple molecular mechanism of presentation that parallels the known mechanism for presentation of peptides, but solves the special problems related to the hydrophobic chemical nature of the lipid antigens. We propose that CD1 binds antigen by accommodating the two lipid tails within the hydrophobic groove of its two membrane distal domains, positioning the rigid hydrophilic cap of the antigen on the solvent-exposed surface of the CD1 protein, where it can directly contact the T-cell antigen receptor. This model provides a molecular basis for recognition of a new and diverse set of T-cell antigens contained within the lipid bilayers of cellular membranes.

Antigen presenting cells (APCs) expressing CD1b mediate the specific T cell recognition of mycobacterial lipid antigens. These lipid antigens require internalization by APCs prior to presentation, but the detailed mechanisms of uptake and intracellular processing are not known. Here we have examined several steps in the presentation of two related classes of CD1b-presented antigens, free and glycosylated mycolates. T cell recognition of glucose monomycolate (GMM) was blocked by agents that fix APC membranes or neutralize the pH of endosomes, indicating a requirement for GMM uptake into an acidic compartment prior to recognition. Different T cell lines responded to free mycolate or GMM without crossreactivity, yet both antigens were taken up by APCs at the same rate. This demonstrated that differential recognition of these antigens resulted from T cell specificity for their hydrophilic caps and that APCs were unable to interconvert these antigens by enzymatic or chemical deglycosylation or glycosylation. APCs were also unable to cleave mycobacterial trehalose dimycolate (TDM) at its most chemically labile linkages to yield antigenic free mycolates or GMM. Our results indicate that these mycolate-containing antigens are resistant to chemical or enzymatic cleavage by APCs, suggesting that molecular trimming is not a universal feature of lipid antigen processing.

The ability of human CD1b molecules to present nonpeptide antigens is suggested by the T cell recognition of microbial lipids and lipoglycans in the presence of CD1b-expressing antigen-presenting cells. We demonstrate the high-affinity interaction of CD1b molecules with the acyl side chains of known T cell antigens, lipoarabinomannan, phosphatidylinositol mannoside, and glucose monomycolate. Furthermore, CD1b-antigen binding was optimal at acidic pH, consistent with the known requirement for endosomal acidification in CD1b-restricted antigen presentation. The mechanism for CD1b-ligand interaction involves the partial unfolding of the alpha helices of CD1b at acidic pH, revealing a hydrophobic binding site that could accommodate lipid. These data provide direct evidence that the CD1b molecule has evolved unique biochemical properties that enable the binding of lipid-containing antigens from intracellular pathogens.

The human CD1b protein presents lipid antigens to T cells, but the molecular mechanism is unknown. Identification of mycobacterial glucose monomycolate (GMM) as a CD1b-presented glycolipid allowed determination of the structural requirements for its recognition by T cells. Presentation of GMM to CD1b-restricted T cells was not affected by substantial variations in its lipid tails, but was extremely sensitive to chemical alterations in its carbohydrate or other polar substituents. These findings support the view that the recently demonstrated hydrophobic CD1 groove binds the acyl chains of lipid antigens relatively nonspecifically, thereby positioning the hydrophilic components for highly specific interactions with T cell antigen receptors.