The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation.

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation.

Stimulation of iNKT cells by α-galactosyl ceramide (α-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells' response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in α-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of α-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.

Incorporation of NKT cell-activating glycolipids enhances immunogenicity and vaccine efficacy of Mycobacterium bovis bacillus Calmette-Guerin.

The attenuated strain of Mycobacterium bovis known as bacille Calmette-Guérin (BCG) has been widely used as a vaccine for prevention of disease by Mycobacterium tuberculosis, but with relatively little evidence of success. Recent studies suggest that the failure of BCG may be due to its retention of immune evasion mechanisms that delay or prevent the priming of robust protective cell-mediated immunity. In this study, we describe an approach to enhance the immunogenicity of BCG by incorporating glycolipid activators of CD1d-restricted NKT cells, a conserved T cell subset with the potential to augment many types of immune responses. A method was developed for stably incorporating two forms of the NKT cell activator alpha-galactosylceramide into live BCG organisms, and the impact of this on stimulation of T cell responses and protective antimycobacterial immunity was evaluated. We found that live BCG containing relatively small amounts of incorporated alpha-galactosylceramide retained the ability to robustly activate NKT cells. Compared with immunization with unmodified BCG, the glycolipid-modified BCG stimulated increased maturation of dendritic cells and markedly augmented the priming of Ag-specific CD8(+) T cells responses. These effects were correlated with improved protective effects of vaccination in mice challenged with virulent M. tuberculosis. These results support the view that mycobacteria possess mechanisms to avoid stimulation of CD8(+) T cell responses and that such responses contribute significantly to protective immunity against these pathogens. Our findings raise the possibility of a simple modification of BCG that could yield a more effective vaccine for control of tuberculosis.

Synthesis and biological activity of alpha-L-fucosyl ceramides, analogues of the potent agonist, alpha-D-galactosyl ceramide KRN7000.

Several L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of alpha-L-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.

NKT cells direct monocytes into a DC differentiation pathway.

Monocytes can differentiate into macrophages or dendritic cells (DCs). The processes that promote their differentiation along one pathway rather than the other remain unknown. NKT cells are regulatory T cells that respond functionally to self and foreign antigens presented by CD1d molecules. Hence, in addition to contributing to antimicrobial responses, they may carry out autoreactively activated functions when there is no infectious challenge. However, the immunological consequences of NKT cell autoreactivity remain poorly understood. We show here that human NKT cells direct monocytes to differentiate into immature DCs. The ability to induce monocyte differentiation was CD1d-dependent and appeared specific to NKT cells. Addition of exogenous antigens or costimulation from IL-2 was not required but could enhance the effect. DC differentiation was a result of NKT cell secretion of GM-CSF and IL-13, cytokines that were produced by the NKT cells upon autoreactive activation by monocytes. NKT cells within PBMC samples produced GM-CSF and IL-13 upon exposure to autologous monocytes directly ex vivo, providing evidence that such NKT cell-autoreactive responses can occur in vivo. These results show that when NKT cells are activated by autologous monocytes, they are capable of providing factors that specifically direct monocyte differentiation into immature DCs. Thus, autoreactively activated NKT cells may contribute to the maintenance of the immature DC population, and microbial infection or inflammatory conditions that activate NKT cells further could stimulate them to promote an increased rate of DC differentiation.

Distinct endosomal trafficking requirements for presentation of autoantigens and exogenous lipids by human CD1d molecules.

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRalphabeta and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR- APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.

The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d.

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-gamma and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, alpha-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.

Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors.

CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Valpha24-negative and 13 canonical Valpha24-positive/Vbeta11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The Valpha24-negative clones expressed Vbeta11 paired with Valpha10, Valpha2, or Valpha3. Strikingly, their Valpha-chains had highly conserved rearrangements to Jalpha18, resulting in CDR3alpha loop sequences that are nearly identical to those of canonical TCRs. Valpha24-positive and Valpha24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3alpha loop is sufficient for recognition of alpha-GalCer despite CDR1alpha and CDR2alpha sequence variation. Unlike Valpha24-positive clones, the Valpha24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by Valpha-encoded TCR regions. Valpha24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jalpha18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCer-reactive, Valpha24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.