Original ArticlesActivation of human Vα24NKT cells by α-glycosylceramide in a CD1d-restricted and Vα24TCR-mediated manner
Introduction
The CD1 surface molecules represent a family of conserved non-polymorphic cell surface glycoproteins distantly related to the MHC (major histocompatibility complex) class I and class II molecules, but encoded outside the MHC. CD1 appears to be more closely related to MHC class Ia and class Ib proteins by sequence homology, association with β2 microglobulin (β2m) and domain organization (α1, α2, α3, and β2m) than to class II molecules 1, 2, 3, 4, 5. The CD1 family can be divided into two groups by sequence homology. Group I consists of CD1a, -b, -c isotypes, and group II contains CD1d; CD1e is proposed to be an intermediate isotype [6]. Only the group II CD1d isotypes are conserved in human, mouse, rabbit, and rat.
Analogous with MHC molecules, CD1 molecules appear to function as specialized antigen presenting molecules [2]. Human and murine CD1 restricted T cell lines and clones that recognize lipid antigens 7, 8, 9 or hydrophobic peptide antigens have been identified [10]. Human CD1b was the first CD1 family molecule to be identified as a restriction element for an immune response to a microbial antigen [7]. It has been clearly demonstrated that for recognition of mycobacterial glucose monomycolate by CD1b-restricted T cells, strict structural requirements have to be met. Glucose monomycolate recognition was extremely sensitive to chemical alternations in its carbohydrate or other polar substitutions [11]. CD1c is also reported to be a restriction element for T cell responses to protease-resistant, mycobacterial antigens [12]. CD1a-reactive T cell clones that exhibit specificity for undefined bacterial antigens have been reported to persist in the peripheral blood of healthy human donors uninfected with known pathogenic microorganisms [13].
Recent studies of murine and human CD1d-reactive T cells demonstrated that these T cells have some distinctive characteristics. Murine T cell lines responding in a CD1d-restricted manner express the invariant Vα14 TCR and a lectin type, NK receptor, NKR-P1C and have been called Vα14NKT cells. Vα 14NKT cells may be either CD4−CD8− double negative (DN) or CD4+CD8− 14, 15, 16, 17, 18. The murine Vα14TCR preferentially pairs with Vβ8, 7 or 2 [19]. Vα24TCR, the human counterpart of murine Vα14TCR, is expressed by DN T cells, is rarely expressed among CD4+T cells and even less often CD8+T cells [20]. The invariant Vα24TCR associates preferentially with Vβ11 20, 21, which is the homologue to murine Vβ8. Vα24+CD4−CD8− T cells coexpressing NKR-P1A, the human homologue of the rodent NKR-P1C [22] are CD1d reactive [23]. Specific glycolipids such as α-galactosylceramide (α-GalCer) and α-glucosylceramaide (α-GlcCer) containing sugar with a longer fatty acyl chain and sphingosine base have been identified as ligand(s) recognized by the invariant T cell antigen receptor on murine Vα14Jα281Vβ8 NKT cells in a CD1d-restricted fashion [24]. In humans, an antigen or a ligand for CD1d-restricted recognition by Vα24+T cells has not yet been identified.
To investigate the possibility that glycolipids such as α-GalCer and α-GlcCer are presented by CD1d molecules in humans and to clarify the role of a specific TCR in this recognition, we generated a T cell line specific for α-glycosylceramides using monocyte derived dendritic cell (DC) like cells (Mo-DCs) in an autologous mixed leukocyte reaction (auto-MLR) system and characterized the T cell line. The T cell line was phenotypically Vα24+Vβ11+NKRP1A+ CD4−CD8− DN T cells and designated as Vα24 NKT cells. The Vα24 NKT cells specifically proliferated in response to α-glycosylceramides in a CD1d-restricted and Vα24TCR-mediated manner. Upon stimulation, the line secreted cytokines including Interferon-γ (IFN-γ) and Interleukin-4 (IL-4). The results demonstrate, for the first time in the human system, that specific glycolipids such as α-GalCer and α-GlcCer are recognized by Vα24TCRs on Vα24 NKT cells in a CD1d-restricted manner. The findings are consistent with the recognition pattern of murine Vα14NKT cells. The marked similarities between murine Vα14 NKT cells and human Vα24 NKT cells suggest that the newly defined human Vα24 NKT cells may play an important role in controlling tumors or preventing autoimmunity in humans, as has been demonstrated in mice. Our results also raise the possibility that naturally occurring α-glycosylceramides, or molecules with similar structures in humans, are ligands or can act as antigens presented by CD1d.
Section snippets
Lipid antigens
Synthetic glycolipids including α-GalCer, α-GlcCer, β-galactosylceramide (β-GalCer), and α-mannosylceramide (α-ManCer), and ceramide were obtained from Kirin Brewery Ltd.
Preparation of peripheral blood Mo-DCs
As we have shown that Mo-DC express CD1d, and are known to be antigen presenting cells, peripheral blood derived Mo-DC were prepared for our T cell stimulation experiments. Normal human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using 50% percoll (Pharmacia, Uppsala, Sweden). Monocytes were cultured
Generation of Vα24 NKT cells and cell activation by α-glycosylceramide
To generate a T cell line specific for α-glycosylceramides and assess the potential of α-GalCer to act as an Ag or a ligand, we used an auto-MLR system. In this system, Mo-DCs which consistently express high levels of CD1 a, b, c molecules [7] and mannose receptor [26] and in our own studies, significant levels of CD1d, were used as antigen presenting cells (Fig. 1). The Mo-DCs were cultured with enriched autologous T cells in the presence of α-GalCer for 14 days. IL-2 was added for the final
Discussion
In this study, we show that specific glycolipids such as α-GalCer and α-GlcCer are recognized by DNVα24+Vβ11+NKRP1A+NKT cells in a CD1d-restricted and Vα24TCR-mediated manner. Importantly we have extended the observations described here to show that αGalCer specific Vα24NKT cells can be isolated from other normal human donors in a reproducible fashion. The precise frequency in normal peripheral blood of CD1d-restricted Vα24 NKT cells specific for α-GalCer is unknown, but it appears that this
Acknowledgements
We are grateful to Dr. M. Takamizawa (Tokyo University Hospital) for helpful suggestions and discussions. We also thank Dr. T. Sakai (Kirin Brewery) for preparation of synthetic glycolipids.
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