Dissociation of NKT Stimulation, Cytokine Induction, and NK Activation In Vivo by the Use of Distinct TCR-Binding Ceramides

Abstract

NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is α-galactosylceramide (αGalCer). However, αGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, β-galactosylceramide (βGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with βGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with αGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, βGalCer (C12), in contrast to αGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to αGalCer, βGalCer (C12) is a poor inducer of IFN-γ, TNF-α, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, αGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while βGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show βGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.

CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system

“…Although its role as the singular, or at least the predominant, selecting ligand has been challenged, it remains the most potent endogenous agonistic antigen described so far. Although other β-linked GSLs such as β-Galcer have been shown to stimulate iNKT cells [74,75], these results could not exclude the potential contamination by α-linked GSLs that can occur during the synthesis of these compounds, and further experiments are needed to clarify whether β-Galcer (and other β-linked GSLs) are bona fide low affinity iNKT cell ligands.…”

In Vivo Hydrodynamic Delivery of cDNA Encoding IL-2: Rapid, Sustained Redistribution, Activation of Mouse NK Cells, and Therapeutic Potential in the Absence of NKT Cells

“…However, the significantly reduced number of metastases seen in wild-type mice treated with ␤GalCer (selectively removes NKT cells; Ref. 27) and CD1d Ϫ/Ϫ mice, which have no NKT cells, suggest that NKT cells may actually actively inhibit the ability of other elements of the immune system to control of newly established metastases in the liver, an observation that requires further study.…”

“…At present there are no specific Abs available with which to selectively deplete NKT cells. However, we have shown recently that ␤-galactosylceramide (GalCer) 3 (C12;␤GalCer) can selectively deplete NKT cells in vivo without activating NK or other leukocytes (27). Therefore, we used C12;␤GalCer to selectively impair NKT cell function in the absence of NK cell activation to determine the possible role of NKT cells in the antimetastatic effects of IL-2 cDNA in the liver (Fig.…”

“…NKT cells have been implicated previously as a contributing element in tumor rejection by virtue of the ability of ␣GalCer (a potent NKT cell-stimulating compound) to induce antitumor effects. However, these results are complicated by the fact that ␣GalCer not only alters NKT function but also strongly activates other effector leukocytes due to the cytokine storm created in vivo (27). Using a renal cell carcinoma model that can selectively establish metastases in either the liver or lung, we combined hydrodynamic administration of IL-2 cDNA with ␤GalCer, an agent that we have shown recently to remove but not activate NKT or NK cells to elucidate the role of these effector cells.…”

“…Cytokines were measured using IFN-␥ and chemokine ELISA kits (R&D Systems) as described previously (5,26,27). In vitro leukocyte stimulations were performed at concentrations of 1-5 ϫ 10 6 cells/ml.…”