An analysis of analogues of the alpha-galactosylceramide KRN7000 as activators of Valpha14-invariant NKT cells

Abstract

Valpha14-invariant (Valpha14i) NKT cells represent a T lymphocyte subset of innate-like function, which express an invariant T cell receptor (TCR) encoded by a Valpha14-Jalpha18 gene rearrangement. In contrast to conventional T cells that are activated by peptides presented by MHC class I or II, Valpha14i NKT cells recognize glycolipid antigens within the lipid-binding groove of the nonclassical MHC-like molecule CD1d. Activation by one agonist, the alpha-galactosylceramide (alpha-GalCer) KRN7000, leads to rapid production by the Valpha14i NKT cell of the cytokines interferon-gamma and interleukin-4, and trans -activation of B, natural killer and dendritic cells. KRN7000 therapy is beneficial in various autoimmune, oncologic, and infectious disease models, but is limited by the strong production of proinflammatory cytokines like interferon-gamma. Hence, there is much interest in KRN7000 analogues that elicit strong T helper type 2 (TH2) responses, with sufficient interleukin-4 and decreased interferon-gamma.;We have synthesized a panel of analogues of KRN7000. These lipids exhibited a range of potencies in activating a Valpha14i NKT cell hybridoma. One alpha-GalCer with a diunsaturated N-acyl chain, C20:2, was a potent TH2-biasing compound. While the proximal Valpha14i NKT cell response to C20:2 was intact, there was lowered natural killer cell interferon-gamma production, and differences in bystander cell trans-activation and Valpha14 i NKT cell expansion.;The Valpha14i TCR recognized C20:2-loaded CD1d similarly to CD1d loaded with KRN7000. Consequently, a difference in TCR avidity to the lipid:protein complex is an insufficient explanation for the differential cytokine response. However, C20:2 had less stringent loading requirements onto CD1d than KRN7000, suggesting this as a mechanism for its TH2 bias. We have now generated monoclonal antibodies that could be used to track the formation of alphaGalCer:CD1d complexes. These could help us study how TH2-biasing analogues are presented to NKT cells differently than glycolipids that are not.;This work describes the potential modulation of the immune system using variant agonists of Valpha14i NKT cells. With a more comprehensive understanding of the mechanisms for generation of TH2-biased immune responses, better design of agents for NKT cell-directed immunotherapy of autoimmune disease is possible.