Effects of structural changes of beta-mannosylceramide on activation of invariant natural killer T cells and stimulation of anti-tumor immunity

Abstract

Invariant natural killer T cells (iNKT) are T cells that recognize a diverse repertoire of structurally distinct lipid antigens, instead of peptides, presented by the non-classical major histocompatibility complex molecule, CD1d. Agonists of iNKT cells that induce strong anti-tumor immunity in vivo include alpha-galactosylceramide (aGC) and beta-mannosylceramide (bMC). Previous studies have shown that altering the lipid structures of aGC resulted in different cytokine profiles and anti-tumor properties. In this study, we aimed to examine the effect of altering the lipid structure of bMC on cytokine profiles and anti-tumor activity. The ceramide structure of bMC was replaced with structures that had been reported to significantly alter the activity of aGC. We examined the analogues of bMC whose acyl chain was altered to a lyso, phenylfluoro, phenyl-O-phenylfluoro, or shorter unsaturated form (C20:2). Alpha-mannosylceramide (aMC) and lyso-alpha-mannosylceramide were also studied. An in vitro stimulation assay of two iNKT cell hybridomas, DN32.D3 and 24.8A, was used to determine whether the analogues can be recognized by TCRs of iNKT cells when presented by CD1d. aMC exhibited 8-fold less activity than bMC in the hybridoma assay. An in vivo CT26 lung metastasis model was used to measure the anti-tumor activity of the analogues. Surprisingly, the analogues, besides aMC, were not recognized by the iNKT hybridoma cell lines and appear to have little or no effect on tumor growth typically seen with aGC and bMC in the CT26 lung metastasis model. These results demonstrate significant differences between aGC and bMC in how the structural changes in the lipid portion affect the anti-tumor biological activity.