A Novel Sulfatide Analog Recognized by Human iNKT Cells Enhances Polyfunctional Antigen-Specific CD8⁺ T Cell...

Invariant natural killer T (iNKT) cells are CD1d-restricted T cells that recognize lipid antigens. Studies of analogs of a prototypic iNKT cell antigen, α-galactosylceramide (α-GalCer), have shown that ceramide structure affects the activities of mouse iNKT cells recognizing the antigen. However, the impact of antigen structure on human iNKT cell activities is less well studied. This study evaluated the antigenicity of eight novel synthetic sulfatide-based lipids with fluorobenzene at the acyl chain terminus and a variety of sphingoid base structures. Using CD1d-tetramers, we evaluated TCR recognition in ex vivo iNKT cells from healthy donors' peripheral blood mononuclear cells (PBMCs). One analog, SAP-3, was commonly recognized by iNKT cells from all donors. SAP-3 increased the proportion of CD69⁺CD25⁺ iNKT cells in a CD1d-dependent manner and triggered IL-2 production. Furthermore, SAP-3-activated ex vivo iNKT cells increased the frequency of polyfunctional antigen-specific CD8⁺ T cells (IFN-γ⁺IL-2⁺TNF-α⁺) in PBMC stimulation culture. Importantly, repeated in vitro stimulation with SAP-3 did not induce iNKT-cell functional anergy, unlike α-GalCer; instead, SAP-3 increased IFN-γ-producing iNKT cells. These findings suggest that the structures of sulfatide analogs critically influence their immunogenicity. SAP-3 emerges as a novel human iNKT agonist that enhances antigen-specific CD8⁺ T cell responses and represents a promising vaccine and adjuvant candidate.