In contrast to other species, α-Galactosylceramide (α-GalCer) is not an immunostimulatory NKT cell agonist in horses

• The equine CD1d has a suitable pocket for α-GalCer ligation.
• α-GalCer failed to stimulate equine NKT cells in vitro or in vivo.
• α-GalCer loaded tetramers did not bind equine cells.
• α-GalCer is an unsuitable adjuvant in horses.
• Requirements for equine NKT cells activation is likely unique

α-GalCer is a potent immunomodulatory molecule that is presented to NKT cells via the CD1 antigen-presenting system. We hypothesized that when used as an adjuvant α-GalCer would induce protective immune responses against Rhodococcus equi, an important pathogen of young horses. Here we demonstrate that the equine CD1d molecule shares most features found in CD1d from other species and has a suitable lipid-binding groove for presenting glycolipids to NKT cells. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi, and α-GalCer did not increase killing by CTL co-stimulated with R. equi antigen. Likewise, α-GalCer did not induce the lymphoproliferation of equine PBMC or increase the proliferation of R. equi-stimulated cells. Intradermal injection of α-GalCer in horses did not increase the recruitment of lymphocytes or cytokine production. Furthermore, α-GalCer–loaded CD1d tetramers, which have been shown to be broadly cross-reactive, did not bind equine lymphocytes. Altogether, our results demonstrate that in contrast to previously described species, horses are unable to respond to α-GalCer. This raises questions about the capabilities and function of NKT cells and other lipid-specific T lymphocytes in horses.