THE ROLE OF LIPID ANTIGENS IN THE PROTECTION AGAINST RHODOCOCCAL PNEUMONIA

Thesis (Ph.D.), Department of Veterinary Microbiology and Pathology, Washington State UniversityRhodococcus equi is an actinomycete bacterium that is closely to Mycobacterium tuberculosis. It is also an important cause of pneumonia in young horses worldwide. Like M. tuberculosis, R. equi persists within macrophages and has a cell wall composed of unique microbial lipids. Previous work in our laboratory demonstrates that immune horses carry cytotoxic T lymphocytes (CTL) that recognize and kill R. equi-infected cells in a MHC-unrestricted fashion. Further evidence showing that these cells recognize R. equi lipids provides a potentially important correlation between immune protection and the CD1 system in horses.In this study, we characterized and mapped the equine CD1 gene cluster. It is composed of 13 CD1 genes; seven genes were classified as homologues of human CD1a, two CD1b, one CD1c, one CD1d, and two CD1e, making it the largest CD1 family to date. All but one of the eqCD1 molecules were expressed in all antigen presenting cells investigated. Because R. equi is able to arrest macrophage phagosome maturation between the early endosome and late phagosome, and based on the presence of a sorting motif that is predicted to direct eqCD1a to the early endosome, we hypothesize that the extraordinarily large number of CD1a molecules in horses reflects their role in immunity to R. equi.Because of the predicted expression of a CD1d homologue and published evidence that horse have NKT cells, we have proposed using the synthetic lipid alpha-Galactosyl Ceramide (α-GC) as an adjuvant in an R. equi vaccine. α-GalCer is a potent NKT cell agonist in other species. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi. Likewise, α-GalCer did not increase killing by CTL co-stimulated with R. equi antigen, nor did it induce the proliferation of equine PBMC or increase the proliferation of R. equi stimulated cells. α-GalCer injected intradermally in horses did not increase vasodilation, the recruitment of lymphocytes, or relevant cytokines/markers (e.g. IFN-gamma, granzyme) at sites of injection. These data provide evidence that the horse is well equipped to present lipid-antigens to T cell, however suggest that α-GalCer is unsuitable as a vaccine adjuvant in horses.Department of Veterinary Microbiology and Pathology, Washington State Universit