Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes

Abstract

Immune adult horses have CD8+cytotoxic T lymphocytes (CTLs) that recognize and lyseRhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize uniqueR. equicell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of solubleR. equiantigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulateR. equi-specific CTLs.R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with anR. equilipid extract. Analysis of theR. equilipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed byR. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-γ) and interleukin-4 (IL-4) was also measured in response toR. equilipids by using real-time PCR; elevated IFN-γ, but not IL-4, was associated with host clearance of the bacteria. The whole-cellR. equilipid and all threeR. equilipid fractions resulted in marked increases in IFN-γtranscription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response toR. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.