Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes

Abstract

Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus 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 unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid 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 by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-gamma) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-gamma, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-gamma transcription, 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 to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.

Fig. 1

Antigen that stimulates R. equi-specific, MHC class I-unrestricted CTLs is not protease-sensitive. PBMCs from immune adult horses were stimulated for 5 days with 2 µg protease-treated SRA ml⁻¹ (a) or untreated SRA (b), rested for 2 days and then tested for CTL activity. Target cells were infected (black bars) with 5 m.o.i. R. equi ATCC 33701 for 9 h prior to the addition of effector cells at effector : target (E : T) ratios of 1 : 1, 3 : 1, 9 : 1 and 27 : 1. An asterisk indicates statistically significant lysis of target cells [>3 sem compared with the corresponding uninfected control (grey bars)]. Animal identification numbers are H14 and H15. These results were repeated in a total of four (a) and two (b) independent experiments.

Fig. 2

R. equi-specific CTLs recognize and lyse MHC class I-mismatched target cells pulsed with R. equi cell-wall lipid. PBMCs were stimulated for 5 days with live, virulent R. equi ATCC 33701, then rested for 2 days before testing in the CTL assay. Target cells (equine macrophages) were pulsed (black bars) with R. equi lipid for 9 h prior to the addition of effector cells at the indicated E : T ratios of 1 : 1, 3 : 1, 9 : 1 and 27 : 1. An asterisk indicates statistically significant lysis of target cells [>3 sem compared with the corresponding non-pulsed target-cell control (grey bars)]. Two representative animals (H13 and H14) are shown. These results have been confirmed in two additional experiments.

Fig. 3

TLC analysis of purified TMM, TDM and CL derived from R. equi ATCC 33701. Unfractionated R. equi lipid (lane 1) and purified TMM, CL and TDM (lanes 2–4, respectively) were developed on TLC plates with the solvent system of chloroform/methanol/acetone/acetic acid (80 : 20 : 6 : 1, v/v), and detected with 20 % H₂SO₄ (a) or Dittmer’s reagent (b).

Fig. 4

Mass spectra of TMM, TDM and CL derived from R. equi ATCC 33701. (a, b) R. equi TMM (a) and TDM (b) were acquired by MALDI-TOF MS using 10 mg 2,5-dihydroxybenzoic acid ml⁻¹ in chloroform/methanol (1 : 1, v/v) as a matrix, and the molecular-related ions were detected as [M+Na]⁺ in positive mode. (c) R. equi CL was acquired by ES-MS and the molecular-related ions were detected as [M−H]⁻ in negative mode. a.u., Arbitrary units.

Fig. 5

Proposed structures of R. equi TMM, TDM and CL. (a) R. equi TMM: trehalose 6-monomycolate, C₄₈H₉₂O₁₃, exact mass 876.65 Da, molecular mass 877.24 Da. (b) R. equi TDM: trehalose 6,6′-dimycolate, C₈₄H₁₆₂O₁₅, exact mass 1411.19 Da, molecular mass 1412.18 Da. (c) R. equi CL: cardiolipin, C₇₉H₁₅₄O₁₇P₂, exact mass 1437.07 Da, molecular mass 1438.01 Da.

Fig. 6

CTLs from immune horses recognize and lyse macrophages pulsed with R. equi TMM and CL, but not TDM. PBMCs (from horses #H13, H14 and H68) were stimulated for 5 days with live, virulent R. equi, then rested for 2 days. Target cells were pulsed with unfractionated R. equi lipid or fractionated lipid antigens for 9 h prior to the addition of rested effectors at an E : T ratio of 30 : 1. An asterisk indicates statistically significant lysis of target cells (>3 sem compared with the corresponding uninfected, non-antigen-pulsed control). These graphs represent a single experiment repeated in three animals.

Fig. 7

Lipid stimulation of PBMCs from immune horses induces IFN-γ, but not IL-4. Real-time PCR is shown for IFN-γ and IL-4 following 20 h stimulation with medium alone, live R. equi bacteria, unfractionated R. equi lipid or R. equi cell-wall lipids TMM, CL and TDM. IFN-γ and IL-4 have been normalized against GAPDH. Following antigen stimulation, all animals (horses #H13, H14 and H68) showed 100–1000-fold increases in the transcription of IFN-γ. These graphs represent a single experiment repeated in three animals.