Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis

Abstract

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-alpha-alpha'-D-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon gamma, efficiently recognize M. tuberculosis-infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Figure 1.

Characterization of the T cell antigen. Negative MALDI-Tof mass spectra of (A) the “acetone-soluble” fraction (fraction 1) and (B) fraction 7 obtained after silicic acid column chromatography containing the stimulatory activity. See Table S1 for peak interpretation. Inset: Structural model of the M. tuberculosis SGL with R corresponding to hydroxyphthioceranoic, palmitic, or stearic acid. Ac₄SGL contains three hydroxyphthioceranoic acids and one palmitic or stearic acid, Ac₃SGL contains two hydroxyphthioceranoic acids and one palmitic or stearic acid, and Ac₂SGL contains one hydroxyphthioceranoic acid and one palmitic or stearic acid. MS peaks are labeled with mass values calculated using C = 12, H = 1, O = 16, P = 31, and S = 32.

Figure 2.

Identification of the active ligand present in fraction 7 as Ac₂SGL. (A) TLC analysis of the components present in fraction 7 after reverse chromatography purification revealed by orcinol. Numbers indicate the subfractions tested in the following panels. (B) Negative MALDI-Tof mass spectra of the subfractions 7.1, 7.3, 7.4, and 7.5. See Table S1 for peak interpretation. (C) Stimulation of the Z4B27 T cell clone by (•) subfraction 7.1 from A, (▵) subfraction 7.3, (⋄) subfraction 7.4, (□) subfraction 7.5, (▪) desulfatated Ac₂SGL, and (○) control active fraction 7 as measured by IFN-γ ELISA. Bars indicate SD.

Figure 3.

Structure determination of Ac₂SGL by 1D ¹H NMR spectrum analysis (δ ¹H: 0–7.8). The structure of Ac₂SGL is shown in the inset. The two glucose units are labeled I and II, as indicated in the inset. R corresponds to hydroxyphthioceranoic, palmitic, or stearic acid.

Figure 4.

Biological activity of a synthetic analogue and structure of the natural active molecule. (A) Activation of the Ac₂SGL-specific T cell clone Z4B27 using synthetic 2,3-dipalmitoyl-α-α'-d-trehalose-2′-sulfate (SGL analogue). Purified Ac₂SGL and synthetic Ac₂SGL analogue were used at 10 μg/ml. Release of IFN-γ was tested by ELISA. The results are representative of two experiments. Bars indicate SD. (B) Control with sulfatide-specific DS1C9b T cell clone. Sulfatide was used at 10 μg/ml. Bars indicate SD. (C) Structure of M. tuberculosis Ac₂SGL. The presented structure corresponds to one of the major Ac₂SGL acyl forms (mol wt 1,250).

Figure 5.

CD1b-restricted presentation of Ac₂SGL. (A) Autologous CD1⁺ APCs and not PBMCs or EBV-transformed lymphoblastoid cell lines present Ac₂SGL. (B) Presentation by CD1⁺ APCs isolated from different individuals and by cell lines of the monocytic lineage. (C) Inhibition of T cell activation by anti-CD1b mAbs. (D) Presentation by CD1b-transfected THP-1 cells. Release of IFN-γ by the Z4B27 T cell clone was measured by ELISA. White bars represent response in the absence of antigen. A second Ac₂SGL-specific and CD1b-restricted T cell clone gave similar results (unpublished data). Bars indicate SD.

Figure 6.

Requirements for presentation of Ac₂SGL. (A) Presentation of Ac₂SGL is inhibited when APCs are pulsed with Ac₂SGL (solid bars) at 4°C, or in the presence of monensin or chloroquine. White bars represent response in the absence of antigen. (C) APCs pulsed and then fixed present Ac₂SGL. (B and D) The same treatments did not affect presentation of sulfatide to the CD1b-restricted and sulfatide-specific DS1C9b T cell clone. (A and C) Release of IFN-γ by the Z4B27 T cell clone measured by ELISA. Similar results were obtained in three additional experiments and also with the Z4A26 T cell clone (unpublished data). Medium indicates response in the absence of treatment. Bars indicate SD.

Figure 7.

Ac₂SGL-specific T cells recognize and kill M. tuberculosis H37Rv-infected cells. CD1⁺ APCs were pulsed with Ac₂SGL or infected with virulent M. tuberculosis and used to stimulate the Z4B27 (A) or the Z4A26 (B) T cell clones. Released IFN-γ was detected by ELISA. The results are representative of four independent experiments. Bars indicate SD. (C) Ac₂SGL-specific T cells kill intracellular M. tuberculosis. CD1⁺ APCs were infected with virulent M. tuberculosis. Z4B27 T cells were added at different ratios as indicated. After 72 h of incubation, living intracellular bacteria were counted as described in Materials and Methods. Average CFU ± SEM calculated from four dilutions, each performed in duplicate are shown. Similar results were obtained in two additional experiments.

Figure 8.

Response of PBMCs to Ac₂SGL ex vivo and inhibition with anti-CD1 and anti-MHC mAbs. (A) PBMCs from healthy donors (PPD⁺ or PPD⁻) and from patients with pulmonary tuberculosis were cultured in the presence of autologous CD1⁺ APCs overnight pulsed with Ac₂SGL. Supernatants were harvested and IFN-γ content was measured by ELISA. Each dot presents an individual donor and the dashed line depicts the detection threshold of 15 pg/ml of this assay. Unstimulated samples served as controls and IFN-γ was regularly below the level of detection. (B) This graph summarizes the results ± SEM from three PPD⁺ healthy donors using fraction 7 (10 μg/ml) as antigen. IFN-γ production in the individual experiments was 473, 357, and 349 pg/ml.