Purification and Chemical Characterization of a Novel M. tuberculosis Lipid Antigen.
To identify the antigenic lipids, a more sophisticated purification was used. First, a pool of lipids was generated by extraction of mycobacterial H37Rv cells with a mixture of chloroform/methanol (1:1, vol/vol). Hydrophilic compounds were removed from this pool by water/chloroform partition and the lipids were then allowed to precipitate in acetone, a solvent known to precipitate phospholipids. An acetone-soluble phase and an acetone-insoluble phase were obtained, indicated as fractions 1 and 2, respectively (Fig. S1). CD8+ αβ T cell clones reacted to the acetone-soluble phase (unpublished data). To gain insights into the molecules present, this latter fraction (fraction 1) was analyzed by MALDI-Tof-MS in negative mode.
The mass spectrum revealed peaks assigned to deprotonated molecular ions [M-H]−
typical of phosphatidyl-myo
-inositol (m/z 851) and PIM (Ac3
at m/z 1,413 and Ac3
at m/z 2,061; A). Furthermore, three families of SGL differing in their acylation degree and fatty acyl appendage structure were also detected. SGL is used to describe the global family of sulfoglycolipids, composed of α-α′-d
-trehalose-2′-sulfate core acylated by two to four fatty acids (, inset). In Acx
SGL, x refers to the total number of acyl groups, whatever the nature of the fatty acids, which could be either palmitic, stearic, hydroxyphthioceranoic, or phthioceranoic acids. SGL were initally described as sulfolipids (37
) acylated by three or four fatty acids. In the high mass range, a set of peaks is dominated by the deprotonated molecular ions [M-H]−
at m/z 2,429 and 2,457, typifying Ac4
SGL with three hydroxyphthioceranoic acid residues and either one palmitic or one stearic acid residue. In the middle mass range, a set of peaks with the major ion species at m/z 1,867 correspond to Ac3
SGL containing two hydroxyphthioceranoic acids and one stearic or one palmitic acid residue. Finally, in the low mass range, a third set of peaks was observed to be dominated by the deprotonated molecular ions at m/z 1,249 and 1,277 typifying Ac2
SGL with one hydroxyphthioceranoic acid and either one palmitic or stearic acid residue, respectively.
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 (more ...)
The acetone-soluble phase was further fractionated on silicic acid column irrigated by chloroform containing increasing amounts of methanol (Fig. S1). CD8+
T cell clones were stimulated by fraction 7 and eluted with a mixture of chloroform containing 20% of methanol. The different fractions were analyzed by negative MALDI-Tof
-MS. Mass spectrum of the stimulatory fraction 7 ( B) is complex and dominated by peaks at m/z 1,249 and 1,277 assigned to Ac2
SGL. The acyl appendages were unambiguously assigned to hydroxyphthioceranoic and palmitic or stearic acids by gas chromatography, gas chromatography-mass spectrometry, and electronic impact-mass spectrometry analysis (unpublished data). The acyl appendages of Ac2
SGL (mol wt 1,250) correspond to palmitic acid and hydroxyphthioceranoic acid with m
= 14 and n
= 7, whereas Ac2
SGL of mol wt 1,278 contains stearic acid and hydroxyphthioceranoic acid with m
= 14 and n
= 7 or palmitic acid residue and hydroxyphthioceranoic acid with m
= 16 and n
= 7 (see Table S1 for their assignment, available at http://www.jem.org/cgi/content/full/jem.20031097/DC1
). Besides these two major Ac2
SGL acyl forms, the mass spectrum shows other peaks distant of 14 mass units, assigned to Ac2
SGL acyl forms differing by the chain length of the hydroxyphthioceranoic acid (Table S1). In summary, all the peaks of the MALDI-Tof
mass spectrum of fraction 7 were assigned to Ac2
SGL acyl forms. The two fractions eluted with 10% of methanol (fractions 5 and 6) were found by MALDI-Tof
-MS to contain Ac3
SGL and Ac4
SGL, respectively (unpublished data). These fractions did not stimulate Z4B27 and Z4A26 T cell clones (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20031097/DC1
Fraction 7 was further purified by reverse phase chromatography and five new subfractions (7.1–7.5) were obtained. Their purity was controlled by silicic acid TLC ( A) and negative MALDI-Tof-MS ( B). Fraction 7.1 is a mixture of several compounds, none of them corresponding to Ac2SGL. In contrast, TLC shows one spot for fraction 7.3 and 7.4 and two spots for fraction 7.5. MALDI-Tof mass spectra of the three fractions highlighted the presence of Ac2SGL typified by the previously assigned [M-H]− ions at m/z 1,249 and 1,277. As expected, this reverse phase chromatography allowed the separation of the Ac2SGL acyl forms according to the chain length of their hydroxyphthioceranoic acid. Indeed, fraction 7.3 is characterized by Ac2SGL containing hydroxyphthioceranoic acid with shorter chains (m/z 1,039, 1,067, 1,123, 1,151, 1,165, 1,193, and 1,249) than those of Ac2SGL fractions 7.4 (m/z 1,249, 1,277, and 1,291) and 7.5 (m/z 1,277, 1,305, 1,319, 1,333, and 1,361). In summary, the sub-fractions 7.3, 7.4, and 7.5 are composed of Ac2SGL acyl forms that differ by the hydrophthioceranoic acid chain length (Table S1).
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. (more ...)
The capacity of the different subfractions to stimulate the CD8+ T cell clones was investigated by measuring INF-γ production. As shown in C, the activity was restricted to the fractions 7.3, 7.4, and 7.5. Taken together, these data demonstrated that Ac2SGL acyl forms are the immunogenic compounds of the CD8+ T cell clones.
To support the antigen structure and the contribution of the sulfate group to immunogenicity, the sulfate group was removed under mild acidic conditions. Desulfatation of Ac2SGL completely abrogated IFN-γ production ( C), supporting the previous assignment of the antigenic activity to Ac2SGL, but also revealing that immunogenic activity of Ac2SGL requires the sulfate residue.
The immunological importance of the sulfate group prompted mass spectrometry and NMR studies aimed to precisely assign the position of sulfate as well as the acyl appendages. We performed a MALDI-MS analysis of Ac2SGL in positive mode using a QSTARPULSAR mass spectrometer (unpublished data). The mass spectrum was dominated by two peaks at m/z 1,295 and 1,323, which were assigned to cationized molecular ions of the sodiated Ac2SGL (M-H+2Na)+. MS/MS spectra of the precursor ions 1,295 and 1,323 showed loss of HOSO3− Na+ for both compounds, in agreement with the presence of a sulfate group. Furthermore, loss of sulfoanhydroGlc and sulfoGlc was detected from both precursor ions, revealing that the two fatty acyl appendages including hydroxyphthioceranoic and palmitic or stearic acids were located on the Glcp moiety, which did not bear the sulfate group (Glcp-I; unpublished data).
This result was in agreement with 1D and 2D 1
H NMR experiments. In the anomeric zone of the 1D 1
H spectrum (), four signals were observed, namely I1
, and II3
. The resonances I1
were assigned to the two anomeric protons of the α-α′-trehalose core. Starting from these protons, the complete spin systems of both Glcp
units could be determined from 1
H correlation spectroscopy and homonuclear Hartmann-Hahn spectroscopy experiments (Table S2, available at http://www.jem.org/cgi/content/full/jem.20031097/DC1
). These data allowed assignment of the resonances II2
to H2 and H3 of the Glcp
-II, respectively. The downfield shifts of the H2 and H3 protons proved that the fatty acyl appendages (hydroxyphthioceranoic acid and palmitic or stearic acids) were located on C2 and C3 of Glcp
-II. In addition, from the comparative analysis of the proton chemical shifts (Table S2,
) of the native and peracetylated SGL, the sulfate residue was located on the C2 of the Glcp
-I. Indeed, variations in chemical shifts of >1 ppm for H3 and H4 indicated that these positions were not substituted in the native molecule. A very weak chemical shift variation was observed for H2 and H3 of Glp
-II (0.27 and 0.45 ppm, respectively) in agreement with the fact that fatty acyl appendages were located on C2 and C3. A high chemical shift variation was observed for H3 and H4 of Glp
-I (1.54 and 1.71 ppm, respectively) and H4 of Glp
-II (2.03 ppm) in agreement with the fact that these positions were not substituted in the native molecule. The sulfate was located on the C2 of Glp
-I as a small variation (0.44 ppm) was observed (, inset). Moreover, the exactness of the proposed structure was definitely proven by comparison of proton chemical shifts of natural Ac2
SGL with a synthetic molecule, i.e., 2,3-dipalmitoyl-2′-sulfate-α-α′-d
-trehalose (not depicted; the synthesis of this molecule will be reported elsewhere).
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, (more ...)
To confirm that Ac2SGL is the recognized antigen, this synthetic molecule was also used to stimulate T cells. A significant stimulation of Ac2SGL-specific T cells was observed ( A), which did not occur when sulfatide-specific T cells were used ( B). The synthetic molecule stimulated the specific T cells with a lower efficacy than the natural Ac2SGL, likely due to the lack of the hydroxyphthioceranoic acyl chain.
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 (more ...)
In conclusion, 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL) is the antigen recognized by CD8+ T cell clones ( C).
Requirements for Presentation of Ac2SGL to T Cells.
To determine the restricting element of Ac2SGL we used APCs from different sources to stimulate CD8+ cells. Autologous CD1+ APCs but not PBMCs nor EBV-transformed lymphoblastoid cell lines presented Ac2SGL to specific T cells ( A). Heterologous CD1+ APCs from multiple donors were almost equally efficient in presenting this ligand, suggesting that the response was restricted by a nonpolymorphic molecule ( B). Furthermore, the promyelocytic HL-60, the histiocytic U-937, and the macrophage MonoMac6 cell lines did not present Ac2SGL. These findings exclude that presentation of this antigen is a property of all cells belonging to the monocytic lineage and suggest that a nonubiquitous presenting molecule is involved. Because CD1 molecules are nonpolymorphic and nonubiquitous we explored the possibility that Ac2SGL was presented by these molecules. Ac2SGL is presented by CD1b as shown by inhibition of T cell activation by anti-CD1 mAbs ( C) and efficient presentation by CD1b-transfected but not mock-transfected THP-1 cells ( D).
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) (more ...)
An important question is in which cellular compartment is the Ac2
SGL ligand loaded on CD1b? The current consensus is that lipids with long acyl chains are loaded in late endosomal compartments in which low pH and partial unfolding of CD1b molecules facilitate insertion of long fatty acids in the CD1 lipid-binding pockets (20
). As Ac2
SGL contains the C32 long hydroxyphthioceranoic acid, we investigated whether intracellular internalization in APCs is necessary for presentation. Pulsing of THP-1 CD1b cells with Ac2
SGL at 4°C or in the presence of monensin, two conditions that prevent internalization of surface-bound molecules, prevented T cell activation ( A), suggesting that internalization is required. This was confirmed by lack of presentation when CD1+
APCs were first fixed and then pulsed with the antigen ( C). On the contrary, the same APCs were fully capable of activating specific T cells when they were first pulsed and then fixed. To further test the requirements for internalization, CD1+
APCs were pulsed in the presence of chloroquine, an agent that blocks endosomal acidification. In addition, this treatment prevented T cell activation ( A), further suggesting that presentation occurs only when CD1b and Ac2
SGL converge in an endosomal compartment undergoing acidification.
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) (more ...)
All of these treatments did not abolish presentation of another glycolipid, sulfatide, to specific T cells, thus excluding possible artifacts due to APC treatment ().