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. 2011 Apr 15;186(8):4771-81.
doi: 10.4049/jimmunol.1000921. Epub 2011 Mar 9.

Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire

Affiliations

Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire

Mélanie Dieudé et al. J Immunol. .

Abstract

Cardiolipin (CL), a major phospholipid in bacterial cell walls, is sequestered from the immune system in mammalian mitochondria and is, therefore, a potential danger signal. Based on growing evidence that phospholipids constitute natural ligands for CD1 and that CD1d-restricted T cells recognize phospholipids, we hypothesized that CD1d binds and presents CL and that T cells in the normal immune repertoire respond to CL in a CD1d-restricted manner. We determined the murine CD1d-CL crystal structure at 2.3 Å resolution and established through additional lipid loading experiments that CL, a tetra-acylated phospholipid, binds to murine CD1d with two alkyl chains buried inside the CD1d binding groove and the remaining two exposed into the solvent. We furthermore demonstrate the functional stimulatory activity of CL, showing that splenic and hepatic γδ T cells from healthy mice proliferate in vitro in response to mammalian or bacterial CL in a dose-dependent and CD1d-restricted manner, rapidly secreting the cytokines IFN-γ and RANTES. Finally, we show that hepatic γδ T cells are activated in vivo by CD1d-bearing dendritic cells that have been pulsed with CL, but not phosphatidylcholine. Together, these findings demonstrate that CD1d is able to bind and present CL to a subset of CL-responsive γδ T cells that exist in the spleen and liver of healthy mice and suggest that these cells could play a role in host responses to bacterial lipids and, potentially, self-CL. We propose that CL-responsive γδ T cells play a role in immune surveillance during infection and tissue injury.

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Figures

Figure 1
Figure 1. CL binds to mCD1d
(a) Synthetic tetramyristoyl CL (TM-CL) (14:0) and native bovine heart CL can bind to mCD1d, as detected by native IEF gel electrophoresis. Analytical gel (left panel): mCD1d (negative control) was incubated with bovine heart phosphatidylcholine (PC) (net charge 0), bovine heart cardiolipin (CL) (net charge −2), hydrogenated bovine heart CL (hCL) (net charge −2), or TM-CL (net charge −2) for 16 hr. Removal of the third (monolysoCL) and fourth (dilysoCL) alkyl chain from CL increases loading efficiency (middle panel). Preparative gel (right panel): mCD1d-TM-CL complexes can be fully separated from mCD1d molecules by anion exchange chromatography (purified CD1d/CL). A positive control is shown, where mCD1d was loaded with di-sulfatide (a nonphospholipid ligand with a net negative charge of −2). The preparative gel depicts the large-scale TM-CL loading and purification of mCD1d-TM-CL used for crystallographic studies. The data in panel a are representative of two to four independent experiments. (b) Chemical structure of the variants of CL used in the binding studies. Interestingly, while bovine heart CL, TM-CL, monolysoCL, and dilysoCL bind to mCD1d, hCL fails to bind to mCD1d under these conditions. This is likely due to reduced solubility of hCL during loading, as a result of the long and fully saturated C18 acyl chains. Note the faint upper double bands in the negative control lane (left panel), which are likely the result of endogenously bound charged lipids.
Figure 2
Figure 2. Schematic representation of the mCD1d-CL complex
(a) Tetra-myristoyl CL (TM-CL) (yellow) is bound in the hydrophobic binding groove between the α1 and α2 helices of the mCD1d heavy chain (grey), which non-covalently associates with β2-microblobulin (β2M, grey). Two N-linked glycosylation sites (N42 and N165) carry well-ordered carbohydrates (green sticks). A spacer lipid (C16, cyan) is present in the binding groove to complement the shorter C14-alkyl chains of the synthetic CL. (b) The 2F0−Fc electron density map is contoured at 1σ and shown as a blue mesh around the CL ligand. The third and fourth acyl chains, as well as the connecting glycerol, are not ordered in the crystal structure, and only 11 carbons of the myristoyl chain that binds in the A′ pocket are ordered. (c) Hydrogen-bond interactions between CD1d residues (grey) and the polar moieties of CL (yellow) are represented. In addition, two water molecules also hydrogen-bond with the two phosphate groups of CL. (d) Comparison between the presentation of TM-CL (yellow) and α-galactosylceramide (α-GalCer, cyan) by mCD1d. (e) Comparison between the presentation of CL (yellow) and sulfatide (green) by mCD1d. The central phosphate-glycerol-phosphate moiety of CL is located farther above the binding groove as compared to the more intimate binding of α-GalCer (the predominant invariant NKT cell Ag). However, the polar groups of both CL and sulfatide occupy similar positions. Notably, the terminal phosphate of CL is in a similar position as the sulfate of sulfatide. (f) Binding comparison between TM-CL (yellow sticks) and PIM2 (thin green sticks), and (g) between TM-CL and PC (thin cyan sticks). Protein backbone is colored accordingly and side chains are shown for R79 and Asp80 for orientation. Surface representation, with electrostatic potential (red, electronegative, and blue, electropositive, contoured from −30 to +30 kT/e), is shown in panels d and e. Several aa residues, which are involved either in the shaping of the mCD1d-binding grooves (b, d, e) or in polar interactions with CL (c), are depicted using the single letter aa code with the relevant residue number. Individual atoms are colored as follows: carbon, yellow, green or cyan; oxygen, red; nitrogen, blue; phosphorus, purple; and sulfur, orange.
Figure 3
Figure 3. Murine splenic and hepatic T cells proliferate in response to CL in a dose-dependent manner and express IFN-γ
Purified T cells from spleen (left panels) or liver (right panels) of C57BL/6 mice were incubated with vehicle or phospholipid (cardiolipin [CL], hydrogenated CL [hCL], or phosphatidylcholine [PC]) in presence of APCs in vitro. Phospholipids were used at the following final concentrations: 0.015–0.2 μM (panel a); 0.2 μM (panel b); and 0.1 or 0.2 μM (panel c)]. (a) Proliferation was evaluated by BrdU incorporation during the last 24 h of treatment. The data show the mean values (OD450) ±SD of triplicate samples from one representative experiment of three independent experiments. Other controls (not shown) included con A and cell medium, which had OD450 (± SD) values of 1.194 (0.037) and 0.063 (0.004), respectively. (b and c) IFN-γ production by CD3+ T cells was evaluated by flow cytometry in treated T cells at 0 h, 24 h, or 48 h. Panel b shows the raw flow cytometry data from a representative experiment, while panel c shows the mean IFN-γ-positive T cells ±SD of three independent flow cytometry experiments (including the one in panel b).
Figure 4
Figure 4. CL-responsive T cells express γδ TCR
Purified T cells from spleen (left panels) or liver (right panels) of C57BL/6 mice were incubated with vehicle or phospholipid (CL, hCL, or PC) in presence of APCs in vitro. Phospholipids were used at the following final concentrations: 0.2 μM (panel a); and 0.1 or 0.2 μM (panel b). αβ (a) or γδ (b) TCR expression by CD3+ T cells was evaluated by flow cytometry in treated T cells at 24 h or 48 h, and cell numbers were established using True-Count beads. (c) IFN-γ production by γδ-expressing splenic and hepatic T cells was evaluated by flow cytometry in treated T cells at 0 h, 24 h, or 48 h. Panels a and b show the raw flow cytometry data from a representative experiment of three independent experiments, while panel c shows the mean IFN-γ-positive T cells ±SD of three independent flow cytometry experiments (including the one in panel b).
Figure 5
Figure 5. CL-responsive γδ T cell expansion is independent of αβ T cells
Purified T cells from spleen (left panels) or liver (right panels) of C57BL/6 (wild type) or TCRβ-deficient (TCRβ KO) mice were treated with vehicle or phospholipid (CL, hCL, or PC) in presence of APCs in vitro. Phospholipids were used at the following final concentrations: 0.2 μM (panels a and b); and 0.1 or 0.2 μM (panel c). (a) Proliferation was evaluated by BrdU incorporation during the last 24 h of treatment. The data show the mean values (OD450) ± SD of triplicate samples from one representative experiment of two independent experiments. (b) γδ TCR expression by CD3+ T cells was evaluated by flow cytometry in treated T cells at 24 h or 48 h, and cell numbers were established using True-Count beads. (c) IFN-γ production by γδ-expressing splenic and hepatic T cells was evaluated by flow cytometry in treated T cells at 0 h, 24 h, or 48 h. Panel b shows the raw flow cytometry data from a representative experiment of two independent experiments, while panel c shows the mean IFN-γ-positive T cells ±SD of two independent flow cytometry experiments (including the one in panel b).
Figure 6
Figure 6. γδ T cells respond to CL in a CD1d-restricted manner
(a and b) Purified hepatic T cells from C57BL/6 mice were incubated in vitro with 0.1 μM CL, or vehicle, and APCs for 48 h in the presence of different blocking Abs (anti-MHC I, anti-MHC II, or anti-CD1d). Relative expansion of the γδ TCR-expressing CD3+ cell population was evaluated by flow cytometry using True-Count beads. Raw flow cytometry data, shown in panel a, are represented graphically in panel b. (c and d) Purified hepatic T cells from either C57BL/6 (WT) or C57BL/6 CD1d−/− (CD1d KO) mice were incubated in vitro with 0.2 μM phospholipid (PC, hCL, or CL), or vehicle, and APCs (from either WT or CD1d KO mice) for 48 h. Relative expansion of the CD3+, γδ TCR+, IFN-γ+ cell population was evaluated by flow cytometry using TruCOUNT beads (panel c) or BrdU proliferation assay (panel d). Panels a and b show data from a representative experiment of three independent experiments, while panels c and d show data from a representative experiment of two independent experiments. Error bars in panels b and d indicate SD of the means of duplicate (panel b) or triplicate (panel d) samples.
Figure 7
Figure 7. CD1d-restricted γδ T cells produce RANTES and IFN-γ in response to CL
Purified hepatic T cells from C57BL/6 mice were incubated for 48 h in vitro with vehicle or 0.2 μM phospholipid (CL, hCL, or PC) in presence of APCs. (a) Supernatants of treated cells were tested for the presence of 22 cytokines and chemokines using an Ab array quantitated by densitometry. Values indicate the signal intensity (%) relative to the signal for the positive control (set at 100%) for each cell treatment. (b) IFN-γ or RANTES in supernatants of treated cells was quantified by ELISA. The data in panels a and b show the mean ± SD of two independent experiments. Panels a and b show data from a representative experiment of two independent experiments. Error bars in panels a and b indicate SD of the means of duplicate samples.
Figure 8
Figure 8. γδ T cells are activated by CL in vivo
C57BL/6 mice, which had received 1 mg BrdU i.p., were injected i.v. with 5 × 105 100 μg/ml phospholipid (CL or PC) or vehicle treated BMDCs. (a) BrdU (left) and BrdU+ (right) populations of CD3+, γδ TCR+ liver mononuclear cells were analyzed for CD25 expression (open histograms) compared to vehicle treated controls (shaded histograms) at 24 h. Histograms are representative plots of 2 independent experiments of 2–4 mice per group. (b) Scatter plot of CD25 MFI of 2–4 mice per group 24 h post injection with phospholipid pulsed BMDCs from a minimum of 2 independent experiments. A statistically significant difference (p < 0.001 indicated with *) was found for CL, compared to PC pulsed BMDCs, using the equal variance Student t test.

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