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. 2014 Nov 1;193(9):4580-9.
doi: 10.4049/jimmunol.1400699. Epub 2014 Sep 26.

Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease

Igor Maricic  1 , Enrico Girardi  2 , Dirk M Zajonc  2 , Vipin Kumar  3
Affiliations

Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease

Igor Maricic et al. J Immunol. .

Abstract

Lipids presented by the MHC class I-like molecule, CD1d, are recognized by NK T (NKT) cells, which can be broadly categorized into two subsets. The well-characterized type I NKT cells express a semi-invariant TCR and can recognize both α- and β-linked glycolipids, whereas type II NKT cells are less well studied, express a relatively diverse TCR repertoire, and recognize β-linked lipids. Recent structural studies have shown a distinct mode of recognition of a self-glycolipid sulfatide bound to CD1d by a type II NKT TCR. To further characterize Ag recognition by these cells, we have used the structural data and screened other small molecules able to bind to CD1d and activate type II NKT cells. Using plate-bound CD1d and APC-based Ag presentation assay, we found that phospholipids such as lysophosphatidylcholine (LPC) can stimulate the sulfatide-reactive type II NKT hybridoma Hy19.3 in a CD1d-dependent manner. Using plasmon resonance studies, we found that this type II NKT TCR binds with CD1d-bound LPC with micromolar affinities similar to that for sulfatide. Furthermore, LPC-mediated activation of type II NKT cells leads to anergy induction in type I NKT cells and affords protection from Con A-induced hepatitis. These data indicate that, in addition to self-glycolipids, self-lysophospholipids are also recognized by type II NKT cells. Because lysophospholipids are involved during inflammation, our findings have implications for not only understanding activation of type II NKT cells in physiological settings, but also for the development of immune intervention in inflammatory diseases.

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Figures

Figure 1
Figure 1
Detailed chemical structures of different lysophospholipids as well as glycolipids used in this study
Figure 2
Figure 2. Stimulation of a type II NKT cell Hy19.3 by self-lysophospholipids in an APC-free CD1d-coated antigen presentation assay
Plates were coated with the recombinant CD1d protein in PBS and incubated at 4°C overnight. Next day plates were washed and loaded with indicated lipids for 5–6 hr at 37°C. IL-2 release at an optimum concentration (5 μg/ml) following incubation with 5 × 104 cells/well of a type II NKT cell Hy19.3 (A) or a type I NKT cell Hy1.2 (B) is shown. A typical dose titration curve of stimulation of Hy19.3 with various concentrations (0.6 – 20 μg/ml) of LPC is shown in Figure 2C. There was no significant amount of IL-2 secretion (0.01–0.03 ng/ml) with 1 μg/well of plate coated CD1d only in these assays. These data are representative of 4 different experiments.
Figure 3
Figure 3. CD1d-dependent stimulation of a sulfatide-reactive type II NKT cell by self-lysophospholipids
(A) A type II NKT Hy19.3 or a type I NKT Hy1.2 were stimulated in the presence of APCs (irradiated splenocytes) from either the wild-type C57BL/6 (CD1d+/+) or CD1d-deficient (CD1d−/−) mice in the presence of different concentration of lipids (0.6 – 20 μg/ml). IL-2 release at optimum concentration (5 μg/ml) of indicated lipids is shown. (B) A typical dose titration of Hy19.3 stimulation in the presence of APC and LPC (0.6 – 20 μg/ml) is shown. These data are representative of at least 3 independent experiments.
Figure 4
Figure 4. Binding of a type II NKT TCR to mCD1d/LPC complexes
(A) Sensograms showing mCD1d-LPC complexes binding to the Hy19.3 TCR. The association and dissociation constants measured show a slightly higher binding affinity of this ligand for the TCR, compared to mCD1d-LSF complexes. (B) Affinity measurements for the lysophospholipid variants measured for this study. Average and SEM of two independent measurements are reported.
Figure 5
Figure 5. Recognition of different isoforms of LPC and LSM by a sulfatide-reactive type II NKT cell but not other type II NKT hybridomas
A sulfatide-reactive type II NKT Hy19.3 as well as other non-sulfatide-reactive type II NKT hybridomas were used in a typical IL-2 release assay in the presence of APC and indicated lipids. IL-2 release at an optimum concentration (5 μg/ml) of lipids is shown. All other hybridomas showed IL-2 release (ranges from 0.7 – 1.3 ng/ml) in response to the plate-bound anti-CD3 stimulation. These data are representative of two independent experiments.
Figure 6
Figure 6. Modification of residues in CD1d molecules around the A′ but not F′ pocket inhibits recognition of LPC by a type II NKT cell hybridoma
(A) In a CD1d coated plate assay Hy19.3 was stimulated in the presence of wild-type CD1d or mutant CD1d molecules with modifications in residues as indicated. IL-2 release at an optimum concentration of either 2 μg/ml of LSF or 5 μg/ml of LPC is shown. These data are representative of 3 independent experiments. (B) A proliferative response of spleen cells from naïve wild-type C57BL/6 (CD1d+/+) or CD1d-deficient (CD1d−/−) mice in response to a typical CD1d coated plate assay as above in the presence of an optimum concentration of LPC (5 μg/ml) is shown. Stimulation index was calculated by dividing CPM in the presence vs. absence of the lipid. These data are representative of 2 independent experiments.
Figure 7
Figure 7. Induction of anergy in type I NKT cells following LPC administration in C57BL/6 mice
(A) Flow cytometric analysis of splenocytes from groups of C57BL/6 mice (n = 3–5) treated i.p. with PBS/vehicle or indicated lipids, LPC (C16:0), LPC (C18:0) or LSF, at 100 μg/mouse. An in vivo expansion of type I NKT cells (αGalCer/CD1d-tetramer+) was measured in splenocytes following administration with αGalCer (2 μg) using two-color staining with αGalCer/CD1d-tetramer and anti-TCRβand flow cytometry. Numbers indicate percentage tetramer+ positive cells in total spleen lymphocytes. (B) A summary of the data from 2 independent experiments indicating LPC-mediated inhibition of type I NKT cell expansion in BL/6 mice (n = 3–5) is shown. Average percentage of αGalCer/CD1d-tetramer positive cells for each group from figure A (mean +/− SEM) is shown. *P value <0.05, **P values <0.01 (C) Inhibition of type I NKT cells in response to an in vitro challenge with αGalCer in splenocytes isolated from the groups (n=3) of control (PBS/vehicle) or different lipids-injected mice. Proliferation following a 90 hr culture in the presence of αGalCer among indicated groups is shown as 3H Thymidine incorporation. The frequency of αGalCer-tetramer+ cells in the beginning of the culture was similar in all the groups. ***P values <0.001
Figure 8
Figure 8. Inhibition of ConA-induced hepatitis in mice treated with LPC
(A) Serum ALT levels from groups of BL/6, Jα18−/− or CD1d−/− mice immunized i.v. with ConA (10 mg/kg of body weight) and treated i.p. with PBS or indicated LPC C:16 (100 μg/mouse) or naïve control are shown. ***P value <0.001, ****P value <0.0001. (B) Inhibition of histological severity of ConA-induced liver damage following treatment with LPC as in A. Groups of BL/6, Jα18−/− or CD1d−/− mice were sacrificed 24 hours after the injection and livers were removed and fixed in 10% formalin solution until H & E staining. H & E stained histological sections at 100 x magnification are shown. (C) A fold increase in serum cytokines in Con A or Con A plus LPC-treated BL/6, Jα18−/− or CD1d−/− mice as in A is shown. Basal values (pg/ml) for IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 were 11.0, 2.6, 2.9, 2.3, and 2.1 respectively.
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References

    1. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol. 2007;25:297–336. - PubMed
    1. Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol. 2004;22:817–890. - PubMed
    1. Godfrey DI, Stankovic S, Baxter AG. Raising the NKT cell family. Nat Immunol. 2010;11:197–206. - PubMed
    1. Arrenberg P, Halder R, Kumar V. Cross-regulation between distinct natural killer T cell subsets influences immune response to self and foreign antigens. J Cell Physiol. 2009;218:246–250. - PMC - PubMed
    1. Kronenberg M, Rudensky A. Regulation of immunity by self-reactive T cells. Nature. 2005;435:598–604. - PubMed

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