Structural features of the acyl chain determine self-phospholipid antigen recognition by a CD1d-restricted invariant NKT (iNKT) cell

Abstract

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

Fig. 1. Bioactivity of methanol soluble lipids separated by two-dimensional thin layer chromatography

Fraction 7, isolated by thin layer chromatography from the methanol (MeOH) fraction of RMA-S cellular lipids, was recognized by the 24.8.A iNKT cell hybridoma using the plate-bound CD1d assay. Fraction 7, the original MeOH fraction, or PBS, were diluted serially and incubated with plate-bound CD1d. The presence of IL-2 was determined by ELISA and the data represent the mean absorbance (405 nm) detected in 24.8.A iNKT cell hybridoma supernatants from three replicate wells. Error bars show S.D.

Fig. 2. Mass spectrometry of bioactive fraction 7

a, analysis of the m/z 716 parent ion by CID tandem mass spectrometry produced two major fragments, m/z 255 and m/z 281, consistent with the presence of 16:0 and 18:1 acyl chains. b, at higher energy, additional fragments were observed at m/z 140 and m/z 196, which were consistent with the presence of the phosphoethanolamine moiety (see text for detail). c, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The schematic diagram of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0 –18:1[cis] PE) with its two acyl chains (one 16:0 and one 18:1[cis]) and polar phosphoethanolamine headgroup. The arrows correspond to fragments observed in the CID mass spectra. The structure of the acyl chains and the position of the double bond represent the typical form of 16:0 –18:1 PE, and were not deduced by MS analysis.

Fig. 3. Recognition of synthetic palmitoyl-oleoyl PE by the 24.8.A iNKT cell hybridoma

Synthetic palmitoyl-oleoyl PE (16:0 –18: 1[cis] PE) was recognized by the 24.8.A iNKT cell hybridoma and its recognition was specifically blocked by antibodies to CD1d, but not by antibodies to MHC I or MHC II. The 16:0 –18:1[cis] PE was diluted serially and incubated with plate-bound CD1d in the presence of antibody. The data represent the mean pg/ml of IL-2 detected in 24.8.A iNKT cell hybridoma supernatants from three replicate wells. Error bars show S.D.

Fig. 4. The 24.8.A iNKT cell hybridoma recognizes microbial, mammalian, and plant PE

Naturally occurring PEs, including PE from E. coli, mammalian tissue, and plants were all recognized by the 24.8.A iNKT cell hybridoma. Phospholipids (PL), or the CHCl₃ control, were diluted serially in PBS and incubated with plate-bound CD1d. The data represent the mean pg/ml IL-2 detected in 24.8.A iNKT cell hybridoma supernatants from three replicate wells. Plant PE was recognized most strongly, followed by bovine brain and egg PEs, heart PE, and liver PE and E. coli PE. The degree of recognition correlated with the percentage of 18:1 + 18:2 + 18:3 acyl chains for the PEs (indicated in parentheses for each source of lipid) for which this information is known.

Fig. 5. Fine specificity of the 24.8.A iNKT cell hybridoma

Phospholipids (PL), or the CHCl₃ control, were diluted serially in PBS and incubated with plate-bound CD1d. All phospholipids shown are synthetic forms of PE. 24.8.A iNKT cell hybridoma cells (10⁵/well) were incubated for 18 –24 h in the CD1d/phospholipid-coated wells. The data represent the mean pg/ml IL-2 detected in 24.8.A iNKT cell hybridoma supernatants from three replicate wells and are representative of two independent experiments. a, structure of synthetic 1,2-diacyl-sn-glycero-3-phosphoeth-anolamines: 1) di-18:0 PE; 2) di-18:1[trans] PE; 3) di-18:1[cis] PE; and 4) di-18:2[cis] PE. b, a series of synthetic 1,2-diacyl-sn-glycero-3-phosphoethanolamines with saturated (di-n:0) acyl chains were examined for their recognition by the 24.8.A iNKT cell hybridoma. c, synthetic PEs containing acyl chains with a single double bond (monounsaturated) were recognized significantly better than PEs with saturated acyl chains of the same length. For example, neither di-18:0 PE nor di-16:0 PE were recognized; in contrast, both di-18:1[cis] PE and di-16:1[cis] PE activated the 24.8 A iNKT cell hybridoma. d, the introduction of double bonds into the acyl chains modulates antigen recgonition by the 24.8 A iNKT cell hybridoma. For acyl chains containing 18 carbons, the sequential introduction of double bonds conferred recognition upon the PE. In this series, di-18:1[cis] PE was recgonized best. Introduction of a third double bond (di-18:3[cis] PE) in each acyl chain did not improve activation of the 24.8.A iNKT cell hybridoma. e, a single double bond in one of the two acyl chains is sufficient to confer recognition. Synthetic PEs containing mixed acyl chains were recognized if one chain was monounsaturated. For example, (16:0-18:1[cis]) PE and (18:0-18:1[cis]) PE were recognized, but di-16:0 PE and di-18:0 PE were not. f, the stereochemistry of the double bond affects recognition. The cis, but not the trans, isomer of di-18:1 PE was recognized by the 24.8.A iNKT cell hybridoma.

Fig. 6. Binding specificity of mCD1d for unsaturated PE

Phospholipids (PL), or the CHCl₃ control, in Me₂SO were diluted serially in assay buffer and incubated with mCD1d. The samples were then added to biotinylated di-18:0 PE bound to streptavidin-coated plates and mCD1d binding was detected with an alkaline phosphatase-conjugated goat anti-mouse IgG. All phospholipids shown are synthetic forms of PE. The data represent the mean OD₄₀₅ values of duplicates and are representative of two independent experiments.