Human T cell response to CD1a and contact dermatitis allergens in botanical extracts and commercial skin care products

Abstract

During industrialization, humans have been exposed to increasing numbers of foreign chemicals. Failure of the immune system to tolerate drugs, cosmetics, and other skin products causes allergic contact dermatitis, a T cell-mediated disease with rising prevalence. Models of αβ T cell response emphasize T cell receptor (TCR) contact with peptide-MHC complexes, but this model cannot readily explain activation by most contact dermatitis allergens, which are nonpeptidic molecules. We tested whether CD1a, an abundant MHC I-like protein in human skin, mediates contact allergen recognition. Using CD1a-autoreactive human αβ T cell clones to screen clinically important allergens present in skin patch testing kits, we identified responses to balsam of Peru, a tree oil widely used in cosmetics and toothpaste. Additional purification identified benzyl benzoate and benzyl cinnamate as antigenic compounds within balsam of Peru. Screening of structurally related compounds revealed additional stimulants of CD1a-restricted T cells, including farnesol and coenzyme Q2. Certain general chemical features controlled response: small size, extreme hydrophobicity, and chemical constraint from rings and unsaturations. Unlike lipid antigens that protrude to form epitopes and contact TCRs, the small size of farnesol allows sequestration deeply within CD1a, where it displaces self-lipids and unmasks the CD1a surface. These studies identify molecular connections between CD1a and hypersensitivity to consumer products, defining a mechanism that could plausibly explain the many known T cell responses to oily substances.

Figure 1.. Balsam of Peru activates T cells via a CD1a-dependent, APC-independent mechanism.

T cell lines with CD1a autoreactivity (BC2, Bgp) or foreign antigen reactivity (CD8–2) were tested for activation to lipids using IFN-γ ELISA in cellular assays with CD1a-transfected K562 cells (K562-CD1a) or mock transfected K562 cells (K562-mock) (a, b, e) or on streptavidin plates coated with biotinylated CD1 proteins (c, d). Data are representative of three or more experiments each with the mean of triplicate measurements shown with standard deviation. The significance of lipid concentration on IFN-gamma release was tested by one-way ANOVA (panel a, c). Relevant pairwise comparisons were tested using Welch’s t-test (panel b). Post-hoc comparison of marginal means after adjustment by the Sidak method was used to group treatments at the specified significance level following a significant result by two-way ANOVA (panel d). Post-hoc comparison by least-squares means after adjustment by the Sidak method was used to group treatments with non-overlapping marginal means and 95% confidence levels into a, b or c at the specified significance level following a significant result by two-way ANOVA (panel e).

Figure 2.. Chemical analysis of antigenic substances in balsam of Peru.

a. Normal phase silica TLC plate resolves balsam of Peru oil (BPO), crude balsam of Peru (BP), synthetic benzyl cinnamate (BC) and synthetic benzyl benzoate (BB). b. Structures of benzyl cinnamate and benzyl benzoate are shown with the expected mass of sodium adducts [M+Na]⁺, which were detected in positive-mode nanoelectrospray ionization mass spectrometry. c. T cell clones that are autoreactive to CD1a (BC2) or foreign antigen (CD8–2) were tested for response to antigens (μg/ml) or sphingomyelin (sphingomy) by IFN-γ ELISA in cellular (e) or CD1a-coated plate (c, d) assays. Data are representative of three or more experiments, each shown as the mean of triplicate samples +/− standard deviation. The significance of lipid concentration on IFN-γ release was tested by one-way ANOVA (panel c). The significance of benzyl cinnamate and benzyl benzoate concentration on IFN-γ release and of the effects of CD1b or CD8–2 T-cells were tested by two-way ANOVA (panels d,e).

Figure 3.. T cell responses to chemically diverse oily substances.

a. Using phosphatidylcholine as an example, CD1 ligands are often composed of head groups and lipid anchors, but (b) recently identified CD1a presented antigens are oils. c. BC2 T cells were tested for cytokine release in response to small hydrophobic molecules pulsed on plate-bound CD1a pre-treated with acidic citrate buffer to strip ligands (31). Tested compounds are classified into groups based on the presence of branched chain unsaturated lipids structurally related to squalene (c), ringed lipids structurally related to benzyl cinnamate (d) or molecules that show branched, polyunsaturated and ringed structure, such as coenzyme Q2 (e). Results of triplicate analyses are shown as mean +/− standard deviation with each compound tested 2 or more times. Post-hoc comparison by marginal means of the interaction term between lipid and concentration after adjustment by the Sidak method was used to group treatments by non-overlapping 95% confidence levels at the specified significance level following a significant result by two-way ANOVA. f. The size of all tested antigens is shown based on the number of carbon atoms (C) or mass (atomic mass units, abbreviated as u), as compared to the volume of the CD1a cleft, which has been measured at 1650 ų, and can accommodate ~ 36 methylene units (C36) (19, 40). g. Purified T cells (CD4⁻ and CD4⁺) were incubated overnight with plate-bound CD1a, either mock treated or pre-treated with the indicated antigens (50μg/ml). Real-time PCR of IFN-γ mRNA relative to β-actin.* P<0.05 Student’s t-test 2-sided, antigen-treated compared to mock-treated CD1a.

Figure 4.. CD1a-farnesol complexes.

a. IFN-γ release by BC2 T cells in response to CD1a-coated plates treated with farnesol was measured. * The significance of lipid concentration on IFN-γ release was assessed by marginal means with adjustment by the Sidak method after a significant result by ANOVA, treating experiments 1 and 2 as blocks. At the highest concentration of farnesol in both experiments, non-overlapping 95% confidence intervals were observed at p < 0.001 b. Affinity measurements (K_D) by surface plasmon resonance in response to the recombinant BC2 TCR binding biotinylated CD1a directly isolated from cells (CD1a-endo), CD1a pre-treated with farnesol (CD1a-farnesol) or CD1a treated with buffer (CD1a-mock). Positive mode HPLC-MS analysis of a farnesol standard (c) and eluents from farnesol-treated CD1a (d) demonstrated ions that matched the expected mass (m/z 205.195) of an indicated dehydration product with a retention time of 2.9 min. e-f. Lipid eluents from CD1a-endo and CD1a-farnesol were analyzed by positive normal phase HPLC-MS QToF mass spectrometry. Ion chromatograms were generated at the nominal mass values of diacylglycerol (DAG), phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylinositol (PI), which are shown as CX:Y, where X is the number of methylene units in the combined lipid chains and Y is the total number of unsaturations. g. Compound identifications were based on the unknown matching the retention time and mass of standards. Further, one compound in the PC, SM and PI families (shown in color) underwent collision-induced dissociation mass spectrometry analysis to generate the indicated diagnostic fragments.

Figure 5.. Crystal structure of CD1a-farnesol complexes.

a. Overview of the binary crystal structure of CD1a (grey)-farnesol (purple)/β2m(cyan). b. Molecular interactions of farnesol (purple) with the hydrophobic residues within CD1a binding cleft (grey surface). The side chains of the residues within 4 Å distance from the lipid are shown. A diagram of trans, trans farnesol with carbon numbering is shown. The A’ pole formed by V12-F70 interaction in the context of oleic acid-bound CD1a pocket (PDB ID: 4X6D) is highlighted in the inset. c-e. Superimposition of CD1a bound to farnesol and sphingomyelin (PDB ID: 4X6F, (35)) (c) lipopeptide (PDB ID: 1XZ0, (40)) (d) and urushiol (PDB ID: 5J1A, (30)) (e).