Research ArticleSKIN INFLAMMATION

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

See allHide authors and affiliations

Science Immunology  03 Jan 2020:
Vol. 5, Issue 43, eaax5430
DOI: 10.1126/sciimmunol.aax5430
  • Fig. 1 Balsam of Peru activates T cells via a CD1a-dependent, APC-independent mechanism.

    (A to E) T cell lines with CD1a autoreactivity (BC2 and 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, and E) or on streptavidin plates coated with biotinylated CD1 proteins (C and D). Data are representative of three or more experiments each with the mean of triplicate measurements shown with SD. The significance of lipid concentration on IFN-γ release was tested by one-way ANOVA (A and C). Relevant pairwise comparisons were tested using Welch’s t test (B). Post hoc comparison of marginal means after adjustment by the Sidak method was used to group treatments at the specified significance level after a significant result by two-way ANOVA (D). Post hoc comparison by least squares means after adjustment by the Sidak method was used to group treatments with nonoverlapping marginal means and 95% confidence levels into a, b, or c at the specified significance level after a significant result by two-way ANOVA (E). IgG, immunoglobulin G.

  • Fig. 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 MS. (C to E) T cell clones that are autoreactive to CD1a (BC2) or foreign antigen (CD8-2) were tested for response to antigens (μg/ml) or SM (sphingomy) by IFN-γ ELISA in cellular (E) or CD1a-coated plate (C and D) assays. Data are representative of three or more experiments, each shown as the mean of triplicate samples ± SD. The significance of lipid concentration on IFN-γ release was tested by one-way ANOVA (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 (D and E).

  • Fig. 3 T cell responses to chemically diverse oily substances.

    (A) Using PC 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 pretreated 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, (D) ringed lipids structurally related to benzyl cinnamate, or (E) molecules that show branched, polyunsaturated, and ringed structures, such as coenzyme Q2. Results of triplicate analyses are shown as means ± SD with each compound tested two 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 nonoverlapping 95% confidence levels at the specified significance level after a significant result by two-way ANOVA. (F) The size of all tested antigens is shown on the basis of the number of carbon atoms (C) or mass [atomic mass units (u)], as compared with the volume of the CD1a cleft, which has been measured at 1650 Å3, 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 pretreated with the indicated antigens (50 μg/ml). Real-time PCR of IFN-γ mRNA relative to β-actin.*P < 0.05, two-sided Student’s t test, antigen-treated compared with mock-treated CD1a.

  • Fig. 4 CD1a-farnesol complexes.

    (A) IFN-γ release by BC2 T cells in response to CD1a-coated plates treated with farnesol was measured. Asterisk (*) indicates that 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, nonoverlapping 95% confidence intervals were observed at P < 0.001. (B) Affinity measurements (KD) by SPR in response to the recombinant BC2 TCR binding biotinylated CD1a directly isolated from cells (CD1a-endo), CD1a pretreated 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 and F) Lipid eluents from CD1a-endo and CD1a-farnesol were analyzed by positive normal-phase HPLC-QToF-MS. Ion chromatograms were generated at the nominal mass values of DAG, PC, SM, and 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 of the retention time and mass of standards. Further, one compound in the PC, SM, and PI families (shown in color) underwent collision-induced dissociation MS analysis to generate the indicated diagnostic fragments. RU, resonance units.

  • Fig. 5 Crystal structure of CD1a-farnesol complexes.

    (A) Overview of the binary crystal structure of CD1a (gray)–farnesol (purple)/β2m (cyan). (B) Molecular interactions of farnesol (purple) with the hydrophobic residues within CD1a binding cleft (gray surface). The side chains of the residues within a 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 [Protein Data Bank (PDB) ID: 4X6D] is highlighted in the inset. (C to E) Superimposition of CD1a bound to farnesol and SM [PDB ID: 4X6F (35)] (C), lipopeptide [PDB ID: 1XZ0 (40)] (D), and urushiol [PDB ID: 5J1A (30)] (E).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/43/eaax5430/DC1

    Fig. S1. Screening human T cells for responses to known contact allergens.

    Fig. S2. CD1a-dependent T cell response to small hydrophobic molecules.

    Fig. S3. Idebenone is recognized by CD1a-restricted T cell line BC2.

    Fig. S4. CD1a-dependent polyclonal T cell responses to contact allergens.

    Fig. S5. CD1a tetramer staining of CD1a-autoreactive T cell line.

    Fig. S6. Electron density for farnesol in CD1a-farnesol binary complex.

    Table S1. Supporting data CD1a-farnesol binary complex.

    Table S2. Van der Waals bonds between CD1a and farnesol.

    Table S3. Raw data sets for main figures (Excel spreadsheet).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Screening human T cells for responses to known contact allergens.
    • Fig. S2. CD1a-dependent T cell response to small hydrophobic molecules.
    • Fig. S3. Idebenone is recognized by CD1a-restricted T cell line BC2.
    • Fig. S4. CD1a-dependent polyclonal T cell responses to contact allergens.
    • Fig. S5. CD1a tetramer staining of CD1a-autoreactive T cell line.
    • Fig. S6. Electron density for farnesol in CD1a-farnesol binary complex.
    • Table S1. Supporting data CD1a-farnesol binary complex.
    • Table S2. Van der Waals bonds between CD1a and farnesol.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Raw data sets for main figures (Excel spreadsheet).

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article