Contact sensitizers trigger human CD1-autoreactive T-cell responses

Abstract

Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.

Keywords: Antigen-presentation; CD1; Contact sensitivity; NKT cells; T cells.

Figure 1

DNCB-pulsed CD1⁺APCs trigger the activation of CD1a- and CD1d-restricted clones. (A-D) B13 cells (A, B) and S33d cells (C, D) were stimulated with (A) DCs, (B) THP-1 CD1a, and (C) THP-1 CD1d cells by pulsing for 24 h with DNCB. (D) S33d cells were stimulated with sulfatide presented by THP-1 CD1d cells previously pulsed with DNCB (6 μM, open circles), or DMSO vehicle (VEH, closed circles). Production of (A, B) GM-CSF and (C, D) IFN-γ was measured by ELISA and shown as mean ± SD, n=3 for B13, n=4 for S33d cells. *p 0.01, t-test with Sidak multiple comparisons. Data shown are from single experiments representative of 3 independent experiments. (E) Heatmap of cytokines produced by B13 cells cultured with THP-1 CD1a cells previously pulsed with DNCB (6 μM) or VEH. Normalized data is expressed as the z-score. Absolute cytokine values are illustrated in Supporting Information Fig. 2. The following cytokines were tested, but were not released by the T-cell clone: IL-15, IL-17a/e/f, IL-21, IL-23, IL-27, IL-28, IL-33, TNF-β and MIP-1α. (F) Heatmap of cytokines produced by S33d cells cultured with THP-1 CD1d cells previously pulsed with DNCB (6 μM) or VEH. Normalized data are expressed as the z-score. Absolute cytokine values are illustrated in Supporting Information Fig. 3. The following cytokines were tested, but were not released by the T-cell clone: IL-5, IL-6, IL-9, IL-15, IL-17a/e/f, IL-21, IL-22, IL-23, IL-27, IL-28, IL-31, IL-33, TNF-β and MIP-1α. (E, F) Data in each column represent an individual replica of the indicated experimental condition.

Figure 2

DNCB activity is mediated by CD1 and TCR. (A, B) Anti-CD1a (α-CD1a, A) and anti-CD1d (α-CD1d, B) or isotype-matched control mAbs (IgG1 and IgG2b, respectively) were added to (A) THP-1 CD1a cells and (B) THP-1 CD1d cells pulsed with DNCB (6 μM) or DMSO vehicle, before the incubation with (A) B13 and (B) S33d cells. Cytokines released by the T cells are expressed in ng/ml (mean + SD, n= 3-4) and results are from single experiments representative of 3 independent experiments. *p ≤0.001 (t-test with Sidak multiple comparisons). (C, D) Flow cytometry analysis of CD69 surface expression by (C) SKW-B13 and (D) SKW-S33d cells following incubation with (C) THP-1 CD1a and (D) THP-1 CD1d cells pretreated with DNCB. Control CD3^-SKW-3 cells are also shown. Median fluorescence intensity (MFI) of CD3⁺ and of CD3^- cells is plotted. Each plot represents data from a single experiment and is representative of 2 independent experiments.

Figure 3

DNCB activity requires newly synthesized CD1 molecules. (A) Kinetic of THP-1 CD1d cell pulsing with DNCB (6 μM) before stimulation of S33d cells. IFN-γ release was measured by ELISA and data from single experiments representative of 3 independent experiments, are expressed as mean ± SD, n=3. (B) IFN-γ response of S33d cells to THP-1 CD1d cells fixed then pulsed with DNCB (6 μM) or pulsed first with DNCB (6 μM) and then fixed. Data are expressed as mean + SD, n=3, and are from a single experiment representative of 3 independent experiments. (C) IFN-γ response of S33d cells to DNCB (6 μM)- or DMSO vehicle (VEH)-pulsed THP-1 CD1d cells pre-treated with brefeldin A or cycloheximide (Cyclohex). Data are expressed as mean + SD, n=4, and are from a single experiment representative of 2-3 independent experiments. *p 0.05, **p 0.01 (t-test with Sidak multiple comparisons).

Figure 4

DNCB potentiates S33d cell activation through endogenous lipids. (A) IFN-γ response of S33d T cells to sulfatide presented by C1R CD1d cells pulsed with DNCB (6 μM, open circles) or DMSO vehicle (VEH, closed circles). (B) Non-stimulatory d18:1 C22:1 sulfatide was added to displace endogenous lipids from THP-1 CD1d cells previously pulsed with DNCB (6μM, open circles) or VEH (closed circles), before assessing S33d T-cell response. (C) Control response of the CD1a-restricted T-cell clone K34B9.1 to d18:1 C22:1 sulfatide presented by THP-1 CD1a cells. Data are expressed as mean ± SD, n=4,. *p≤0.05, t-test with Sidak multiple comparisons. Data are from single experiments representative of 2-3 independent experiments.

Figure 5

A wide range of contact sensitizers induce CD1-mediated T-cell activation. (A-C) The IFN-γ response of S33d cells to THP-1 CD1d cells pulsed with (A) resorcinol, (B) isoeugenol and (C) cinnamaldehyde. (D) S33d cell response in the presence of anti-CD1d (α-CD1d) or irrelevant (IgG2a) mAbs. Resorcinol was solubilized in medium and used at 2 mM, isoeugenol was solubilized in DMSO and used at 250 μM, cinnamaldehyde was solubilized in DMSO and used at 40 μM. (E) Response of B13 cells to THP-1 CD1a cells pulsed with 1,4 benzoquinone (10 μM in medium) and in the presence of anti-CD1a or irrelevant (IgG1) mAbs (α-CD1a). Data are expressed as mean ± SD, n=2-3, and are from single experiments representative of 2-3 independent experiments. *p 0.01 vs. the relevant vehicle controls, t-test with Sidak multiple comparisons.