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. 2021 Feb 26;180(1):136-147.
doi: 10.1093/toxsci/kfaa184.

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells

Alistair Bailey  1   2 , Ben Nicholas  1   2 , Rachel Darley  2 , Erika Parkinson  1 , Ying Teo  3 , Maja Aleksic  4 , Gavin Maxwell  4 , Tim Elliott  2 , Michael Ardern-Jones  3 , Paul Skipp  1
Affiliations

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells

Alistair Bailey et al. Toxicol Sci. .

Abstract

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-ω. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Keywords: DNCB; HLA; HaCaT; keratinocyte; peptidome.

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Figures

Figure 1.
Figure 1.
No evidence for altered peptidome by DNCB modulation of the proteome: A and B, Upset plots (Conway et al., 2017) of intersections of distinct HLA-I peptides observed in 3 biological replicates of control and DNCB treated HaCaT and HaCaT HLA-A2 cells. The total number of distinct 8 − 15mer peptides observed for each condition are shown on the side bars. C and F, Cell component gene ontology classification of distinct genes from which peptidome originates using PANTHER (Mi et al., 2019) from 3 biological replicates of control and DNCB treated HaCaT and HaCaT HLA-A2 cells.
Figure 2.
Figure 2.
No evidence for an altered peptidome by DNCB modification of HLA molecules: A, C, E, G, HLA molecule 9-mer binding motifs ±DNCB treatment (Bassani-Sternberg and Gfeller, 2016; Gfeller et al., 2018; Jessen, 2018). B, D, F, H, HLA bound peptide length distributions ±DNCB treatment (Bassani-Sternberg and Gfeller, 2016; Gfeller et al., 2018).
Figure 3.
Figure 3.
DNCB modified HLA peptide peptide-spectrum matches: A, B, C, D, Peptide-spectrum matches for RFC(+DNP)PFAERTR DNCB modified HLA-A31 peptide from glutathione-S-transferase omega-1 (Uniprot: P78417), AETEC(+DNP)RYAL DNCB modified HLA-B40 peptide from Keratin, type I cytoskeletal 13 (Uniprot: P13646), GLLDPQC(+DNP)RL DNCB modified HLA-A2 peptide from Ribonuclease inhibitor (Uniprot: P13489) and KLLEGEEC(+DNP)RL DNCB modified HLA-A2 peptide from Keratin, type II cytoskeletal 5 (Uniprot: P13647). E, F, G, H, Peptide-spectrum matches for RFC(+DNP)PFAERTR, AETEC(+DNP)RYAL, GLLDPQC(+DNP)RL, KLLEGEEC(+DNP)RL synthetic peptides.
Figure 4.
Figure 4.
DNCB modified HLA peptides model structures and functional assay: A, B, C, D, Homology models of DNP modified peptides: Putative structures were created of the DNP modified peptides using USCF Chimera (Pettersen et al., 2004; Shapovalov and Dunbrack, 2011) using PDB: 1IM3. E, F, Representative images from ex vivo IFN-γ ELISpot assay or enumerated spots after short term culture. Freshly isolated PBMC from an HLA-A*02:01 individual IMS1 were incubated with dinitrophenylated HLA-A*A02:01 GLL-D, KLL-D, or matched nondinitrophenylated peptides (GLL, KLL) before activation in an ELISpot assay with nil, peptides, or phytohemagglutinin.
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