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. 2015 Dec 14;212(13):2203-12.
doi: 10.1084/jem.20151093. Epub 2015 Nov 30.

Melanocyte antigen triggers autoimmunity in human psoriasis

Akiko Arakawa  1 , Katherina Siewert  2 , Julia Stöhr  1 , Petra Besgen  1 , Song-Min Kim  1 , Geraldine Rühl  2 , Jens Nickel  1 , Sigrid Vollmer  1 , Peter Thomas  1 , Stefan Krebs  3 , Stefan Pinkert  4 , Michael Spannagl  5 , Kathrin Held  2 , Claudia Kammerbauer  1 , Robert Besch  1 , Klaus Dornmair  2 , Jörg C Prinz  6
Affiliations

Melanocyte antigen triggers autoimmunity in human psoriasis

Akiko Arakawa et al. J Exp Med. .

Abstract

Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific _target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.

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Figures

Figure 1.
Figure 1.
HLA-C*06:02-positive melanocytes are skin-specific _target cells of the Vα3S1/Vβ13S1 TCR. (A and B) TCR hybridoma (round cells) activation by HLA-C*06:02–positive (left) but not HLA-C*06:02–negative (right) primary human melanocytes (elongated) in the presence of IFN-γ, as determined by combined UV-light microscopy (A) or flow cytometry and effect of melanocyte preincubation with IFN-γ or an HLA class I–blocking antibody (B). Data represent three HLA-C*06:02–positive (healthy n = 2, psoriasis n = 1) and nine HLA-C*06:02–negative subjects (healthy n = 7, psoriasis n = 2) as mean ± SD. Numbers give stimulating/total samples. *, P < 0.05; ***, P < 0.005; two tailed Fisher’s exact. Bars, 20 µm. (C) Western blot analysis of HLA-C expression in primary melanocytes or WM278 cells cultured without/with IFN-γ or HLA-C*06:02–negative WM9 cells without/with HLA-C*06:02 transfection. (D and E) Hybridoma activation by an HLA-C*06:02–positive (WM278) or HLA-C*06:02–negative (WM9) melanoma cell line transfected with HLA-C*06:02 or HLA-A*02:01 and effect of a blocking HLA antibody. (F and G) Hybridoma activation by HLA-C*06:02–negative primary keratinocytes (F) or the HLA-C*06:02–negative epithelial carcinoma cell line A431 (G) transfected with HLA-C*06:02 or HLA-A*02:01 or cotransfected with HLA-C*06:02 and mimotope #6. Data represent three (C, E, and F), five (D), and two (G) independent experiments as mean ± SD.
Figure 2.
Figure 2.
Melanocytes are _targets of a noncytotoxic CD8+ T cell response in psoriasis lesions. (A and B) Representative immunohistologic images of CD8+ T cells (green) and melanocytes (red) stained for c-Kit (A) or MART-1 (B) in healthy skin (n = 5), nickel contact eczema (n = 5), and psoriasis (n = 23). Asterisks designate CD8+ T cells contacting melanocytes. Overlay of red and green shows as yellow and of red, green, and blue as white. Dashed lines indicate basal membrane. (C) Incidence of CD8+ T cells (left) and percentage of CD8+ T cells in contact with c-Kit+ melanocytes (right) in normal skin, nickel eczema, and psoriasis. Each dot represents one subject, and bars mark the median; *, P < 0.05; ***, P < 0.005; ****, P < 0.001; two tailed Mann-Whitney U test. (D) Percentage of CD8+ T cells in contact with c-Kit+ melanocytes in three HLA-C*06:02–negative and seven HLA-C*06:02–positive psoriasis patients. Bars indicate median. (E and F) CD8+ T cells (blue) polarizing lytic granules (Granzyme B: green; overlay with red: yellow; overlay with red and blue: white; see arrowheads) toward contact sites with c-Kit+ (E) or MART-1+ (F) melanocytes (red) in psoriasis. Data are representative of eight psoriasis lesions. (G) Staining for MART-1 (red) and cCASP3 (green) in normal skin, nickel eczema, and psoriasis (n = 3). Only keratinocytes in nickel eczema focally stained positive for cCASP3 (arrowheads). Dashed lines indicate basal membrane. Bars: (A and G) 40 µm; (B) 20 µm; (E and F) 5 µm.
Figure 3.
Figure 3.
ADAMTSL5 is an HLA-C*06:02–presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. (A) Design of PECPLs #1–3. Predefined amino acid residues are in pink; X indicates randomized. (B and C) Mimotopes derived from PECPLs #1–3 (B) and natural human TCR peptide ligands stimulating the Vα3S1/Vβ13S1 TCR (C). HLA-C*06:02 anchor positions are labeled in yellow, and other conserved amino acids in green, blue, or pink. (D) TCR hybridoma stimulation by plasmid-encoded human peptide epitopes cotransfected with HLA-C*06:02 into COS-7 cells as normalized to CD3 stimulation. (E and F) Changes in TCR hybridoma activation by full-length proteins (E) or by wild-type, epitope-mutated (Ala58-65) or epitope-deleted (Del58-65) full-length ADAMTSL5 transfected into WM278 cells preincubated with IFN-γ as compared with vector control (F). Data represent three (D and E) and five (F) independent experiments as mean ± SD; *, P < 0.05; ***, P < 0.005; two tailed Mann-Whitney U test. (G) TCR hybridoma activation by WM278 cells transfected with two different control siRNAs or siRNAs _targeting ADAMTSL5 and incubated with IFN-γ as compared with mock control. Data represent three biological triplicates as mean ± SD. (H) Validation of ADAMTSL5 knockdown by qPCR in triplicates and normalized to PBGD. Data are given as mean ± SD.
Figure 4.
Figure 4.
ADAMTSL5 is a psoriasis autoantigen. (A) Representative flow cytometry dot plots for IL-17A– and IFN-γ–producing CD8+ T cells as the proportion of total CD8+ T cells in a healthy and psoriasis subject. (B and D) Change in the percentage of IL-17A– and IFN-γ–positive CD8+ T cells after ADAMTSL5 peptide stimulation of PBMCs from healthy and psoriasis subjects over medium control (B) and differentiated according to HLA-C*06:02 status (D). Data were assessed by multiparametric flow cytometry analysis after CD8 and intracellular cytokine staining and compared by two-tailed Mann-Whitney U test. Each dot represents one subject; *, P < 0.05; ***, P < 0.005. (C and E) Change in IL-17A and IFN-γ secretion of PBMCs stimulated with ADAMTSL5 or control peptide over medium control as evaluated by ELISA (C) and differentiated according to HLA-C*06:02 status (E). Dashed lines indicate the positive threshold. *, P < 0.05; ***, P < 0.005; two tailed Fisher’s exact test.
Figure 5.
Figure 5.
ADAMTSL5 is selectively expressed by melanocytes in epidermis. (A) ADAMTSL5 mRNA expression in healthy skin (n = 2), primary melanocytes (n = 3), and WM278 cells was assessed by qPCR in triplicates and normalized to PBGD. Data are given as mean ± SD. (B) Immunostaining of ADAMTSL5 (green; overlay with red: yellow) and MART-1 (red) in healthy skin (n = 3) and psoriasis (n = 3). Dashed lines indicate basal membrane, and arrowheads indicate ADAMTSL5 expression by melanocytes. (C) Representative image of a CD8+ T cell (blue) contacting a melanocyte (red) expressing ADAMTSL5 (green; overlay with red: yellow) in psoriasis (n = 2). Bars: (B, left) 20 µm; (B [right] and C) 10 µm.
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