ANTIGENIC PEPTIDES FOR THE DIAGNOSIS, THERAPY MONITORING AND/OR TREATMENT OF PSORIASIS VULGARIS

Abstract

The present invention relates to peptides with a conserved amino acid motif and pharmaceutical compositions comprising these peptides. The present invention further relates to the use of the peptides as biomarker, the medical use of the peptides, in particular in the diagnosis, prevention, monitoring and/or treatment of Psoriasis. The present invention relates to complexes of the peptides of the invention with HLA-C monomers or multimers and their use as biomarker and their medical uses, in particular in the treatment of Psoriasis and in monitoring such treatment. The present invention furthermore relates to means and methods for the prevention and/or treatment of Psoriasis, that comprise inhibiting or blocking the interaction of TCR and HLA-C.

Claims

1. A peptide comprising an amino acid sequence of the general formula I
X1.sub.n-Arg-X2-X3-Y1-X4-Y2-Arg-Z(I) wherein X1 is selected from Phe, Arg, Gly, Met, Ala, Ser, Leu and Val; n is 0 or 1. X2 is any amino acid; X3 is any amino acid; X4 is selected from Thr, Tyr, Val, Cys, Ser, and Ala; Y1 is selected from Arg, Lys, Gln, and Asn; Y2 is selected from Arg, Leu or and Ser; and Z is selected from Leu, Met, Ile or and or wherein X1 is selected from Phe, Arg, Gly, Met, Ala, Ser, Leu, Val, His, Tyr, Lys and Trp; n is 0 or 1. X2 is any amino acid; X3 is any amino acid; X4 is selected from Thr, Tyr, Val, Cys, Ser, Ala, Gly and Arg; Y1 is selected from Arg, Lys, Gln, and Asn; Y2 is selected from Arg, Leu and Ser; and Z is selected from Leu, Met, Ile, Val, Thr and Tyr.

2. The peptide of claim 1, wherein the peptide has a length of 8 to 150 amino acids.

3. The peptide of claim 1, further comprising one or more further components selected from: N- and/or C-terminal modifications; labels; tags; drugs or their respective prodrugs; carriers or depots for drugs, prodrugs or labels; immunogenic epitopes; cytotoxins; radionuclides, and/or combinations thereof.

4. The peptide of claim 1, wherein X2 is selected from Ser, His, Cys, Trp, Asn, Ala, Tyr, Gln, Phe, Thr, and Pro; and/or X3 is selected from Tyr, Arg, Trp, Ser, Ala, His, Phe, Val, Gly, Cys and Glu; and/or X1, if present, is selected from Phe, Arg, Gly, Met, Ala, Ser, Leu, Val, His, Tyr, Lys and Trp; and/or X4 is selected from Thr, Tyr, Val, Cys, Ser, Ala, Gly and Arg and/or Y2 is selected from Arg, Leu and Ser; and/or Z is selected from Leu, Met, Ile, Val, Thr and Tyr.

5. The peptide of claim 1, comprising an amino acid sequence selected from the following formulas
X1-Arg-X2-X3-Y1-X4-Y2-Arg-Leu(Ia)
X1-Arg-X2-X3-Y1-X4-Arg-Arg-Z(Ib) and
X1-Arg-X2-X3-Y1-X4-Arg-Arg-Leu(Ia) wherein X1, X2, X3, X4 and Y1 are as defined in claim 1; Y2 is selected from Arg, and Leu; and Z is selected from Leu, Met, Ile and Val; and/or wherein the peptide comprises or consists of an amino acid sequence selected from the group of SEQ ID NOs. 1 to 13, 17 to 19 and 32 to 57.

6. A peptide-HLA-C complex selected from: a) a peptide-HLA-C monomer complex, comprising (i) at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and (ii) a HLA-C monomer that binds to a T-cell receptor (TCR); and b) a peptide-HLA-C multimer complex, comprising at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and at least two HLA-C monomers that bind to a TCR.

7. (canceled)

8. The complex of claim 6, wherein the HLA-C comprises one or more further components selected from: labels; tags; drugs or their respective prodrugs; carriers or depots for drugs, prodrugs or labels; immunogenic epitopes; cytotoxins; radionuclides; coupling moiety/moieties; and combinations thereof.

9. The complex of claim 6, wherein the at least one peptide is altered for modifying the TCR binding affinity of the peptide or the peptide-HLA-C complex.

10. A pharmaceutical composition, comprising (A) at least one peptide of claim 1 and/or a a peptide-HLA-C complex selected from: a) a peptide-HLA-C monomer complex, comprising (i) at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ IDNOs: 14-16, and (ii) a HLA-C monomer that binds to a T-cell receptor (TCR); and b) a peptide-HLA-C multimer complex, comprising at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and at least two HLA-C monomers that bind to a TCR; and a pharmaceutically acceptable carrier and/or excipient.

11. The pharmaceutical composition of claim 10, comprising two or more of said peptides.

12. A method for performing at least one of the following: monitoring Psoriasis disease activity; monitoring efficacy of Psoriasis treatment; determining Psoriasis disease risk; and/or determining the frequency of autoreactive T cells in samples of subjects having Psoriasis; wherein said method comprises the use of: a peptide of claim 1; a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16; a peptide-HLA-C complex selected from: a) a peptide-HLA-C monomer complex, comprising: (i) at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and (ii) a HLA-C monomer that binds to a T-cell receptor (TCR); and b) a peptide-HLA-C multimer complex, comprising at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and at least two HLA-C monomers that bind to a TCR; or a protein comprising an amino acid sequence of SEQ ID NOS: 22 to 31 or 60 to 76;

13. (canceled)

14. A method for the prevention and/or treatment of Psoriasis wherein said method comprises administering, to a subject in need of such prevention and/or treatment: a peptide of claim 1; a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16; a peptide-HLA-C complex selected from: a) a peptide-HLA-C monomer complex, comprising (i) at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and (ii) a HLA-C monomer that binds to a T-cell receptor (TCR); and b) a peptide-HLA-C multimer complex, comprising at least one peptide selected from a peptide of claim 1 or a peptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 14-16, and at least two HLA-C monomers that bind to a TCR; or a protein comprising an amino acid sequence of SEQ ID NOs: 22 to 31 or 60 to 76.

15-16. (canceled)

17. The method according to claim 14, wherein the prevention and/or treatment of Psoriasis, comprises (a) inhibiting or blocking the interaction of TCR and HLA-C, and/or (b) suppressing the expression of HLA-C.

18. The method according to claim 14, wherein the prevention and/or treatment of Psoriasis comprises: creating immunotolerance against autoantigens; affecting, tolerating or blocking the activation of autoreactive T cells for therapeutic purposes; eliminating pathogenic CD8.sup.+ T cells; and/or down-regulating a pathogenic immune response by specifically blocking HLA-C/T-cell-interactions.

19. (canceled)

20. A method for the prevention and/or treatment of Psoriasis, comprising (a) inhibiting or blocking the interaction of TCR and HLA-C, and/or (b) suppressing the expression of HLA-C.

21. The method according to claim 20, wherein (a) inhibiting or blocking the interaction of TCR and HLA-C comprises interfering with the contact between TCR and HLA-C and/or (b) suppressing the expression of HLA-C comprises suppressing the expression of HLA-C on cell surfaces.

22. The method, according to claim 20, which comprises the use of a compound that is directed against HLA-C, wherein said compound is an anti-HLA-C antibody or fragment thereof; a small molecule inhibitor; or a small interfering RNA.

23. The peptide-HLA-C complex, according to claim 6, wherein the HLA-C is HLA-C*06:02.

24. The method, according to claim 20, wherein the HLA-C is HLA-C* 06.02.

25. The method, according to claim 21, wherein suppressing the expression of HLA-C comprises suppressing the expression of HLA-C on cell surfaces by small interfering RNAs or other molecule that reduces HLA-C transcription, HLA-C translation or HLA-C transport to the cell surface.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0384] FIG. 1. V3S1/V13S1-TCR hybridoma activation by CD3-antibody.

[0385] Shown is activation of hybridoma cells by a CD3 monoclonal antobody, that leads to the production of green fluorescent protein. This can be visualized by fluorescence microscopy or determined by FACS analysis.

[0386] FIG. 2. HLA-C*06:02-Restricted Activation of the CD8.sup.+ V3S1/V13S1 TCR Reporter Hybridoma Cell Line by Melanocytes.

[0387] (A) TCR-hybridoma activation by primary human melanocytes (MC, elongated cells). Merged light and fluorescence microscopy images of V3S1/V13S1-TCR hybridoma cells (round shapes) that were activated to express sGFP upon co-culture with HLA-C*06:02-positive (left) but not HLA-C*06:02-negative melanocytes (right).

[0388] (B) TCR-hybridoma activation by HLA-C*06:02-positive (left) or HLA-C*06:02-negative (right) primary human melanocytes (MC) in the absence or presence of IFN- or a HLA-class I blocking antibody. Frequency of NFAT-sGFP.sup.+ hybridoma cells was assessed by flow cytometry and normalized to the maximum value induced by CD3 activation in the same experiment. Data are representative of three HLA-C*06:02-positive (healthy n=2, psoriasis n=1) and nine HLA-C*06:02-negative individuals (healthy n=7, psoriasis n=2).

[0389] (C) Western blot analysis of HLA-C (upper panel) and -actin expression (loading control, lower panel).in lysates from primary human melanocytes (MC, left) or the melanoma-derived cell line WM278 (right) cultured in the absence or presence of IFN-.

[0390] (D) TCR-hybridoma activation by co-culture with the HLA-C*06:02-positive melanocytic cell line (MCL) WM278 preincubated without or with IFN- or a HLA-class I blocking antibody. Data were assessed as in (B).

[0391] (E) TCR-hybridoma activation by HLA-C*06:02-negative melanocytic cell line (MCL) WM9 transfected with HLA-C*06:02 or HLA-A*0201 and incubated with an anti-HLA-class I antibody. Data were assessed as in (B).

[0392] (F) Western blot analysis of HLA-C (upper panel) and -actin expression (loading control, lower panel) in lysates of WM9 cells transfected with pRSV-HLA-C*06:02.

Data represent 3 (C), >3 (D), and 3 (E) independent experiments.

[0393] FIG. 3. Screening Strategy to Determine the V3S1/V13S1 TCR Reactivity. Schematic of screening strategy to identify cellular targets (left) or antigens (right) of the V3S1/V13S1-TCR. Target cell screening involved co-cultured with different skin cell types. MC, primary human melanocytes. Antigen screening by nonamer peptide libraries was followed by transfection of candidate peptide epitopes and full-length proteins into HLA-C*06:02-transfected HEK293FT or COS-7 cells, or HLA-C*06:02-positive melanoma cell lines (MCL).

[0394] FIG. 4. Identification of Peptide Ligands of the V3S1/V13S1 TCR.

[0395] (A) Design of plasmid-encoded combinatorial nonamer peptide libraries (PECPL) #1-3 with predefined amino acid residues in single letter amino acid code. X: randomized residue.

[0396] (B) Mimotopes derived from PECPL#1-3 activating the V3S1/V13S1-TCR hybridoma. HLA-C*06:02 anchor positions are labelled in yellow, conserved amino acids of mimotopes independent from anchor positions in green, blue or red. See also FIG. S3.

[0397] (C) TCR-hybridoma activation by mimotopes co-transfected with HLA-C*06:02 into COS-7 cells. Frequency of NFAT-sGFP.sup.hybridoma cells was assessed by flow cytometry and normalized to the maximum value induced by CD3 activation in the same experiment.

[0398] (D) Natural human peptide ligands activating the V3S1/V13S1-TCR hybridoma. Subscript numbers give amino acid positions in parent proteins. Labelling as in (B).

[0399] (E) TCR-hybridoma activation by plasmid-encoded natural human peptide epitopes co-transfected with HLA-C*06:02 into COS-7 cells. Data were assessed as in (C).

[0400] FIG. 5. Stimulation of V3S1/V13S1 TCR hybridoma cells by mimotopes 1-9 co-transfected with HLA-C*06: 02 into COS-7 cells.

[0401] Flow cytometry analysis of NFAT-sGFP.sup.+ V3S1/V13S1 TCR hybridoma cells following co-culture with COS-7 cells co-transfected with plasmids containing mimotopes and HLA-C*06:02.

[0402] FIG. 6. Stimulation of V3S1/V13S1 TCR hybridoma cells by natural human peptide ligands co-transfected with HLA-C*06:02 into COS-7 cells.

[0403] Flow cytometry analysis of NFAT-sGFP.sup.+ V3S1/V13S1 TCR hybridoma cells following co-culture with COS-7 cells co-transfected with plasmids containing natural human peptide epitopes and HLA-C*06:02.

[0404] FIG. 7. Staining of peripheral blood CD8.sup.+ T cells with peptide-HLA-C*06:02 dextramers.

[0405] (A) HLA-C*06:02/control peptide

[0406] (B) HLA-C*06:02/RASSF10 peptide

[0407] (C) HLA-C*06:02/HEPACAM peptide

[0408] (D) HLA-C*06:02/IL24del3 peptide

[0409] Shown is the FACS analysis of CD8.sup.+ T cells stained with labelled peptide/HLA-C*06:02-dextramers. HLA-C*06:02/control peptide refers to an unrelated peptide formerly isolated from HLA-C*06:02.

[0410] FIG. 8. Cytokine Induction by Stimulation of PBMC with ADAMTSL5 Peptide.

[0411] (A) Intracellular staining of IFN- and IL-17A in CD8.sup.+ T cells following stimulation of PBMC with synthetic ADAMTSL5 peptide (right) or medium control (left). Representative histograms show flow cytometry analysis of each a representative experiment for a healthy control (HC) and a psoriasis patient (PV).

[0412] (B) Changes in frequencies of IFN-.sup.+- and IL-17A.sup.+CD8.sup.+ T cells after ADAMTSL5 peptide stimulation of PBMC from healthy controls (HC, n=11) and psoriasis patients (PV, n=47) after normalization to medium control. Each dot represents one individual. ***p<0.005 (Mann-Whitney U test).

[0413] (C) Changes in IFN- (left) and IL-17A concentrations (right) in culture supernatants of PBMC from healthy controls (HC, n=11) or psoriasis patients (PV, n=37) following stimulation with synthetic ADAMTSL5 peptide as determined in triplicates by ELISA. Graphs depict changes by ADAMTSL5 peptide over medium control, each dot represents one individual. Dashed line indicates threshold. ***p<0.005, **p<0.01 (Chi square test).

[0414] FIG. 9. Alanine scan of peptide positions in the ADAMTSL5 peptide.

[0415] Exchange of amino acids at positions 1 to 9 (a) of the ADAMTSL5 peptide against Ala. Activation of V3S1/V13S1 TCR hybridoma by mutated peptides expressed together with HLA-C*06:02 in COS-7 (b) or WM278 cells (c) is determined by FACS analysis of green fluorescent hybridoma cells.

[0416] FIG. 10. Validation of amino acids at P2 (R) and P9 (Z) in the ADAMTSL5 nonameric peptide.

[0417] Exchange of amino acids at position 2 (a) and position (c) of the ADAMTSL5 peptide. Activation of V3S1/V13S1 TCR hybridoma by mutated peptides co-transfected with HLA-C*06:02 in COS-7 cells is deteimined by FACS analysis of green fluorescent hybridoma cells (b and d).

[0418] FIG. 11. Definition of amino acids at position 1 to 9 of nonamer peptides stimulating the V3S1/V13S1 T-cell receptor.

[0419] (A) Amino acids at position 1 to position 9 of 12 nonamer mimotopes identified by library screening.

[0420] (B) Amino acids at position 1 to position 9 of 27 octamer or nonamer peptides identified by database screening. Numbers give the frequency of amino acids identified at a particular position.

EXAMPLES

[0421] 1. Experimental Procedures

[0422] 1.1 Human Subjects

[0423] Lesional biopsies were obtained from patients with chronic plaque psoriasis. Blood samples were obtained by antecubital venous puncture. All participants gave written infoiiiied consent. The study was performed in accordance with the Helsinki Declaration and approved by the Ethics committee of the university hospital 1.2 Generation of V3S1/V13S1-TCR CD8.sup.+ reporter T-hybridoma

[0424] Identification of the matching V3S1/V13S1-TCR chains of a CD 8.sup.+ T-cell clone (V3S1-NN-J 45.1: CA TDAL YSGG, VP13S1-N(D)N-J 3 1.1: CASSY SEGED EAFF; Arden nomenclature, see Arden et al. 1995) has been described (see Kim et al. 2012). TCR-hybridoma generation was perfoinied as described (Seitz et al., 2006; Siewert et al., 2011). Briefly, V and V-regions were cloned into expression plasmids pRSV-hygro (-chain) and pRSV-neo (-chain) using restriction sites SalI-PvuII or SalI-BIpI. The resulting plasmids were linearized (XmnI and NdeI, respectively) and electroporated into 58 .sup..sup. T hybridoma cells. Functional clones were supertransfected with pLPCX-CD8-IRES- and pcDNA-NFAT-sGFP. TCR-activation induced NFAT-sGFP-expression was determined by CD3 crosslinking with anti-mouse CD3 antibody (clone 17A2, eBioscience), flow cytometry and fluorescence microscopy (AxioVert200M, Zeiss, 520/35 BrightLine filter, Semrock and 605/70 filter). Clones with highest frequencies of responding cells (usually >30% NFAT-sGFP-positive cells after CD3 stimulation) were expanded in T-hybridoma medium (see Primary cells and cell lines section). Hybridoma batches were frequently recloned to minimize sporadic sGFP expression and decrease of activation rates. The resulting cell line is teimed V3S1/V13S1-TCR hybridoma.

[0425] 1.3 Construction of Plasmid-Encoded Combinatorial Peptide Libraries (PECPL) and Identification of V3S1/V13S1-TCR Mimotopes

[0426] Completely randomized nonameric PECP library #1, PECPL #2 with predetermined amino acids Leu at P9 or #3 with Phe/Tyr at P1, Arg at P2 and Leu/Ile at P9 were prepared as described.sup.4. Screening strategy and library designs and are shown in FIG. 3 and FIG. 4. COS-7 cells were co-transfected with PECP-libraries and HLA-C*06:02, co-cultured with V3S1/V13S1-TCR hybridoma and screened by UV-microscopy for the induction of sGFP. Mimotope-containing plasmids were isolated and sequenced.

[0427] PECPL screening was performed as described (Siewert et al., 2011). COS-7 cells were cotransfected with PECPLs and HLA-C*06:02 and cocultured with V3S1/V13S1-TCR hybridoma cells. After 16 h, COS-7 cells in close contact to fluorescent hybridoma cells were isolated and library peptides were amplified by nested PCR. PCR products were cloned into pcDNA.3.1DN5-His-TOPO and transformed into E. coli. The mimotope-enriched library plasmids were cotransfected with pRSV-HLA-C*06:02 into COS-7 cells and the mimotope-containing plasmid was isolated by subcloning and sequencing.

[0428] Amino acid sequences of mimotopes were aligned and used in different search strategies to identify corresponding peptides from human proteins in the taxa-specific homo sapiens [9606] UniProt database or melanocytic transcriptome. cDNA corresponding to 180 candidate peptide antigens or six corresponding full-length proteins were cloned into pcDNA3.1DN5-His-TOPO.

[0429] For this purpose, forward and reverse oligonucleotides coding for octamer or nonamer candidate peptides were annealed in 1 Pwo buffer at a concentration of 10 pmol/l by heating to 95 C. for 5 min and then slowly cooled to room temperature. Diluted annealing products were ligated into pcDNA3.1D/V5-His-TOPO using the Directional TOPO Expression Kit. Forward primers carried a 5-CACCATG overhang and a stop codon at 3-end of the target sequence. Resulting constructs were sequence verified. Oligonucleotides for cloning of ADAMTSL5 are given as example for the design.

TABLE-US-00013 ADAMTSL5-peptidefor: [SEQIDNO.77] 5-caccatggtgcggagccggcggtgcctgcggctgtga-3 ADAMTSL5-peptiderev: [SEQIDNO.78] 5-tcacagccgcaggcaccgccggctccgcac-3

[0430] For Ala scans we generated plasmids encoding the sequence of the ADAMTSL5 peptide with exchanges of each one amino acid of the natural peptide sequence at position P1 to P9 against nucleotide triplets encoding the amino acid Ala. To verify amino acids at P2 and P9, the amino acids at P2 and P9 were exchanged against nucleotide triplets encoding the amino acids Ala, Gly, Thr, Pro or Tyr (P2) or against Ala, Gly, Met, Thr, Val or Tyr (P9) accordingly.

[0431] Ectopic protein expression was verified by Western blotting. Mutations of ADAMTSL5 epitope.sub.58-65 were introduced by PCR using internal reverse primers. siRNAs were transfected into WM278 cells for ADAMTSL5 knock down. Knock down efficacy was examined by triplicate quantitative PCR using PBGD as an internal standard.

[0432] 1.4 Cells, Cell Lines and V3S1/V13S1-TCR Hybridoma Activation Assays

[0433] Primary human melanocytes were cultured from skin samples (Hsu and Herlyn, 1996). Peripheral blood mononuclear cells were prepared by density gradient centrifugation. Human neonatal epidermal keratinocytes were obtained commercially. The following cell lines were used: COS-7, HEK293FT, HaCaT, A431, WM278, WM9, WM239A, 1205Lu.

[0434] Activation of V3S1/V13S1-TCR hybridoma cells was assessed in co-culture experiments by NFAT-sGFP induction and determined by flow cytometry and UV-fluorescence microscopy. IFN- was added to increase HLA-class I expression of HLA-C*06:02-positive cells/cell lines. HLA-C*06:02-negative cells or cell lines were transfected with pRSV-HLA-C*06:02 or pRSV-HLA-A*0201. Cell lines were transfected with plasmid-encoded peptides or full-length proteins. HLA-class I antibody was used to block antigen-mediated TCR ligation.

[0435] sGFP induction in V3S1/V13S1-TCR hybridoma cells was examined after 24 h coculture with antigen-presenting cells by UV-fluorescence microscopy and/or flow cytometry. As controls, V3S1/V13S1-TCR hybridoma cells were cultured in anti-mouse CD3 antibody-coated (clone 17A2, 2 g/ml in PBS, eBioscience) culture plates. pRSV-GFP or pcDNA-GFP served as transfection controls.

[0436] HLA-C*06:02-negative antigen-presenting cells or cell lines were transfected with pRSV-HLA-C*06:02 or pRSV-HLA-A*0201. Cell lines were transfected with plasmid-encoded peptides or full-length proteins. HLA-class I antibody was used to block antigen-mediated TCR ligation.

[0437] 1.5 Identification of Natural Candidate Peptide Antigens Based on Mimotope Sequences

[0438] Two different search strategies were used to identify human candidate peptide epitopes based on the conserved amino acid motifs of the mimotopes. We employed two tools from the The European Molecular Biology Open Software Suite (http://emboss.sourceforge.net/): PROPHECY (http://emboss.sourceforge.net/apps/release/6.6/emboss/apps/prophecy.html) was used to create a frequency matrix from sequences of positively tested antigenic peptides. With this matrix and the PROFIT tool (http://emboss.sourceforge.net/apps/release/6.6/emboss/apps/profit.html), the taxa-specific Homo sapiens [9606] UniProt database was scanned. The list of results was further refined with infoiniation on anchor amino acids of HLA-C*06:02 ligands (see Dionne et al., 2004; Falk et al., 1993) (Syfpeithi database at http://www.syfpeithi.de/). The matrix was constantly adjusted according to newly isolated mimotopes or tested candidate antigen peptides

[0439] 1205Lu transcriptome data were used for a selective search in melanocytic proteins. The MOTIF Search web server (http://www.genome.jp/tools/motif/MOTIF2.html) was searched for human genes in the KEGG Genes dataset containing peptide motifs that were defined according to mimotope sequences and general HLA-C*06:02 anchor positions.

[0440] To identify peptides from pathogens or food components which could activate the V3S1/V13S1 TCR we searched the sequences of the mimotopes and the sequences of the human peptides ligating the V3S1/V13S1 TCR against all known protein sequences or sequence specified according to bacterium sp. (taxid:77133), viruses (taxid:10239), fungi (taxid:4751) using the Protein BLAST search tool at: blast.ncbi nlm nih.gov/Blast.cgi?PAGE=Protein. Candidate peptides were cloned into pcDNA3.1DN5-His-TOPO as described above and tested in cotransfection experiments with HLA-C*06:02 for their ability to activate the V3S1/V13S1 TCR hybridoma. Hybridoma activation was verified by measuring induction of green fluorescence protein in flow cytometry.

[0441] 1.6 Dextramer Staining

[0442] Fluorescent dextramers were prepared of HLA-C*06:02 and antigenic or control peptides. The control peptide has been originally described as an HLA-C*06:02-ligand by Falk et al. (1993). PBMC from psoriasis patients were incubated with standard dilutions of the HLA-C*06:02-peptide dextramers and a phycoerythrin-labelled CD8 antibody and the frequency of double stained cells subsequently determined by flow cytometry on a FACScan flow cytometer. Data were analyzed by FlowJo software 887.

[0443] 1.7 Peptide Stimulation of PBMC

[0444] PBMC were stimulated with synthetic peptides (10 ng/ml) for 48 hrs. Intracellular cytokine staining (Fujii et al., 2011) employed antibodies against CD8, IL-17A and IFN-. Data were acquired by flow cytometry and analysed by Flow Jo software. INF- and IL-17A levels in culture supernatants were determined by ELISA.

[0445] 1.8 Statistics

[0446] Kruskal-Wallis H-test was used for multiple comparisons and Bonferroni correction was applied. Whenp-value of Kruskal-Wallis H-test was significant, unpaired data of two groups were compared using Mann-Whitney U-test. Differences in the two groups were compared with Fisher's exact test. Two-tailed p-values<0.05 were considered significant.

[0447] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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