Down-regulation Of Autoreactive T Cells By Psoriasis-specific APLs

Psoriasis is a common dermatological disorder, affecting approximately 1% to 3% of the general population. Although the pathogenesis of the disease has not been definitively understood, recent evidence suggests that activated CD4-positive helper T-lymphocytes of the Thl phenotype play an important role in the progression of psoriasis under a polygenic inheritance for the predisposition. A number of HLA class II antigens have shown a positive association with psoriasis, including HLA-DRB1*07. CKRTl 7 is believed to play an important role in the pathogenesis of the disease based on our study. CKRT17, which is not expressed in normal skin, is over-expressed in psonatic epidermis and shares α -helical coiled coil structure with M-protein and an extensive amino acid homology has been reported between protein M6 and CKRTl 7. CKRTl 7 is of the ability to stimulate psoriatic T cells in the context of HLA DRB1, measured by cell proliferation and IFN- γ production. It has moreover been demonstrated that the expression of CKRTl 7 can be induced in vitro by IFN- γ. This is the only keratin so far reported to be induced by IFN-γ . Such a vicious circle may contribute to the pathogenic process of the disease, although the mechanism how CKRTl 7 is presented to psoriatic T cells by antigen-presenting cells is not clear yet. In conclusion, infection of streptococcus is the key factor to the onset of psoriasis and theanomalo-expression of CKRT17 contributes to the maintenance, aggravation,chronic progression and recurrence of the disease.OBJECTIVE:To identify the HLA DRB1*07-restricted T cell epitopes on CKRT17. With that we are to change the key amino acid in the epitopes with the method of altered peptide ligand and screen out anti-autoreactive T cell peptides, which need to be verified in the level of cell, organ and model. Further studies based on these peptides would provide a more complete understanding of the immunological basis and a more safe, more specific strategy to the treament of psoriasis. METHODS:1. The HLA DRB1-restricted T cell epitopes were predicted with matrix-based algorithm by the servers of SYFPEITHI and ProPred.The hydrophobicity and hydrophilicity profile, amino acid composition and secondary structure of CKRT17 were also taken into consideration.Epitope regions were devided base on the density of predicted epitopes.2. Total RNA was extracted from epidermis of psoriatic patients using Trizol reagent and was reversely transcribed with RT-PCR kit.The predicted epitope regions were expressed and purified with glutathione sepharose 4B affinity column.ELISA and Western Blot were performed to identify the expression and purification.3. All objects were HLA-DRB1*07 positively qualified for the study after genotyped by PROTRANS 96-well HLA DRB1 alleles typing cyclerplates.4. T cells were isolated and purified by gradient centrifugation and AET-SRBC (2-aminoethylosothiouronium bromide-treated sheep red blood cells) . The MTT and direct cell counting method were used to detect the proliferation. The concentration of IFN- γ was measured by ELISA.The epitope regions showed stronger effect as agonists of T cell proliferation and IFN-γ expression were selected.5. The two nucleic acid strains of each predicted epitope with restriction endonuclease sites of BamH I and EcoRI were synthesized and then expressed at N-terminus of GST. These recombinant epitopes were purified and identified with ELISA and Western Blot and then were detected the level of T cell proliferation and the concentration of IFN- γ in the culture.The epitopes showed stronger effect as agonists of T cell proliferation and IFN- γ expression were selected via Positive selection and Negative selection.6. The altered peptide ligangds(APLs) were prepared via mutation of key amino acids by PCR with TaKaRa MutanBEST kit.The corresponding peptides were expressed, purified and identified with ELISA abd Western Blot and then were detected the level of T cell proliferation and the concentration of IFN- Y in the culture via Positive selection and Negative selection.In this part we selected the suppressive APLs.7. After APLs were presented to T cells,from which genome DNA were extracted, apoptosis ladder were deteced by gel electrophoresis.The apoptosis ratios were analyzed by flow cytometry.8. After APLs were presented to T cells,the supernatants were collected and added to the keratinocytes,which were cultured in the KC-SF medium. MTT method was uesd to analyzed degree of th proliferation of keratinocytes.9. After APLs were presented to T cells,the supernatants were collected and added to nest dishes,in which psoriatic lesion organs were culued.The levels of transcription and expression of CKRT17 were analyzed by RT-PCR,Western Blot and immunohistochemistry respectively.10. Psoriatic lesion-like model was prepared by application of 5% propranolol emulsion on back skin of Guinea pigs' ears. Supernatants were collected after APLs were presented to T cells.Ointments were prepared with main component of the supernatants.The ointments were applied to the back skin of Guinea pigs' ears. Pathological section were prepared by HE staining.The mean thickness of the epidermis before and after therapy werecompared. RESULTS:1. The HLA DRB1*07-restricted T cell epitopes were predicted with matrix-based algorithm by the servers of SYFPEITHI and ProPred.Five epitope regions were devided base on the density of predicted epitopes,which consist of 80-140AA,150-180AA,190-230AA,320-390AAand 400-430AA.2. The full-length cDNA of CKRT17 was cloned from epidermis of psoriatic patients.The sequence was identical to the one coded GenBank BC000159.The predicted epitope regions were expressed,with the molecular weights of 28KD,32KD,30KD,31.5KD,33.8KD,31KD and 73 KD (GST, S1-S5 and CKRT17 respectively) ,and purified with glutathione sepharose 4B affinity column with the concentrations of 230ug/mL,110ug/mL,89ug/mL,112 ug/mL,72ug/mL,95ug/mL and 50ug/mL ( GST , S1-S5 and CKRT17 respectively) .ELISA and Western Blot were performed positively to identify the expression and purification.3.15 patients of psoriasis guttata,37 patients of psoriasis plaque,8 healthy volunteers and 10 patients of chronic eczema were qualified for this study after genotyped by PROTRANS 96-well HLA DRB1 *07 typing.4. The epitope regions of SI and S4 showed stronger effect as agonists of psoriatic T cell proliferation and IFN- Y expression compared to other regions in the concentrations of 10 ug.mL-1 and 100 ug.mL-1. They have weak effect on the T cells of healthy volunteers and patients of chronic eczema.Meanwhile, we found that SI and S4 showed immunodominant ability to induce psoriatic T cells to form colonies and blastoformation.5. The following epitopes, Sl(118-132), Sl(101-115), Sl(115-129), S4(358-372), S4(323-337) and S4(348-362)can stimulate the proliferation and IFN-γ production of psoriatic T cells more effectively than other epitopes.Among them, Sl(118-132), S4(323-337) and S4(348-362) react weakly with the T cells from healthy volunteers and patients of chroniceczema.The results showed that the epitopes of Sl(l 18-132), S4(323-337) and S4(348-362) are psoriasis-specific, powerfully reactive epitopes.Among them, SI (118-132) contains a region of ALEE AN, sharing a part of conserved C-terminal of the streptococcal M6 protein, which is consistent in part with what Sigmundsdottir H described, while the other three ones have not been reported up to now.6. APLs of S12ECVRALEEANTELEVKI) and 4T8I(ENRYCVQLSQIQGLI)actS asantagonists,which suppress psoriatic T cell proliferation and the expression of IFN- Y in the culture.S12E and 4T8I react weakly with the T cells from healthy volunteers.7. After S12E and 4T8I were presented to T cells,from which genome DNA were extracted,apoptosis ladder were positively deteced by 1.2% gel electrophoresis.The apoptosis ratios analyzed by flow cytometry showed that S12E and 4T8I can induce apoptosis of psoriatic T cells in the concentrations of 1ug/mL,10ug/mL and 100ug/mL,especially the latter.They have weak such effect on the the T cells from healthy volunteers.8. After APLs were presented to T cells,the supernatants were collected and added to the keratinocytes,which were cultured in the KC-SF medium. MTT result showed that the supernatants of S12E and 4T8I can suppress the proliferation of keratinocytes in the concentration of 1ug/mL, 10ug/mL and 100ug/mL with dilution ratio of 1:10 and l:100,especially in the concentration of IOOug/mL with dilution ratio of 1:10.9. After APLs were presented to T cells,the supernatants were collected and added to nest dishes,in which psoriatic lesion organs were culued.The results showed that he supernatants of S12E and 4T8I can suppress the levels of transcription and expression of CKRT17 in the concentration of 100ug/mL with dilution ratio of 1:10 analyzed by RT-PCR,Western Blot and immunohistochemistry respectively.10. Psoriatic lesion-like model was prepared by application of 5%...