Studies On The T Cell Epitopes Of BPAG2 Antigen And The Epitope-based Pathogenesis Of Bullous Pemphigoid

Bullous pemphigoid(BP) is a autoimmune blistering skin disease commonly seen in elderly individuals. It is clinically characterized by persistent blisters or bullae. The main histopathologic characters are subepidermal blisters and the local infiltration of neutrophils and eosinophils, linear deposition of IgG autoantibody and complement C3 are the immunohistological features of BP. The course of BP is fairy long, and it is famous of intractable and being tend to attack repeatedly, which has a great impact on both human health and spirit. Since the pathogenesis of BP has not been elucidated completely, at present systematic application of glucocorticoid and immunosuppressants are the main treatment which can just control progress and postpone the recurrence of BP, a number of problems about efficacy and side effects of drugs are still remained. So further researches are needed to understand the pathogenesis of BP and seek new and effective treatment strategies. Objectives:The purpose of this study is to identify the T cell epitope of BPAG2, the major antigen of BP; To analyze HLA restriction of the T cell epitope of BPAG2; to understand the course of humoral immune response in BP, to explore the pathogenesis of BP and to seek new therapies. Methods:(1) 77 amino acids(490-566aa) including the whole length of BPAG2-NC16 A as the target area for screening, 22 overlapping peptides were synthesized which had 12 amino acids overlapped in turn from N-terminal to C-terminal.(2) To screen the Th1 cell epitope, ELISPOT was used to detect the cytokine IFN-γ levels after the PBMCs from BP patients were stimulated by 22 overlapping peptides.(3) To screen the Th2 cell epitope, ELISPOT was used to detect the cytokine IL-4 levels after the PBMCs from BP patients were stimulated by 22 overlapping peptides.(4) PBMCs from BP patients who show positive response stained by CFSE in advance were stimulated by positive peptides of T cell epitopes, then were cultured for 7 days. The proliferation levels of CD4+T cells were analyzed by flow cytometry.(5) PBMCs from BP patients who show positive response and normal controls were stimulated by positive peptides of T cell epitopes, then were cultured for 7 days, the levels of sCD23, the activation marker of B cells in the cell culture supernatant were detected by ELISA.(6) PBMCs from BP patients who show positive response and normal controls were stimulated by positive peptides of T cell epitopes, then were cultured for 7 days, the antibody titers of anti-BPAG2-NC16 A in the cell culture supernatant were detected by ELISA.(7) To observe whether there exists “epitope spreading”, PBMCs collected twice from the same BP patients during progression stage and recovery stage of disease respectively were stimulated by 22 overlapping peptides, then ELISPOT was used to detect the change of cytokine levels between the progression stage and the recovery stage of BP.(8) HLA-ABC、HLA-DP、HLA-DQ and HLA-DR neutralizing antibodies were added before the positive peptides to the PBMCs from BP patients, then the IL-4 levels were detected by the same method of ELISPOT. Results:(1) After the PBMCs of 25 BP patients being stimulated by the 22 overlapping peptides, the peptide No.21 can induce the PBMCs of 8 BP patients(accounted for 32% of all BP patients) to secrete much IFN-γ, the peptide No.18 can induce the PBMCs of 9 BP patients(accounted for 36% of all BP patients) to secrete much IFN-γ, the peptide No.14 and the peptide No.13 can induce the PBMCs of 10 BP patients(accounted for 40% of all BP patients) to secrete much IFN-γ, resulting in strong Th1 cell response. So the peptide No.21:492-506aa:VRKLKARVDELERIR, No.18:501-515aa:ELERIRRSILPYGDS, No.14:513-527aa:GDSMDRIEKDRLQGM and No.13: 516-530aa:MDRIEKDRLQGMA PA were the Th1 cell epitope.(2) After the PBMCs of 26 BP patients being stimulated by the 22 overlapping peptides, the peptide No.21 can induce the PBMCs of 15 BP patients(accounted for 57.7% of all BP patients) to secrete much IL-4, the peptide No.20 can induce the PBMCs of 9 BP patients(accounted for 34.6% of all BP patients) to secrete much IL-4, the peptide No.18 and the peptide No.14 can induce the PBMCs of 7 BP patients(accounted for 26.9% of all BP patients) to secrete much IL-4, resulting in strong Th2 cell response. So the peptide No.21:492-506aa:VRKLKARVDELERIR, No.20:495-509aa:LKARVDELER IRRSI, No.18:501-515aa:ELERIRRSILPYGDS and No.14:513-527aa:GDSMDRIEKDRL QGM were the Th2 cell epitope.(3) There was no obvious correlation between the immune response widths of Th1 cell epitope and Th2 cell epitope and the anti-BPAG2-NC16 A antibody titers in the serum of BP patients.(4) The immune response intensity of BP patients who show positive response after being stimulated by the peptide No.21, the peptide No.20 and the peptide No.14 were correlated positively with the anti-BPAG2-NC16 A antibody titers in the serum of BP patients.(5) Compared with the blank control, obvious proliferation of CD4+T cells was seen after PBMCs of BP patients being stimulated by the the peptide No.21, the the peptide No.18 and the peptide No.14.(6) Stimulating the PBMCs of BP patients and the normal control with the peptide No.21, the peptide No.18 and the the peptide No.14, by detecting the levels of sCD23 in the cell culture supernatant, we found that: the B cell activation levels of BP patients were higher than that of the normal controls; The B cell activation levels of BP patients after being stimulated by positive peptides were higher than that of BP patients after being stimulated by negative peptides.(7) Stimulating the PBMCs of BP patients and the normal control with the peptide No.21, the peptide No.18 and the peptide No.14, by detecting the levels of anti-BPAG2-NC16 A antibody titers in the cell culture supernatant, we found that: The anti-BPAG2-NC16 A titers of BP patients were higher than that of the normal controls; The anti-BPAG2-NC16 A titers of BP patients after being stimulated by positive peptides were higher than that of BP patients after being stimulated by negative peptides.(8) Comparing the positive peptides which stimulated the IL-4 secretion screened from the same BP patients during progression stage and recovery stage of disease respectively, we found that the number of positive epitopes of the progression stage was more than that of the recovery stage; The positive epitopes of the progression stage and the recovery stage were not consistent. There exists “epitope spreading”.(9) HLA-ABC, HLA-DP, HLA-DQ and HLA-DR neutralizing antibodies being added before the positive peptides to the PBMCs from BP patients, we found that the IL-4 levels were decreased when HLA-ABC and HLA-DR neutralizing antibodies being added in advance, but the IL-4 levels did not change notably when HLA-DP neutralizing antibodies or HLA-DQ neutralizing antibodies being added in advance. It was indicated that the positive peptides screened were HLA-ABC and HLA-DR restricted. Conclusion:In conclusion, there were specific Th1 cell epitope and the Th2 cell epitope in the NC16 A region of the BPAG2, the self-antigen of BP. The strengths of immune response of the positive peptide of Th2 cell epitopes were correlated positively with the anti-BPAG2-NC16 A antibody titers in the serum of BP patients. Stimulating PBMCs from BP patients with positive peptides, we found the proliferation of CD4+T cells,the activation of B cells and the production of anti-BPAG2-NC16 A antibodies. The Th2 epitope of BP patients were different in the different stage of disease, and we found there existed “epitope spreading” phenomenon of the Th2 cell epitopes. HLA restrictions of the positive peptides screened being analyzed, the positive epitope we screened before were found to be all HLA-ABC and HLA-DR restricted. Our findings contribute to clarify the immunological pathogenesis of BP, providing new ideas and the scientific evidences about the treatment of BP.