Screening And Identification Of HLA-A*0201-restricted CTL Epitope In Chinese Vitiligo Patients

Vitiligo is a common depigmentation disorder caused by progressive destruction ofepidermal melanocytes, with a world prevalence of0.5%-2%. The cellular immunitytowards autoantigens has been demonstrated to play a key role in the destruction ofmelanocytes in vitiligo. While the recognition and presentation of autoantigens areconsidered to initiate the immune response, the identification of autoantigens, especiallythe HLA-A*0201-restricted autoantigens, seems important in our investigation of theautoimmune pathogenesis in vitiligo. Tyrosinase, gp100and MART-1, which are allmelanocyte differentiation antigens, have been considered as autoantigens not only inmelanoma-related immune response but also in the pathogenesis of vitiligo. However, no previous studies have investigated the autoantigens in Chinese vitiligopatients. It still remains a question whether the autoantigens identified in Caucasians playa part in Chinese vitiligo patients. Additionally, there is still lack of evidence whethersome other autoantigens are involved in the pathogenesis of Chinese vitiligo patients. Weapplied ELISPOT assays to determine the cellular immunity against the identified andpredicted autoantigens. The results will deepen our understanding of autoimmunity invitiligo etiology and shed new light in the immune therapy of vitiligo patients.MethodsWe administered the autoantigenic peptides that have been confirmed in Caucasians todetect the cellular immunity in Chinese vitiligo patients. These peptides includetyrosinase369-377，3D: YMDGTMSQV、gp100209-217，2M: IMDQVPFSV、gp100280-288，9V：YLEPGPVTV、MART-126-35，2L： ELAGIGILTV、MART-126-35：EAAGIGILTV. Additionally, we compared the patients’ responses to the above peptidesbefore and after therapy to detect the correlation of the immune response and diseaseprogression.The online software was applied to predict the possible HLA-A*0201-restrictedantigenic peptides originated from tyrosinase, gp100, and MART-1. The peptides andperipheral blood monocytes from HLA-A*0201patients were incubated in the ELISPOTplates and IFN-γ-secreting T cells were detected. Through this, we screened the positivepeptides. We also investigated the peptides’ affinity to HLA-A2molecules throughT2-binding assays, the secretion of cytokines after the stimulation of peptides towardsperipheral blood monocytes of vitiligo patients, and the patients’ immune responsetowards peptide-HLA tetramer through flow cytometry and the laser scanning confocalmicroscope.Results1. The mean T cell frequency of48HLA-A*0201vitiligo patients towards the identifiedpeptides1-5were4.63/2×105PBMC、7.44/2×105PBMC、13.42/2×105PBMC、5.60/2×105PBMC、4.71/2×105PBMC. And37of them demonstrated decreased immuneresponses towards peptide gp100280-288，9V（YLEPGPVTV） after1month’s therapy. 2. We screened the prediction results of the BIMAS and SYFPEITHI softwares, andfinally chose51peptides from tyrosinase,106from gp100and13from MART-1as“possible antigenic peptides”. We sorted out about every10peptides as a “peptide pool” tostimulate the peripheral blood monocytes from HLA-A*0201vitiligo patients. Thepositive peptide pools we detected were peptide pools3and5from tyrosinase, peptidepools12and13from gp100and none from MART-1.3. We applied every single peptide from the positive peptide pools to stimulate theperipheral blood monocytes from HLA-A*0201vitiligo patients. We confirmed thefollowing as positive peptides in ELISPOT assays:①Gp100P-112QILKGGSGT；②tyrosinase P-41AMVGAVLTA；③Gp100P-118QLIMPGQEA；④tyrosinase P-24SSADVEFCL；⑤tyrosinase P-28TLEGFASPL；⑥Gp100P-119TLEGFASPL.4. We discovered that peptide Gp100P-112QILKGGSGT could lead to a higher amountof IFN-γsecretion through ELISA, while other peptides failed to do so. Additionally, theT2-binding assays, the peptides’ response towards peptide-HLA tetramer through flowcytometry and the laser scanning confocal microscope all failed to reveal any positiveresults.Conclusion1. The autoantigens that have been identified in Caucasians can elicit cellular immunityin Chinese vitiligo patients. The cellular responses are correlated with the patients’ diseaseprogression.2. The possible autoantigenic peptides in Chinese vitiligo patients we identified include:①Gp100P-112QILKGGSGT；②tyrosinase P-41AMVGAVLTA；③Gp100P-118QLIMPGQEA；④tyrosinase P-24SSADVEFCL；⑤tyrosinase P-28TLEGFASPL；⑥Gp100P-119TLEGFASPL.