Synthesis And Screening Of HLA-A*0201 Supertyperestricted CTL Epitopes Derived From The T1D Associated Antigen ChgA

Objective:Type 1 diabetes(T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic islet β cells. It has been commonly accepted that CD4+ T cells play an important role in initiation and progression of T1 D. However, autoreactive CD8+ T cells have been proved to play an indispensable key role in the destruction of islet βcells and development of T1 D.The identification of epitop-e s from islet β cell antigen recognized by CD8+ T cells in humans not o nly can provide new tools for a more accurate prediction of T1 D, but al so is important for the elaboration of new immunotherapeutic strategies. most β-cell antigens, such as insulin, glutamic acid decarboxylase(GAD),and islet-specific glucose-6-phosphatase catalytic subunit-related protein(I GRP), can be recognized by both islet-specific CD4+ T cells and CD8+ T cells. Chromogranin A(Chg A) has recently been identified as an aut-o antigen for diabetogenic CD4+ T cells in both NOD mice and human T1 D subjects. However, it remains unknown whether Chg A can be recogn-i zed by autoreactive CD8+ T cells in T1 D. Human histocompatibility leukocyte antigen-A*0201(HLA-A*0201) is the most commonly expressed HLA class Ⅰ allele in Caucasians and Asians(50%), and contributes to the susceptibility to T1 D. T1 D onset is significantly accelerated in HLAA*0201 transgenic NOD mice with HLA-A*0201-restricted CD8 T cells appearing in early, prediabetic insulitic lesions. Therefore, several studies have used humanized NOD.β2mnull.HHD mice to identify β cell autoanti-g ens-derived peptides that are recognized by HLA-A*0201-restricted CD8+ T cells with potential clinical relevance to human T1 D. However limited peptides have been identified as targets recognized by HLA-A2-restricted CD8+ T cells in human T1 D.Prediction and identification of an HLA-A*0201 supertype restricted CTL epitope derived from the T1 D associated an tigen Chg A.Methods: The Prediction of HLA-A*0201 restricted CTL epit ope derived from the T1 D associated antigen Chg A was based on the we b of SYFEPITHI,IEDB and NETMHC.We identified thecandidate epitop es through HLA-A*0201 binding assaysand characterized the epi-topes i n NOD.β2m nullHHD mice by measuring interferon gamma(IFN-γ) secretion after the activation of splenic lymphocyte cells using Elispot as-say.We check the epitope-specific CD8+CTLs response by measuring IFN-γ, IL-17 and perforin secretion using intracellular cytokine staining. We evaluate the ability of proliferation of the splenic lymphocyte cells by 3H-thymid ine.Results:1. To screen candidate HLA-A*0201 binding pepti-des, an int egrated approach combining three online T-cell epitope predict-ion algorit hms including SYFPEITHI,IEDB and Net MHCwas used.Fo-ur peptides were acquired(Chg A10-19, Chg A43-52, Chg A8-16, Chg A75-84). The purity of pep tides was greater than 95% through HLPC testing and the formula weigh t agreed with the theoretical value. 2. We got the fluo-rescence intensity of the peptides through HLA-A*0201 binding assay.Compared with IGRP206-214 group(40.70±0.07), Chg A10-19(2.74±0.37) and Chg A43-52(3.56±0.41) gr oups had statistical significance, P<0.05. Chg A8-16(0.83±0.20)and Chg A75-84(0.77±0.04)groups had no statistical significan-ce, P>0.05. Compared wi th HIVpol476-484(2.33±0.08) group, Chg A10-19 gr-oups had no statistical sig nificance, P>0.05. Chg A43-52, Chg A8-16 and Chg A75-84 groups had statistic al significance, P<0.05. 3. We evaluated the Splenocytes proliferative res ponses to m Chg A peptides through 3H-th-ymidine uptaking assay. We got the number of counts per minute(cpm) of the peptides. Compared with medium(3116±195), Chg A10-19(7807±524)and Chg A43-52(8166±714) groups had statistical significance,P<0.05. Chg A8-16(3556±360) and Chg A75-84(3766±334) groups had no statistical si-gnificance, P>0.05.Compared with In A2-10 group(8931±768), Chg A10-19 and Chg A43-52 groups had no statistical significance, P>0.05. Chg A8-16 and Chg A75-84 groups had statistical signifi cance, P<0.05. 4. We charact-erized the epitopes by measuring IFN-γsecr etion after the activation of splenic lymphocyte cells using Elispot assay and got the number of cells producing IFN-γ. Compared with medium(23.00±4.00), Chg A10-19(71.00±7.81) and Chg A43-52(54.00±4.58) groups had s tatistical significance,P<0.05. Chg A8-16(20.00±1.73) and Chg A75-84(25.67±1.20) groups had no statisti-cal significance, P>0.05. Compared with In A2-10(80.67±5.81) group, Ch- g A8-16, Chg A10-19 and Chg A43-52 groups had no statistical significance, P>0.05. Chg A75-84 group had statistical significanc e, P<0.05. 5. We got the frequencies of CD8+T cells producing IFN-γ,I L-17 and perforin through flow cytometry. IFN-γ group: Compared with medium(0.93±0.27), Chg-A10-19(-)(22.23±5.04)and Chg A43-52(-)(15.13±3.99)groups had statistical si-gnificance, P<0.05. Chg A10-19(+)(0.80±0.58) and Chg A43-52(+)(0.90±0.06) groups had no statistical significance, P>0.05. Compared with Chg A10-19(-), Chg A10-19(+) had statistical significance, P<0.05.Compared with Ch-g A43-52(-), Chg A43-52(+) had statistical significanc e, P<0.05. IL-17 groups: Compared with medium(6.63±0.59), Chg A10-19(-)(52.23±7.98) and Chg A43-52(-)(45.13±5.23) groups had statistical significan ce,P<0.05. Chg A10-19(+)(6.73±0.12) and Chg A43-52(+)(6.40±0.25) groups ha d no statistical signific-ance, P>0.05. Compared with Chg A10-19(-), Chg A10-19(+) had statistical s-ignificance, P<0.05.Compared with Chg A43-52(-), Chg A43-52(+) had stat-istical significance, P<0.05. Perforin group: Comp ared with medium(3.50±0.49), Chg A10-19(-)(17.27±2.69) and Chg A43-52(-)(15.13±1.73) groups had statistical significance,P<0.05. Chg A10-19(+)(4.63±0.38) and Chg A43-52(+)(0.77±0.09) groups had no statistical significance, P>0.05. Compared wi-th Chg A10-19(-), Chg A10-19(+) had statistical significa nce, P<0.05.Compa-red with Chg A43-52(-), Chg A43-52(+) had statistical si gnificance, P<0.05. Conclusions: Based on thepeptide-specific CD8+ CTLs frequence and re-sponse againstpeptides in spleniclymphocytes from NO D.β2m nullHHD mi-ce, we considered Chg A10-19 and Chg A43-52 immunodomin anter than Chg-A8-16 and Chg A75-84.