Identification Of MHC Class ⅠH-2～b-restricted Epitopes Within Retinal Antigens

Purpose:To identify MHC class I H-2b-restricted epitopes within retinal antigens. Methods:Using computer-based prediction strategy to predict MHC class I H-2b-restricted epitopes within IRBP ( Interphotoreceptor retinoid-binding protein ),S-Ag ( Soluble retinal antigen ),Recoverin,Phosducin and PEDF ( Pigment epithelium-derived factor ). The binding abilities of selected peptides for MHC class I H-2b molecules were analysed by peptide binding assay with flow cytometry. C57BL/6 ( H-2b background ) mice were subcutaneously immunized with selected peptides in IFA ( Incomplete Freund's adjuvant ) respectively. 8 days later after immunization,Spleens were removed to prepare splenocytes suspension. Splenocytes of the primed mice were restimulated in vitro for 5 days with indicated peptide loaded syngeneic splenocytes irradiated to proliferate cytotoxic T lymphocytes ( CTLs ). The indicated peptide loaded-RMA-S (H-2b positive TAP-deficient) incubated with 5ICr for 1 hour to be as target cells. Chromium-release assay was used to detect the cytotoxicity of the CTLs. Results:All the selected peptides can upregulate and stabilize expression of MHC class I on the RMA-S cells in vitro. 5 Peptides of them,IRBP-derived peptides:Q-8-L ( QAPVLTNL ),I-8-L ( ISYEPSTL ) and PEDF-derived Peptides:L-9-L (LNQPFLFVL),S-9-L ( SRIVFERKL),S-8-L ( SIIFFLPL ) can induce specific CTLs responses in vivo,while the other 17 peptides,including S-Ag-derived,Recoverin-derived and Phosducin-derived peptides can not generate specific CTLs activities. Conclusion:The IRBP-derived peptides,Q-8-L,I-8-L and PEDF-derived peptides,L-9-L,S-9-L,S-8-L can generate CTLs activities in C57BL/6 mice,which indicate that they can act as immunogenic MHC class I H-2b-restricted peptides. Thesefindings of the specific CTLs epitopes in IRBP and PEDF may lead to improvedunderstanding of the pathogenesis of uveitis. Our study also provides a strategy toidentify specific CTLs epitopes within tumour antigen,which is helpful to make tumourvaccine for the patients.