Predicition Of T Cell Epitope Of RBP3 In Tree Shrew And Aheir Ctivity On Uveitis

Background and ObjectiveRetinol-binding protein?RBP?,also known as interphotoreceptor retinoid-binging Protein?IRBP?,is a type of glycoprotein produced and secreted mainly by retinal rod cells.It locates in the photoreceptor matrix and exerts the role of transport and release of vitamin A between the photoreceptors and the retinal pigment epithelium.In addition,IRBP also has strong antigenic and uveitogenic activity.The experimental autoimmune?EAU?model established by IRBP-induced susceptibility animals is the main tool for the study of human autoimmune uveitis.Currently,scientists mainly study human uveitis with EAU in immunological susceptibility animals induced by human and bovine IRBP-derived peptide.For example,the EAU in B10.RIII mice immunized by human IRBP_161-180 peptide,EAU obtained by inducing C57BL/6 mice with human IRBP_1-20 or human IRBP_651-670,Lewis rats were immunized with bovine IRPB_1177–1191?R16?antigen polypeptide and human IRPB_1169-1191?R14?antigen peptide.The mechanism of IRBP-induced EAU remains unclear.However,the possible mechanism is the immunodominant polypeptide fragments have strong affinity with the major histocompatibility complex?MHC?on antigen-presenting cells.That is,the immunodominant epitope of IRBP binds to an important site on the MHC molecule on the antigen presenting cell,activates the antigen presenting cell,and the activated antigen presenting cell can induce the differentiation of Naive T cells into effector Th1 cells and Th17 cells.Then,those cells cause the occurrence of diseases by producing inflammatory cytokines such as IFN-?and IL-17.Recently,tree shrews,the closest relatives of primates,have a high degree of similarity with primates in terms of visual systems,immune systems,and have successfully used tree shrews to establish disease models such as myopia and hepatitis.Moreover,studies have shown that the major histocompatibility complex?MHC?class II molecules involved in antigen presentation during the immune response contain human corresponding MHC class II molecules.Whole genome sequencing reported that the tree reed interstitial retinol-binding protein RBP3?ELW66657.1?,but its protein T cell epitopes and whether it has potential for uveitis activity have not been reported.Based on the above background,we conducted the following three aspects of the study:?1?Analysis of homology of amino acid sequences of tree shrew RBP3 protein with human IRBP protein,papio IRBP,bovine IRBP,rat IRBP and mouse IRBP by DNAMAN software.Predicts T cell epitopes,Th cell epitopes,and CD4⁺T epitopes.According to the prediction results,antigen peptides with high homology to IRBP_161-180 induced in the classical B10.RIII mouse EAU model were screened and synthesized,and the immunogenicity and activity of murine uveitis induced by mice were evaluated in order to predict the tree shrew RBP3 protein antigen peptide with immunogenicity and activity;?2?EAU was induced in B10.RIII mice by screening with tree shrew RBP3 protein antigen peptides and compared with IRBP_161-180-induced B10R.III mouse EAU;?3?Tree shrew RBP3_1197-1211 peptide with high homology with human R14 antigen and human R14 antigen were synthesized and injected in tree shrews to investigate clinical manifestations and pathological features of disease,through the above research,we hope to provide the basic theoretical basis for the establishment of a new type of EAU model,and provide an ideal animal model for the study of uveitis.Part ?Method:Comparison of amino acid sequences of tree protein RBP3protein with human IRBP,papio IRBP,bovine IRBP,rat IRBP and mouse IRBP protein amino acid sequences using DNAMAN software.The Protean software was used to predict and analyze Th cell antigenic epitopes of tree shrew RBP3 protein and T cell antigenic sites that interact with murine MHC class II molecules.RBP3_182-192 peptide was synthesized based on the predicted results,and 200?l of a 100?g RBP3_182-192 polypeptide emulsified complete freund's adjuvant?CFA?was subcutaneously injected into B10.RIII mice.The clinical manifestations and histopathological changes of ocular inflammation in mice were evaluated by slit lamp microscope and H&E staining in order to evaluate the uveitis activity of mice induced by RBP3_182-192 peptide;MTT assay was used to detect the proliferation of spleen cells of RBP3_182-192 peptide at the peak of inflammation to evaluate the immunogenicity of the selected RBP3_182-192peptide.Results:Blast results showed that RBP3 had the highest homology with human IRBP?83.50%?,then it was followed by rat IRBP?82.94%?papio IRBP?82.81%?,mouse IRBP?82.63%?and bovine IRBP?82.23%?;The results of predictive analysis showed that RBP3 protein in tree shrews contained 47 potential epitopes of Th cell antigen,accounting for approximately 44%of the amino sequence,and also had 34immunologically relevant antigen binding sites for murine MHC-II molecules.The disease in B10.RIII mice developed on 10 day after immunization and peaked on 13 day postimmunization.The mice were found corneal opacity,ciliary hyperemia,abnormal pigmentation of the iris,hypopyon,and irregular pupils,as observed by a clinical slit lamp microscope.Histopathological examination of HE staining revealed that the ocular tissues showed full uveitis,with a large number of inflammatory cells in the anterior segment and posterior segment of the eye,retinal structures collapsed,granuloma formed in the retina,and extensive cellulose in the subretinal space was exuded.No inflammation was observed in the control group.At the same time,RBP3_182-192 peptide significantly induced splenic lymphocyte proliferation in mice at the peak of inflammation.Conclusion:There are more Th cell epitopes and CD4⁺T epitopes in the RBP3 protein of tree shrews.The predicted and screened RBP3_182-192peptide has the immunogenicity of inducing T cell proliferation and the activity of mouse uveitis disease,and provides a theoretical basis for the application of RBP3 protein and establishment of a novel EAU model.Part ?Method:200?l of CFA emulsion containing 50?g of RBP3_182-192 or IRBP_161-18061-180 peptide was induced by subcutaneous injection of B10.RIII mice,which were TS-EAU group and HU-EAU group respectively.HU-EAU group was used as positive control.200?l CFA-PBS solution was used to immunize B10.RIII mice as a normal control group.Application of slit lamp microscopy and H&E staining to evaluate the clinical manifestations and histopathological changes in inflammation,development and disappearance of mice eyes;after the EAU model was established,RNA from spleen tissue was isolated and extracted at the onset of inflammation,peak period,and remission period,q-PCR was used to detect the expression levels of T-bet mRNA and Th17 cell specific transcription factor ROR?t mRNA;after the establishment of EAU model,serums isolated at the onset of inflammation,peak period and regression period were detected,and the levels of IFN-?Secretion by Th1 and Th17cells secrete IL-17 were measured by ELISA;after the establishment of EAU model at the peak of inflammation,the proliferation of T lymphocytes was detected using the MTT cell proliferation assay kit.Results:Inflammation started on the 10th day after induction in TS-EAU group,which was 3 day later than that of HU-EAU group?7d?,and the inflammation peaked at the 13th day,then the inflammation gradually decreased,and there was still slight inflammation on the 46th day.The inflammation in HU-EAU group basically disappeared on 35d after immunization.Clinical manifestations and histopathological changes are basically the same,the main clinical manifestations of mouse corneal opacity,ciliary hyperemia,abnormal pigmentation of the iris,anterior chamber empyema,pupillary irregularities and defects.Histopathological changes were mainly retinal and choroidal inflammatory cell infiltration,retinal structural disorder folding,granuloma formation in the retina,and extensive cellulose exudation in the subretinal space.No inflammation was seen in the normal control group.q-PCR results showed that the level of ROR?t in HU-EAU group was highest at the beginning of inflammation and then decreased,and T-bet expression was highest at the peak of inflammation,while the ROR?t and T-bet in the TS-EAU group were highest at the peak of inflammation and low at the beginning of inflammation.The results of ELISA showed that the expression of IL-17 in HU-EAU group was highest at the beginning of inflammation,then decreased,and the expression of IFN-?was highest at the peak of inflammation,while the IL-17 and IFN-?in the TS-EAU group were highest at the peak of inflammation and low at the beginning of inflammation.T cell proliferation assays showed that both RBP3_182-192 and IRBP_161-180 antigens significantly stimulated the proliferation of lymphocytes.Conclusion:TS-EAU in B10.RIII mouse induced by RBP3_182-192peptide has the similar characteristics in the clinical manifestations and histopathological changes as IRBP_161-180 induced B10.RIII mouse HU-EAU model.However,the immune mechanism of TS-EAU has its own characteristics and can be used as a new potential EAU model for studying uveitis.Part ?Method:300?g human R14 peptide and 300?g tree shrew RBP3_1197-1211 peptide emulsified with 1:1 CFA supplemented with 2.5mg/ml of H37Ra tuberculosis in a total of 200?l were subcutaneously injected into the tree shrew.At the same time,intraperitoneal injection of500ng of PTX was conducted.Tree shrew immunized by those peptide were designated as group A and group B respectively.200?l of CFA-PBS solution was used to immunize tree shrew was used as a normal control group.Slit lamp microscope and ophthalmoscope were used to observe the changes of ocular inflammation in tree shrews.Results:Slit lamp microscopy showed that inflammation appear on 30day after induction in group A,manifested as local mild corneal opacity,iris depigmentation,and iris depigmentation at 5 months;In group B,there was a slight local corneal opacity and iris depigmentation around 20 days.Ophthalmoscope observation showed that after 5 months of immune tree shrew in group A,local vasculitis was exuded;In group B,no obvious inflammation was observed for 30 days.The histopathology showed that the retina layers in group A were thickened,the inflammatory cells in the retina were infiltrated,and the retinal pigment layer,photoreceptor cell layer,and ganglion cell layer were folded and distorted.The retina layers in the B group were thickened.No inflammation was seen in the normal control group.Conclusion:The tree shrew EAU model can be established by using human R14 antigen fragment and tree shrew RBP3_1197-1211 polypeptide fragment.However,further studies are needed to investigate the optimal conditions for inducing tree shrew EAU model.It is expected to provide a new EAU model for uveitis research after further study.