Human Leukemia Antigen-A~*0201-restricted Epitopes Of Human Endogenous Retrovirus W Family Envelope(HERV-W Env)Induce Strong Cytotoxic T Lymphocyte Responses

Objective:Human Endogenous Retroviruses(HERVs)are widely found in the human genome sequence,accounting for 8%-12%of the human genome sequence.The HERV-W family is a member of the HERVs which has similar elements to the gamma retrovirus,and it belongs to the Class ? category.It has been reported that the envelope protein(env)of HERV-W is closely related to many neuropsychiatric and autoimmune diseases.HLA-A*02 is one of the most common HLA class ? molecules expressed in the Han population.Many studies have shown that HLA-A*02 molecules are involved in many viral infections.HERV-W env has been found to induce the immune response.But whether HERV-W env induces an immune response with the restriction of HLA-A*0201 has not been reported yet.The purpose of this article is to study the immunological potential of HERV-W env HLA-A*0201-restricted CTL epitopes.Our research provides experimental basis for HERV-W env polypeptide vaccine research,and contributes a new idea for the treatment of neuropsychiatric diseases.Methods(1)The HLA-A2.1+restricted CTL peptides of HERV-W env were predicted by using SYFEPITHI and BIMAS,and synthesized.(2)HLA-A2 positive blood samples were screened.The whole blood of genomic DNA was extracted by Blood Genomic DNA Mini kit according to the manufacturer's instructions.And then the polymerase chain reaction(PCR)was performed as follow:95? for 5 min,96? for 50s,63.7? for 50s,and 72? for 50s.The positive samples were selected for the following experiment.(3)T cell proliferation was analyzed by CCK-8.PBMCs were distributed in 96-well plates at a density of 6×104 T cells/well in 100?L medium.Peptides were added at a terminal concentration of 20?g/mL.T cells were harvested every 24h.Vital T cells were then tested by Cell Counting Kit(CCK-8).(4)The frequency of peptide-specific stimulation was analyzed by IFN-? ELISPOT assays,PBMCs were stimulated with peptides at a final concentration of 20?g/mL every 24h,constant stimulated 3 times,the PBMCs were harvested for IFN-? ELISPOT assay,spot-forming cell(SFCs)would be counted by Dakewe Biotechnology Company,and culture supernatants were for ELISA assay.(5)PBMCs were cultured with the env peptides(20?g/mL)and ?2-micro globulin(3?g/mL)at 37? in a 5%CO2cell incubator.Stimulated for 16h,peptide-specific T cells were harvested as effector cell,after transfected a pCMV-env for 48h,U251(HLA-A2.1+)and A172(HLA-A2.1-)were used as target cells,pCMV was used as control.Peptide-specific T cells and target cells were co-cultured for 6h at the ratio of 50:1(effective cells to target cells).And then cells were treated by an Annexin V-FITC/PI apoptosis detection kit following the manufacturer's protocol,cells were analyzed via flow cytometry,cytotoxicity mediated by lymphocytes was measured using LDH.Results(1)Among them,five epitope peptides were chosen:TLQDQLNSL(named as T);WILPFLGPL(named as W);CLPSGIFFV(named as C);RVADSLVTL(named as R);QLNSLAAVV(named as Q);EYLVSFGVW was used as negative.(2)The T cell started proliferating on the third day.On the eighth day,the proliferation of T cell of stimulating with peptide W increased hightest by 2.2 times,P<0.001,and peptides(Q and T)increased by nearly 1.8 times as much compared by negative control,P<0.001.In conclusion,T cell would proliferate when stimulating with peptides(W,Q,T and C),but peptides R wouldn't induce T cell proliferating.(3)The SFCs stimulatiing with peptides(W,Q and T)were about 82,66,76 peptide-specific spots,increased 4,3,3.7 times,peptide C increased 0.8 times compared with negative peptide,P<0.0001.The supernatant of the T cell specifically secreted IFN-y determined by ELISA.In this,stimulation with peptide W IFN-y rose nearly one times,P<0.05,stimulation with peptide Q IFN-y rose 0.8 times,P<0.001,and stimulation with peptide T IFN-y increased 0.8 times,P<0.001.(4)The effector cells stimulation with peptides(W,Q and T),would cause the death rate of the target cells which transfected a plasmid pCMV-env increasing by 2.2±0.2 times compared to pCMV,P<0.0001,the apoptosis of the target cells(U251)stimulation with peptides(W,Q,T and R)increased 2.2±0.2 times compared to plasmid pCMV,P<0.0001.In conclusion,peptides(W,Q and T)were HERV-W env antigenic epitopes,and they have both antigenicity and immunogenicity,so that they could cause strong T-cell immune responses.From these results,we suspect that HERV-W env may act as an autoantigen induced autoimmunity in people with neuropsychiatric disease.Conclusion(1)Peptides(W,Q and T)have the immunogenicity,and they can product IFN-?(2)Peptides(W,Q and T)specifial CTLs could cause env+ U251 cell apoptosis in in an HLA-A*0201-restricted manner,but not env-U251 or env+/env-A172 cells.(3)Peptides(W,Q and T)are the HERV-W env epitope.