| Many diseases such as tumor,ischemia and inflammation cause acidic extracellular microenvironment at the site of the lesion,This extracellular acid microenvironment also provides new opportunities for the diagnosis and treatment of these diseases.Acid-responsive peptide(The pH low-responsive peptides,p HLIP)can be spontaneously folded into a transmembrane helix and spanned the cell membrane in an acidic microenvironment,which facilitates to target these acidic tissues.PD-1(Programmed death-1,CD279)is an inhibitory receptor,which is induced in T cells upon activation.The engagement of PD-1 with its two ligands PD-L1 and PD-L2 maintains peripheral immune tolerance.Objective:In this study,m PD-L1-WT p HLIP is constructed by ligation of WT p HLIP to ectodomain of PD-L1 via(G4S)3 linker and then its function is validated,which will fuel the utilization of the protein to treat autoimmune diseases in the future.Methods:m PD-L1-WT p HLIP and related proteins are constructed,followed by eukaryotic expression using HEK293 cell lines.The identity and purity is determined by SEC and SDS-PAGE respectively.ELISA and FACS are used to determine the recognition of anti-PD-L1 antibody and recombinant m PD-1 protein as well as membrane-bound PD-1 by m PD-L1-WT p HLIP.The acid-responsive property is validated by FACS and immunofluorescence.Finally,the immunosuppressive function of m PD-L1-WT p HLIP is determined by mouse lymphocyte isolation and culture.Results:(1)m PD-L1-WT p HLIP and related proteins were obtained successfully;(2)The engagement of m PD-L1-WT p HLIP with anti-PD-L1 antibody and recombinant m PD-1 protein as well as membrane-bound PD-1 was very well;(3)m PD-L1-WT p HLIP had excellent property to responding low p H;(4)Under immobile or acidic conditions,m PD-L1-WT p HLIP potently inhibited lymphocyte expansion and IFN-γ secretion through PD-1-PD-L1 pathway.Conclusions:m PD-L1-WT p HLIP harbors superior features of responding low pH and immunosuppressive function. |
