IDENTIFICATION OF NATURALLY PROCESSED 'SELF' AND HIV DERIVED MHC CLASS I LIGANDS PRESENTED BY HEALTHY AND HIV-INFECTED CELLS

摘要

Class I MHC molecules display to CD8 T cells short peptides derived from intracellular processing of host and pathogen derived proteins. The endogenous class I peptide repertoire presented by healthy or pathogen-infected cells remains largely unknown. The identification of peptides uniquely presented by infected cells that could trigger most potent immune responses remains of utmost importance for efficient design of vaccine strategies. To identify endogenous class I peptide repertoire presented by human healthy and HIV infected cells, we developed two complementary MS-based approaches: one to identify peptides eluted from immunoaffinity-isolated MHC class I molecules, and one to identify all surface peptides directly eluted from live cells. This strategy was applied to analyze "self" peptides eluted from peripheral blood cells (PBMC) of anonymous healthy blood donors. To discover HIV-derived ligands, a human B cell line was infected with a HIV-derived lentiviral vector expressing GFP and pseudotyped with VSV-G envelope (LV-GFP-VSVg). In addition, we applied MS/MS method to determine HLA-A and HLA-B isotypes of membrane-isolated MHC class I molecules from PBMC and B cells along with the identification of their corresponding bound peptide ligands. In this study, we reliably identified a repertoire of over 300 "self" MHC class I peptides eluted from PBMC and/or from a human B cell line, originating from 55 respective proteins. 80% of the identified "self" peptide ligands were predicted as possible epitopes, based on the presence of anchor aa residues for MHC-I binding, by the epitope prediction algorithm. The comparison of surface peptides presented by infected and uninfected cells demonstrate that HIV infection altered presentation of host "self" peptidome in B cell line expressing HLA-A03, A11, B35 and B51 isotypes. In total, 62 of the identified "self" class I peptide ligands were differentially presented on infected B cells. These "self" peptide ligands, uniquely presented by infected B cells, originated from 42 common host cell proteins and from 20 host cell proteins encoded only following HIV infection. We identified 8 HIV-derived peptides presented by HLA class I molecules of LV-GFP-infected human B cell line. All corresponded to HIV-Gag sequence. One of the identified peptide ligand, naturally processed by human B cell line, corresponded to an already reported HLA-B35-restricted epitopes documented to elicit immune responses in HIV-infected HLA-B35 individuals. Class I peptide epitopes identification displayed by HIV-infected cells is crucial for understanding unbiased antigen processing and vaccine immunogens development.