title: Identification of HLA-C restricted, HIV-1-specific CTL epitopes by peptide induced upregulation of HLA-C expression.
abstract: HIV-1 negative regulatory factor (Nef) can inhibit CTL recognition by downregulation of HLA-A and HLA-B on the cell surface. In contrast, HLA-C is not affected by Nef and a growing number of studies demonstrate an important role of HLA-C for the control of HIV-1. So far, only a limited number of HLA-C restricted CTL epitopes are known. As the mapping of new CTL epitopes is time and labor intensive, we investigated a novel method for the identification of HLA-C restricted CTL epitopes. B-lymphoblastoid cell lines (B-LCLs) and T2-cells were incubated with HIV-1 specific peptides and subsequently stained for HLA-C surface expression using the HLA-C specific antibody DT9. Peptides that led to increased HLA-C surface expression were used for stimulation of PBMC from HIV-1-infected patients. Subsequently, outgrowing cells were tested for peptide recognition in IFN-γ ELISPOT assays and HLA restriction of the recognized peptides was analyzed in ELISPOT assays using HLA-matched B-LCL. We observed that known HLA-C binding peptides increase HLA-C surface expression on T2-cells and on HLA-C*0102 and HLA-C*0702 homozygous B-LCL. Moreover, screening of HIV-1 Nef with overlapping peptides for potential C*0702 restricted epitopes using this method revealed a total of 8 peptides which considerably increased cell surface expression of HLA-C. By epitope mapping and functional analysis of peptide-stimulated T-cell lines we were able to define the peptide YPLTFGWCY as a new C*0702-restricted CTL epitope. These results show that the analysis of peptide induced HLA-C upregulation on B-LCL and T2-cells enables the efficient identification of new HLA-C restricted CTL epitopes.
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Andrej
authors: A
authors: Stoll
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Silke
authors: S
authors: Bergmann
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Christiane
authors: C
authors: Mummert
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Sandra M
authors: SM
authors: Mueller-Schmucker
authors: Department of Internal Medicine 5, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Bernd M
authors: BM
authors: Spriewald
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors: Ellen G
authors: EG
authors: Harrer
authors: Infectious Diseases Unit, Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany. Electronic address: thomas.harrer@uk-erlangen.de.
authors: Thomas
authors: T
authors: Harrer
mesh_terms: D002478:Cells, Cultured; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D015236:HLA-C Antigens / Q000096:biosynthesis* / Q000276:immunology*; D006801:Humans; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D015854:Up-Regulation
chemical_list: D018984:Epitopes, T-Lymphocyte; D015236:HLA-C Antigens
keywords: CTL epitope; DT9; HIV-1; HLA-C