title: Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.
abstract: Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.
authors.affiliation: Laboratory of Cellular Physiology and Immunology and Chris Browne Center, The Rockefeller University, New York, New York, United States of America.
authors.forename: Leonia
authors.initials: L
authors.lastname: Bozzacco
authors.forename: Haiqiang
authors.initials: H
authors.lastname: Yu
authors.forename: Jörn
authors.initials: J
authors.lastname: Dengjel
authors.forename: Christine
authors.initials: C
authors.lastname: Trumpfheller
authors.forename: Henry A
authors.initials: HA
authors.lastname: Zebroski
authors.forename: Nawei
authors.initials: N
authors.lastname: Zhang
authors.forename: Victoria
authors.initials: V
authors.lastname: Küttner
authors.forename: Beatrix M
authors.initials: BM
authors.lastname: Ueberheide
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.forename: Ralph M
authors.initials: RM
authors.lastname: Steinman
authors.forename: Svetlana
authors.initials: S
authors.lastname: Mojsov
authors.forename: David
authors.initials: D
authors.lastname: Fenyö
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D000956:Antigens, Viral / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology* / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000276:immunology* / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D016655:HIV Core Protein p24; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments