title: Effect of peptide pools on effector functions of antigen-specific CD8+ T cells.
abstract: Peptide pools are routinely used to study antigen specific T cell responses, both in epitope discovery as well as immune monitoring. However, optimal assay conditions such as concentration of peptides or the best possible number of peptides per pool have not been defined. Thus, we examined whether different peptide concentrations or varying number of peptides per pool influence effector functions of antigen-specific human T-cells. PBMC isolated from HLA-A2-positive individuals with known responses to frequently recognised dominant CD8+ T cell epitopes derived from four different viruses (influenza virus, CMV, EBV, or HCV) were studied. PBMC were cultured with one of these HLA-A2 restricted peptides and varying concentrations of overlapping peptide pools derived from unrelated viruses specific for the hepatitis D and E viruses, the subjects have not been exposed to. Importantly, unrelated peptide pools inhibited the proliferation of IV-M1(58), CMVpp65(495-503), EBV-BMLF(1259-267) and HCV NS3(1073)-specific CD8 T-cells in a dose dependent manner. Similarly, an increase in the number of peptides per pool also impaired antigen specific CD8+ T cell proliferation. In contrast, secretion of cytokines such as IL-2, IL-10, IFN-gamma, TNF-alpha or IP-10 as well as cytotoxicity was not affected by these unrelated peptide pools. The inhibition of proliferation could be restored by blocking PD-1/PDL-1 interaction and was not dependent on DMSO when DMSO concentration was <or=0.5%. Thus, peptide-specific CD8 T-cell proliferation but not cytokine production may be largely underestimated when using a peptide pool which warrants caution in immunomonitoring during clinical trials and in epitope discovery studies.
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Pothakamuri Venkata
authors.initials: PV
authors.lastname: Suneetha
authors.forename: Verena
authors.initials: V
authors.lastname: Schlaphoff
authors.forename: Chun
authors.initials: C
authors.lastname: Wang
authors.forename: Kerstin Anne
authors.initials: KA
authors.lastname: Stegmann
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Fytili
authors.forename: Shiv Kumar
authors.initials: SK
authors.lastname: Sarin
authors.forename: Michael Peter
authors.initials: MP
authors.lastname: Manns
authors.forename: Markus
authors.initials: M
authors.lastname: Cornberg
authors.forename: Heiner
authors.initials: H
authors.lastname: Wedemeyer
mesh_terms: D000956:Antigens, Viral / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D000939:Epitopes / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000276:immunology; D003698:Hepatitis Delta Virus / Q000276:immunology; D016752:Hepatitis E virus / Q000276:immunology; D006801:Humans; D010455:Peptides / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D016207:Cytokines; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D010455:Peptides; D004121:Dimethyl Sulfoxide