title: Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases.
abstract: To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gamma-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-gamma upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-gamma-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-gamma staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-gamma upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RA(low) Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-gamma ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.
authors.affiliation: Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital, Lausanne, Switzerland.
authors.forename: M J
authors.initials: MJ
authors.lastname: Pittet
authors.forename: A
authors.initials: A
authors.lastname: Zippelius
authors.forename: D E
authors.initials: DE
authors.lastname: Speiser
authors.forename: M
authors.initials: M
authors.lastname: Assenmacher
authors.forename: P
authors.initials: P
authors.lastname: Guillaume
authors.forename: D
authors.initials: D
authors.lastname: Valmori
authors.forename: D
authors.initials: D
authors.lastname: Liénard
authors.forename: F
authors.initials: F
authors.lastname: Lejeune
authors.forename: J C
authors.initials: JC
authors.lastname: Cerottini
authors.forename: P
authors.initials: P
authors.lastname: Romero
mesh_terms: D000951:Antigens, Neoplasm; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018984:Epitopes, T-Lymphocyte / Q000032:analysis; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000032:analysis / Q000276:immunology; D006801:Humans; D018189:Immunomagnetic Separation; D016130:Immunophenotyping; D007231:Infant, Newborn; D009980:Influenza A virus / Q000276:immunology; D007371:Interferon-gamma / Q000097:blood / Q000378:metabolism*; D018655:Lymphocyte Count; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D015166:Monitoring, Immunologic / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000032:analysis / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; C098952:influenza virus membrane protein (58-66); D007371:Interferon-gamma