Article: article from journal or magazin.
Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases.
Journal of Immunology
Publication types: Journal Article ; Research Support, Non-U.S. Gov't - Publication Status: ppublish
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.
Antigens, Neoplasm, CD8-Positive T-Lymphocytes/cytology, CD8-Positive T-Lymphocytes/immunology, Cell Differentiation/immunology, Cytomegalovirus/immunology, Enzyme-Linked Immunosorbent Assay, Epitopes, T-Lymphocyte/analysis, Flow Cytometry, HLA-A2 Antigen/analysis, HLA-A2 Antigen/immunology, Humans, Immunomagnetic Separation, Immunophenotyping, Infant, Newborn, Influenza A virus/immunology, Interferon-gamma/blood, Interferon-gamma/secretion, Lymphocyte Count, Melanoma/immunology, Melanoma/pathology, Monitoring, Immunologic/methods, Neoplasm Proteins/analysis, Neoplasm Proteins/immunology, Peptide Fragments/analysis, Peptide Fragments/immunology, Sensitivity and Specificity, Viral Matrix Proteins/analysis, Viral Matrix Proteins/immunology
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