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: M J Pittet, A Zippelius, D E Speiser, M Assenmacher, P Guillaume, D Valmori, D Liénard, F Lejeune, J C Cerottini, P Romero
• Journal: Journal of immunology (Baltimore, Md. : 1950) (J Immunol) Vol. 166 Issue 12 Pg. 7634-40 (Jun 15 2001) ISSN: 0022-1767 [Print] United States
• PMID: 11390521 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antigens, Neoplasm
• Epitopes, T-Lymphocyte
• HLA-A2 Antigen
• MART-1 Antigen
• MLANA protein, human
• Neoplasm Proteins
• Peptide Fragments
• Viral Matrix Proteins
• influenza virus membrane protein (58-66)
• Interferon-gamma

Topics

• Antigens, Neoplasm
• CD8-Positive T-Lymphocytes (cytology, immunology, metabolism, pathology)
• Cell Differentiation (immunology)
• Cytomegalovirus (immunology)
• Enzyme-Linked Immunosorbent Assay
• Epitopes, T-Lymphocyte (analysis)
• Flow Cytometry
• HLA-A2 Antigen (analysis, immunology)
• Humans
• Immunomagnetic Separation
• Immunophenotyping
• Infant, Newborn
• Influenza A virus (immunology)
• Interferon-gamma (blood, metabolism)
• Lymphocyte Count
• MART-1 Antigen
• Melanoma (immunology, metabolism, pathology)
• Monitoring, Immunologic (methods)
• Neoplasm Proteins (analysis, immunology)
• Peptide Fragments (analysis, immunology)
• Sensitivity and Specificity
• Viral Matrix Proteins (analysis, immunology)