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Ex Vivo IFN-γ Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases

2001; American Association of Immunologists; Volume: 166; Issue: 12 Linguagem: Inglês

10.4049/jimmunol.166.12.7634

1550-6606

Mikaël J. Pittet, Alfred Zippelius, Daniel E. Speiser, Mario Assenmacher, Philippe Guillaume, Danila Valmori, Danielle Líénard, Ferdy J. Lejeune, Jean‐Charles Cerottini, Pedro Romero,

Immunotherapy and Immune Responses

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-γ. Magnetic cell sorting of IFN-γ-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-γ 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-γ-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-γ staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-γ upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RAlow 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-γ 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.