Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: Implicationsof a novel approach for T cell monitoring in infectious and malignant diseases

elucidate the functional heterogeneity of Ag-specific T lymphocyte populati ons, we combined labeling of lymphocytes with MHC/peptide tetramers and a c ell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gam ma -positive lymphocytes allowed the selective enrichment and identificatio n of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, me mory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells se creted IFN-gamma upon challenge with cognate peptide, consistent with an ef fector/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 (cyt ospot). CD8(+) T cells specific for the melamoma-associated Ag Melan-A/MART -1 did not produce IFN-gamma upon challenge with cognate peptide, reminisce nt with their naive functional state in healthy individuals. In contrast, C D45RA(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 different iation into competent effector/memory T lymphocytes in vivo. Notably, a siz able fraction of Melan-A/MART-1-specific cells from a patient secreted IFN- gamma ex vivo following peptide-based vaccination. Thus, the high sensitivi ty of the assay provides a valuable tool to monitor effector T cell respons es in different clinical situations.