Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: Comparison with the MHC-tetramer technology

Cell therapy with antigen-specific T cells holds promise for various diseas es including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)-tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surfac e affinity matrix technology that allows direct identification and enrichme nt of life antigen-specific T cells based on cytokine secretion was evaluat ed in this respect. To this end, CD8(+) T cells directed against the HLA-A* 0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generate d by combining stimulation of peptide-pulsed autologous dendritic cells (DC ) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal ant ibodies (MoAb). Antigen-specific cytotoxic T lympocyte (CTL) were detected based on stimulation-induced interferon (IFN)-gamma and interleukin (IL)-4 secretion and enriched > 100-fold using the cell surface affinity matrix te chnology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cy totoxic activity against Melan-A expressing target cells that was significa ntly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the resu lts obtained from the cell surface affinity matrix technology and those obt ained from tetrameric complexes. Altogether, our study demonstrates that cy tokine-driven enrichment based on the cell surface affinity matrix technolo gy enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.