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 diseases 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 surface affinity matrix technology that allows direct identification and enrichment of life antigen-specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8(+) T cells directed against the HLA-A(*)0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generated by combining stimulation of peptide-pulsed autologous dendritic cells (DC) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies (MoAb). Antigen-specific cytotoxic T lymphocyte (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 technology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan-A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine-driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.
Oelke
M
M
Department of Hematology/Oncology, Freiburg Medical University Center, Hugstetterstr. 55, D-79106 Freiburg, Germany.
Kurokawa
T
T
Hentrich
I
I
Behringer
D
D
Cerundolo
V
V
Lindemann
A
A
Mackensen
A
A
D000951:Antigens, Neoplasm; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D064987:Cell- and Tissue-Based Therapy; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D058965:MART-1 Antigen; D008285:Major Histocompatibility Complex; D009363:Neoplasm Proteins / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*
D000951:Antigens, Neoplasm; D016207:Cytokines; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D015847:Interleukin-4; D007371:Interferon-gamma