Separation and growth of human CD4+ and CD8+ tumor-infiltrating lymphocytes and peripheral blood mononuclear cells by direct positive panning on covalently attached monoclonal antibody-coated flasks.
A direct positive panning technique was developed in order to achieve highly purified CD4+ and CD8+ cells. Fresh peripheral blood mononuclear cells or tumor-infiltrating lymphocytes derived from bulk cultures were applied to modified polystyrene surfaces to which anti-CD4 or anti-CD8 monoclonal antibodies were covalently attached. Adherent cells were allowed to grow in the original flask and were then harvested and cultured in IL-2-containing medium. This positive immunoselection technique resulted in CD4+ and CD8+ cell subsets with high cell viability and a high degree of purity. In several samples, the isolated cell subsets were subsequently subjected to a negative immunomagnetic bead selection in order to remove reciprocal cell subset contamination or double-positive CD4+/CD8+ cells. The isolated cells maintained their ability to proliferate, kept their phenotypic profiles, and remained functionally intact after long-term growth in culture without the further addition of mitogenic or allogeneic cell stimulation. This approach is a simple, rapid, and reproducible technique that might be useful on a large scale to isolate and to grow T-cell subsets for research and for clinical use.
Morecki
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S
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Topalian
S L
SL
Myers
W W
WW
Okrongly
D
D
Okarma
T B
TB
Rosenberg
S A
SA
D000911:Antibodies, Monoclonal; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002455:Cell Division / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D016246:Lymphocytes, Tumor-Infiltrating / Q000166:cytology* / Q000276:immunology; D008280:Magnetics; D008863:Microspheres; D011137:Polystyrenes; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D002860:Chromium Radioisotopes; D011137:Polystyrenes