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.

Abstract

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.