STUDIES ON NK CELL PURIFICATION BY NEGATIVE SELECTION IN HUMAN PERIPHERAL-BLOOD

We have attempted to improve negative selection procedures for the large scale purification of human CD3- CD56+ NK cells. In a series of experiments, purifications of NK cells from 10(8) PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3+ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10(8) cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10(9) PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior to in vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.