Abstract
Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.
Highlights
Natural killer (NK) cells play an important role in the immune response against leukemia or tumor cells after stem cell transplantation
We evaluated the samples for NK cell purity, cell viability, CD56dimCD16+ and CD56brightCD16dim/− NK cell subpopulations, residual T cells, monocytes, dendritic cells (DCs) including the subtypes myeloid DCs and plasmacytoid DCs, and NK cell cytotoxic activity
Both, starting numbers of absolute white blood cells (WBC) and percentage of NK cells did not differ between the three groups with median numbers of 1.9 × 1010, 1.9 × 1010, and 2.1 × 1010 for WBCs and 8.5, 7.5, 7.2% for CD56+CD3− cells
Summary
Natural killer (NK) cells play an important role in the immune response against leukemia or tumor cells after stem cell transplantation They represent a promising therapeutic option for patients with various types of malignant disease (Passweg et al, 2006; Rubnitz et al, 2010). NK cells are able to exert a graft-vs.leukemia/tumor (GvL/T) effect without concomitant severe graft vs host disease (GvHD) (Ruggeri et al, 2002) They express CD56, an isoform of the neural cell adhesion molecule, on their surface simultaneously lacking the CD3 antigen. They can be further divided into a CD56dimCD16+ population of about 90% that has cytotoxic activity, and a CD56brightCD16dim/− subpopulation with immunoregulatory properties. Compared to resting NK cells, cytotoxicity of IL-2 activated NK cells is enhanced by up-regulation of the natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp46, and the NK group 2D (NKG2D) receptor (Moretta et al, 2001; Farag et al, 2002; Lanier, 2005; Ljunggren and Malmberg, 2007)
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