Purified mouse IgG1, mouse anti-DNAM-1, NKp46, NKp44, NKp30 or all four together (all at 10 μg/ml) were added to defined wells during 4 hours of cytotoxicity in order to assess specific activating NK cell receptor-tumor ligand interactions. Reduction in cytotoxicity was calculated based on

percentage cytotoxicity in the presence of indicate blocking mAb(s) versus percentage cytotoxicity in the presence of mouse control mAb. The % reduction in ADCC was calculated with percentage cytotoxicity in the presence of human IgG1 set at 100%. To minimize changes that may occur when cell lines are established from primary tumors, the gastric cell lines used in these studies were cultured for less than 10 passages after isolation from the primary tumor tissue. Statistics Paired two-tailed Student’s t tests were used to calculate p values. P < 0.05 was considered to be significant. Results

Cytotoxic Evofosfamide NK cells are efficiently expanded from PBMC from normal individuals and patients with various solid tumors without the need of primary enrichment protocols To achieve large-scale expansion of human NK cells, PBMC were co-cultured in a 1 to 1.5 ratio with lethally irradiated K562 cells expressing membrane-bound IL-15 and 4-1BBLigand (K562-mbIL15-4-1BBL) in culture media containing 200 units IL2/ml. After 14 days of culture, NK cells (CD56+CD3- as defined by flow cytometry) expanded greater than 2 orders of magnitude from PBMC (mean 165 fold; range 4-567 fold, n = 6) and cell products became significantly enriched in NK cells (day 0 with mean 7%, range 3.2%-12.6% versus day 14 with mean 45.6%, range 7.4%-76.4%; P = 0.0140). Staurosporine At the same time, NKT cells (CD56+CD3+ as defined by flow cytometry) expanded at an average Metformin supplier of 57 fold (range 7-234), although no significant enrichment (day 0 with mean

3.8%, range 0.8%-8.1% versus day 14 with mean 11.4%, range 2.3%-17.9%; P = 0.1907) was observed. In contrast, a significant decrease in T cells (CD3+ as defined by flow cytometry) was noted after 14 days of expansion (day 0 with mean 54.5%, range 39.9%-71.2% versus day 14 with mean 30.0%, range 4.2%-58.4%; P = 0.0436) with an absolute expansion of 7 fold (range 2-19). The distribution of NK cells and NKT cells in PBMC after expansion is shown in Figure 1A. Figure 1 Cytolytic NK cells are efficiently expanded from PBMC. In the presence of K562-IL15-41BBL (A) expanded cells become significantly enriched (P = 0.0307) in NK cells (defined by CD56+CD3- cells) after 14 days of culture. Expanded cells were evaluated for cytolytic activity using 4 hour51Cr release assays. Ex-vivo expanded cells from PBMC (■ donor 1 and △ donor 2), but not freshly purified non-expanded NK cells (◇), efficiently lysed allogeneic tumor cell lines derived from breast (MCF-7) and prostate (LNCaP) cancers but not allogeneic or autologous PBMC derived from donor 1 (B).