Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma

Figure 1 Dose-response relationships of ⁵¹Cr release assay and the two crystal violet staining assays. PBMCs were detected after 15-d culture with irradiated HFWT cells. A: ⁵¹Cr release assay, in which the effector lymphocytes and the fresh target cells pre-labeled with ⁵¹Cr were incubated for 4 h. B and C: crystal violet staining, in which effector lymphocytes and fresh target HFWT cells were incubated for 4 and 24 h, respectively. Each point and bar represent mean and SD (3-6 replicates), respectively.

Figure 2 Killing by T cell- and NK-enriched lymphocytes. Lym-phocytes derived from PBMC of the Subject-1 (taken from Experiment-1 of Table 1) were used in this 24-h crystal violet staining with various E/T ratios. Eight columns on left side are the results from assays of lymphocytes grown on irradiated TKB-17RGB cells (MHC class I positive). Major lymphocyte population was CD3+CD56- (T cell-enriched, see Table 1). For the killing assay, fresh TKB-17RGB (left-end 4 columns) or cells (mid-left 4 columns) were submitted as the target. Right side 8 columns are the results from assays of lymphocytes grown on irradiated HFWT cells (MHC class I negative). Major lympho-cyte population was CD3-CD56+ (NK-enriched, see Table 1). For the killing assay, fresh TKB-17RGB (mid-right 4 columns) or HFWT cells (right-end 4 columns) were used as the target. Each column represents the mean value of triplicate measurements.

Figure 3 Killing activity of the NK-enriched population on MHC class I-positive and -negative cell lines. PBMCs of HCC patient were expanded on irradiated T cells and submitted to the 24-h crystal violet staining. Target cells were MHC class I-negative HFWT and HCC-2 cells, MHC class I-positive HCC- 1 and TKB-17 RGB cells. HCC-1 and HCC-2 cells were derived from the same human hepatocellular carcinoma tissue.