Int. J. Radiat. Biol., Vol. 82, No. 5, May 2006, pp. 355 – 361
Irradiated KHYG-1 retains cytotoxicity: Potential for adoptive immunotherapy with a natural killer cell line
G. SUCK1,2, D. R. BRANCH2,3 & A. KEATING1,2 1
Department of Medical Oncology and Hematology, Princess Margaret Hospital/Ontario Cancer Institute, Toronto, 2Division of Cell and Molecular Biology, Toronto General Research Institute, Toronto, and 3Research & Development, Canadian Blood Services, Toronto Centre, Toronto, Ontario, Canada (Received 28 September 2005; accepted 20 February 2006) Abstract Purpose: To evaluate g-irradiation on KHYG-1, a highly cytotoxic natural killer (NK) cell line and potential candidate for cancer immunotherapy. Methods and materials: The NK cell line KHYG-1 was irradiated at 1 gray (Gy) to 50 Gy with g-irradiation, and evaluated for cell proliferation, cell survival, and cytotoxicity against tumor targets. Results: We showed that a dose of at least 10 Gy was sufficient to inhibit proliferation of KHYG-1 within the first day but not its cytolytic activity. While 50 Gy had an apoptotic effect in the first hours after irradiation, the killing of K562 and HL60 targets was not different from non-irradiated cells but was reduced for the Ph þ myeloid leukemia lines, EM-2 and EM-3. Conclusions: g-irradiation (at least 10 Gy) of KHYG-1 inhibits cell proliferation but does not diminish its enhanced cytolytic activity against several tumor targets. This study suggests that KHYG-1 may be a feasible immunotherapeutic agent in the treatment of cancers.
Keywords: KHYG-1, natural killer cell line, irradiation, adoptive immunotherapy, cytotoxicity
Introduction Novel therapies are emerging that involve cells of the innate immune system, such as natural killer (NK) cells (Gong et al. 1994, Tam et al. 2003). A major advantage clinically is that their response is immediate and does not require prior immunization. Exploiting the innate immune response enables development of therapies that potentially also can target many types of cancers. A novel approach to the adoptive immunotherapy of cancer involves the permanent NK cell line NK-92, which can be easily expanded and maintained in vitro (Tonn et al. 2001, Tam et al. 2003). NK-92 has superior cytotoxic potential compared with endogenous NK cells. Clinical phase I/II trials are currently under way in Europe and North America. NK-92 cells are irradiated before intravenous administration to prevent proliferation of these transformed cells in vivo (Tonn et al. 2001, Tam et al. 2003). After
irradiation, the cells are tested for their ability to lyse standard tumor target cells, such as K562. A dose of 10 gray (Gy) suppresses proliferation of NK-92 cells but retains their full cytotoxic potential (Klingemann et al. 1996, Tam et al. 1999, Tonn et al. 2001). Lower doses of g-irradiation are either insufficient to control proliferation (2.5 Gy) or cell numbers remain constant over 3 days (5 Gy; Tam et al. 1999). We have recently identified another NK cell line, KHYG-1, which exceeds the cytotoxicity of NK-92 against K562 (Suck et al. 2005). Moreover, the cytolytic activity of KHYG-1 is stable under different culture conditions and our molecular data indicate that KHYG-1 remains in a novel activated state. Furthermore, KHYG-1 seems to trigger tumor cell apoptosis by a novel mechanism that involves granzyme M, but not granzymes A or B (Suck et al. 2005), which could enable these cells to overcome tumor cell resistance mediated by the only known naturally occurring granzyme B inhibitor, proteinase
Correspondence: Garnet Suck, PhD, Department of Oncology and Hematology, Princess Margaret Hospital/Ontario Cancer Institute, 610 University Avenue, Suite 5-211, Toronto, ON M5G2M9, Canada. Tel: þ1 416 946 4595. Fax: þ1 416 946 4530. E-mail:
[email protected] Current address: Division of Biomedical Sciences, John Hopkins in Singapore, 31 Biopolis way, #02-01, The Nanos, Singapore 138669. Tel: þ65 6874 0104. Fax: þ65 6874 0177. E-mail:
[email protected] D. R. Branch and A. Keating contributed equally to this work. ISSN 0955-3002 print/ISSN 1362-3095 online Ó 2006 Taylor & Francis DOI: 10.1080/09553000600649653
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inhibitor-9, which is expressed by several cell types (Mahrus et al. 2004). We consider the KHYG-1 cell line, like NK-92, of potential utility in adoptive immunotherapy. Here, we examine the effect of girradiation on KHYG-1 proliferation, survival and cytotoxicity.
Material and methods Cell lines and culture K562 and HL60 were purchased from the American Type Culture Collection (ATCC, Manassass, VA, USA) and maintained in Roswell Park Memorial Institute Medium (RPMI) 1640 medium/glutamine (RPMI, Invitrogen, Grand Island, NY) supplemented with 10% or 20% (v/v) heat inactivated fetal bovine serum (FBS, HyClone, South Logan, UT, USA), respectively. EM-2 and EM-3 were cultured according to standard methods as previously published (Keating et al. 1983, Raskind et al. 1987). KHYG-1 (Yagita et al. 2000) was purchased from The Human Science Research Resources Bank (JCRB0156, HSRRB, Tokyo, Japan) and cultured in RPMI with 2% (v/v) human low-toxicity AB serum (ABS, provided by a single healthy volunteer donor after informed consent) and 450 U/ml recombinant human interleukin 2 (rIL-2, Chiron, QC, Canada). All cell lines were negative by polymerase chain reaction (PCR) for mycoplasma contamination using a Mycoplasma Detection Kit (ATCC).
colony-forming assays (following manufacturer’s instructions) using semisolid methylcellulose medium (StemCell Technologies, Seattle, WA, USA) supplemented with FBS and 450 U/ml rIL-2. Inoculation levels (in duplicates) were equal for control cells and irradiated cells at all doses, 105 cell/ml. Colonies consisting of at least 40 cells colony forming cells (CFCs) per 105 cells were enumerated using an inverted microscope after 5 days incubation at 378C/5% CO2. For studies of apoptosis, cells were incubated with Annexin V-FITC (Biovision) for 4 h at 378C/5% CO2 or directly stained as indicated in the text and Figure legends. For repeated cytotoxicity tests cells were re-cultured after irradiation and then subjected to the assays as described below under ‘Flow cytometric cytotoxicity assay’. Flow cytometry Viable cells were stained with CD2-PE in PBSbuffer, containing 1% (v/v) FBS, 5 mM ethylenediamine tetraacetic acid (EDTA, Flow buffer), and incubated 30 min. on ice, in a 30 ml volume. Viability and early apoptosis were determined with Annexin V-FITC (Biovision) and 7AAD (SigmaAldrich, Oakville, ON, Canada). Acquisition (10000 cells/reaction) was performed using a flow cytometer FACS Calibur (BD, Mississauga, ON, Canada), calibrated with CaliBRITE Beads (BD), and data were analyzed with CellQuest Software (BD). Flow cytometric cytotoxicity assay
Reagents Phycoerythrin (PE)-labeled MoAb anti-CD2 (CD2PE) was purchased from BD Biosciences Pharmingen (San Jose, CA, USA). Fluorescein isothiocyanate labeled Annexin V (Annexin V-FITC) was purchased from Biovision (Mountain View, CA, USA). G-irradiation Exponentially growing KHYG-1 cells were suspended at a concentration of 0.5 6 106 – 106 cells/ ml in phosphate buffered saline (PBS)/0.1% BSA. 5 – 50 ml aliquots in 15 – 50 ml conical centrifuge tubes were irradiated at room temperature using the Gammacell 3000 Elan (Nordion International Inc., Vancouver, BC, Canada) containing a Cesium-137 source using doses of 1 Gy, 5 Gy, 10 Gy, 25 Gy, and 50 Gy, respectively. After irradiation, cells for proliferation/viability studies were plated into 12-well plates (1.5 6 105 cells/ml) and incubated at 378C/5% CO2 and monitored from 1 – 4 days, by counting viable cells in the presence of trypan blue. Potential clonogeneic growth was analysed in standard
The flow cytometric cytotoxicity assay has been previously described (Suck et al. 2005). Briefly, effector NK cells and target cells (2 6 104 target cells per well) were plated in triplicate into 96-well Ubottom plates, at ratios indicated in the figure legends, in RPMI/0.1% bovine serum albumin (BSA) and 225 U/ml rIL-2 and incubated for time periods as indicated in the Figure legends at 378C/5% CO2. As controls for the 0 h time point, the effector and target cells were plated in parallel at identical concentrations, also in triplicate, but separated from each other, and pooled together at the time of harvest. Cell mixtures were stained (see above) for 100% detection of effector NK cells with anti-CD2-PE (BD) for KHYG-1, with K562, EM-2, EM-3, and HL60, previously tested negative for this antibody (data not shown). The cells were then stained with Annexin V-FITC (Biovision) and 7AAD (Sigma-Aldrich) according to the manufacturer’s instructions, and subjected to flow cytometry as described above. A total of 104 cells/ reaction were analysed for each sample at each time point. Percent lysis was reverse calculated from the viable cells at a given time point compared to time 0 h, excluding 7AAD and Annexin V-FITC positive cells,
Cytotoxicity of irradiated KHYG-1
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according to the formula [time 0 h (mean) – time point h (mean)/time 0 (mean) 6 100] (Ozdemir et al. 2003) and the standard error of the mean (SEM) was calculated. Significance for the results is expressed as p-values determined by Student’s t-test for pairwise comparisons. Statistical analysis was performed with Excel software.
Results g-irradiation fails to inhibit cytolytic activity of KHYG-1 To evaluate the effects of irradiation on KHYG-1, we used 10 Gy, 25 Gy and 50 Gy, respectively, by additive application of 10 Gy, 15 Gy, and 25 Gy (all tubes were irradiated together and removed at the corresponding doses). As shown in Figure 1a, all doses of g-irradiation up to 50 Gy inhibited KHYG-1 proliferation. Cell numbers decreased using 10 Gy from 1.5 6 105 cells/ml on day 0 to 6.7 6 104 cells/ ml on day 1, to 3 6 104 cells/ml on day 2 and to 1.3 6 104 cells/ml on day 3. With a dose of 25 Gy cell numbers decreased from 1.5 6 105 cells/ml on day 0 to 4 6 104 cells/ml on day 1, to 1.7 6 104 cells/ ml on day 2 and to 1.7 6 103 cells/ml on day 3. Using a dose of 50 Gy resulted in a decrease in cell counts on day 1 to 5 6 104 cells/ml, on day 2 to 6.7 6 103 cells/ml with no viable cells detected on day 3. This result may indicate a more severe effect of a 50 Gy dose on cell viability. Indeed, as shown in Figure 1b, in the first hours after irradiation, 50 Gy induced a 3.3-fold increase in apoptotic cells vs. control cells, from 13.5 – 45% (Figure 1b) of the whole cell population. None of the other doses led to an increase in the frequency of apoptotic cells compared with control cells. Cell viability in this experiment was monitored for 3 days and measured by trypan blue exclusion (Figure 1c). At day 1, we found an increase of cell death from 8.5% of control cells, to 43%, 59%, and 61%, for 10 Gy, 25 Gy, and 50 Gy, respectively. Whereas control cell viability did not significantly change over time (7.5% at day 3), the irradiated cells underwent significant cell death to 89% (10 Gy), 99% (25 Gy), and 100% (50 Gy) at day 3. We next evaluated the cytotoxicity of irradiated KHYG-1 cells compared with control cells against K562 using our previously established flow cytometric cytotoxicity assay (Suck et al. 2005). We used a 28-hour assay to assess early and late events. Target cells were lysed to 96% by control cells and to 97% (10 Gy), 97% (25 Gy), and 90% (50 Gy) by irradiated cells (Figure 2). Statistical analysis using the Student’s t-test revealed that the minor reduction in cytotoxicity (6%) observed for cells irradiated at the highest dose (50 Gy) compared
Figure 1. Effect of 10 Gy, 25 Gy, and 50 Gy on KHYG-1 proliferation and viability. KHYG-1 cells were irradiated with 10 Gy, 25 Gy, and 50 Gy, respectively, by additive application of 10 Gy, 15 Gy, and 25 Gy (all tubes were irradiated together and removed at the corresponding doses) and compared to untreated KHYG-1 cells (control). Error bars represent SEM. (A) Viable cell numbers were assessed by trypan blue exclusion for 3 days; (B) Annexin V positive cells were determined after 4 h incubation at 378C/5% CO2 by Flow cytometry; (C) Percentage of trypan blue positive cells is shown for 3 days.
with non-irradiated control cells is not significant (p-value ¼ 0.16). These results demonstrate that irradiation with doses up to 50 Gy does not diminish KHYG-1 cytotoxicity. However, lower doses inducing less toxicity within the first day and extending the lifespan of KHYG-1 in vivo would be preferable for AIT. We therefore studied the effect of lower radiation doses on KHYG-1 function. Effects of lower dose g-irradiation on KHYG-1 We irradiated KHYG-1 cells with 1 Gy, 5 Gy, and 10 Gy and monitored cell numbers over 4 days. As shown in Figure 3a, there are no differences in proliferation between control cells and cells irradiated
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Figure 2. Different doses of irradiation do not diminish KHYG-1 cytotoxicity against K562. Flow cytotoxicity assay results comparing the cytotoxic potential of irradiated (additive application of 10 Gy, 15 Gy, and 25 Gy to achieve 10 Gy, 25 Gy, and 50 Gy doses, respectively) and untreated KHYG cells (control), against K562 at a 10:1 E:T ratio and 28 h coincubation; percent target lysis is calculated. Error bars represent SEM. (*p ¼ 0.16 compared to control cells, Student’s t-test).
Figure 3. Effects of lower dose treatment on KHYG-1 proliferation and viability. KHYG-1 cells were irradiated with 1 Gy, 5 Gy, and 10 Gy and compared to untreated KHYG-1 cells (control). Error bars represent SEM. (A) Viable cell numbers were assessed by trypan blue exclusion for 4 days; (B) Annexin V positive cells were determined directly after irradiation by Flow cytometry; (C) Percentage of trypan blue positive cells is shown for 4 days.
with 1 Gy for the first two days. A minor inhibitory effect becomes obvious at day 3 as control cell counts increased 1.6-fold from day 2 to day 3, and treated cells by only 1.2-fold over the same time period. A 5 Gy dose prevented an increase in cell numbers at day 2, but allowed a 1.6-fold increase in cell counts at day 1, compared to a 2.3-fold increase for control cells (Figure 3a). Again, no increase in cell number was observed with a 10 Gy dose. This indicates that irradiation with 5 Gy does not fully inhibit proliferation within the first day. As expected from our experiments using higher doses, no apoptotic effect, as measured by Annexin V, was observed for lower doses directly after irradiation (Figure 3b). At Day 1, 91.5%, 86%, and 66%, of 1 Gy, 5 Gy, and 10 Gy treated cells, respectively, were viable compared with a viability of 95% for control cells (Figure 3c). No differences were apparent in cytotoxicity against the standard target cell line K562, in a 4.5 h assay, at a 10:1 effector to target ratio, with 82% (control), 90% (1 Gy), 89% (5 Gy), 83% (10 Gy) target cell lysis (Figure 4). We next evaluated the cytolytic activity of irradiated KHYG-1 cells over time. KHYG-1 control cells and irradiated cells (1 Gy, 5 Gy, and 10 Gy) were tested for cytotoxicity against K562 in 4.5 – 5 h assays at days 0, 1 and 2. As shown in Figure 5a, the irradiated cells maintained their high cytolytic potential, however, at day 2 cytolytic activity of cells irradiated with 10 Gy significantly (Student’s t-test, p-value of 9.5 6 1075) decreased compared to control cells. Nevertheless, 50% of targets were still lysed by KHYG-1 irradiated with 10 Gy compared to 89% by control cells (Figure 5a). Since cells irradiated with 5 or 10 Gy doses underwent significant cell death on days 3 and 4 (Figure 5b), cytotoxicity was not assessed after day 2. Proliferation (Figure 5c) was monitored by taking cell counts as above (Figure 3a).
Figure 4. KHYG-1 cytolytic capacity against K562 is undiminished after lower dose irradiation. Flow cytotoxicity assay results comparing the cytotoxic potential of irradiated (1 Gy, 5 Gy, and 10 Gy doses) and untreated control KHYG cells against K562 10:1 E:T ratio and 4.5 h coincubation; percent target lysis is calculated. Error bars represent SEM.
Cytotoxicity of irradiated KHYG-1 In order to detect potential outgrowth of individual irradiation resistant clones, we subjected KHYG-1 control cells and cells irradiated at doses 1 Gy, 5 Gy, and 10 Gy to standard colony forming assays. We plated KHYG-1 cells at high densities (105 cells/ml) to score even minimal numbers of CFCs at respective irradiation doses. After five days culture, we did not detect any CFC per 105 cells at the 10 Gy irradiation dose. Using lower irradiation doses, 3 – 5 CFCs per 105 cells (0.003 – 0.005%)
Figure 5. KHYG-1 maintains its cytotoxic potential for several days after irradiation. KHYG-1 cells were irradiated with 1 Gy, 5 Gy, and 10 Gy and compared to untreated KHYG-1 cells (control). Error bars represent SEM. (A) Flow cytotoxicity assays comparing the cytotoxic potential of irradiated (1 Gy, 5 Gy, and 10 Gy doses) and untreated control KHYG cells against K562 at 10:1 E:T ratios and 4.5-5 h coincubation on days 0, 1, and 2; percent target lysis is calculated (*p ¼ 9.5 6 1075 compared to control cells on day 2 determined by Student’s t-test); (B) Percentage of trypan blue positive cells is shown for 4 days; (C) Viable cell numbers were assessed by trypan blue exclusion for 4 days.
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were counted at the 5 Gy dose and 3050 – 3200 CFCs per 105 cells (3 – 3.2%) at the 1 Gy dose, compared to 5200 – 6200 CFCs per 105 cells (5.2 – 6.2%) for control cells (data not shown). Effect of g-irradiation on KHYG-1 cytotoxicity against different tumor targets We chose the highest dose of 50 Gy used in our study to evaluate the effect of irradiation on KHYG-1 cytotoxicity against several target cell lines with relevance to leukemia, lymphoma and myeloma. We previously showed that KHYG-1 efficiently lyses EM-2 (CML), EM-3 (CML), and HL60 (AML, Suck et al. 2005). KHYG-1 was irradiated with 50 Gy, which fully inhibited proliferation on day 1 (data not shown). The frequency of apoptotic cells increased from 11% of control cells to 47% irradiated cells as measured by Annexin V directly after irradiation (data not shown). Irradiated cells were compared to control cells in a 4 h cytotoxicity assay at a 10:1 effector to target ratio (Figure 6). No differences between cytolysis of irradiated and nonirradiated KHYG-1 cells were observed against K562, with 87.5% (control) and 88% (50 Gy) lysis and a minor non-significant difference (Student’s ttest, p-value of 0.15) was found for HL60, with 37.5% (control) and 43% (50 Gy) lysis. However, significant (p-values of 0.04, Student’s t-test), but minor differences were detected for EM-2 with 38% (control), 29% (50 Gy), and EM-3 with 32% (control), and 26% (50 Gy) lysis (Figure 6). These data demonstrate that KHYG-1 cytotoxicity is not substantially affected by doses of g-irradiation as high as 50 Gy. Thus, KHYG-1 cells would be amenable for use in AIT at doses of g-irradiation that would prevent proliferation in vivo while maintaining the
Figure 6. KHYG-1 maintains its cytotoxic potential after irradiation against cell lines with relevance to leukemia. Flow cytotoxicity assay results comparing the cytotoxic potential of irradiated and control (ctrl) KHYG cells against K562, EM-2, EM-3, and HL60 at a 10:1 E:T ratio and 4 h coincubation; percent target lysis is calculated. Error bars represent SEM; p-values between control and irradiated samples for each cell line are determined by pairwise comparisons (Student’s t-test).
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superior cytotoxicity of this cell line compared to other NK cell lines. Discussion Highly cytotoxic permanent NK cell lines are an attractive option for adoptive immunotherapy provided they are safe to administer. At least one NK cell line, NK-92, is in clinical trials (Tonn et al. 2001) and studies with other highly cytolytic cell lines are warranted. We previously showed that KHYG-1 is highly cytotoxic, likely has an unusual molecular pathway triggering target cell killing and warrants further study for possible clinical application. Furthermore, KHYG-1 has been tested negative for mycoplasma, bacterial and fungi contamination by the JCRB (HSRRB) cell bank as well as Epstein-Barr virus infection (Yagita et al. 2000, Matsuo & Drexler 2003), and the ‘host patient’ tested negative for hepatitis B and C, human T cell leukemia virus-1, human immunodeficiency virus-1 and cytomegalovirus-1 and -2 (Yagita et al. 2000). In this context it is essential to demonstrate that irradiation of the cells prevents proliferation but retains cytotoxicity. For potential clinical applications a master cell bank established from KHYG-1 must be reassessed including tests for sterility, mycoplasma contamination, endotoxin, purity of the population, optimal irradiation dose, and functionality. Previous work with NK92 showed that proliferation was prevented but cytotoxicity unimpaired after g-irradiation with 10 Gy (Klingemann et al. 1996, Tam et al. 1999). We, therefore, evaluated KHYG-1, for its proliferation, viability and cytotoxic potential after g-irradiation using a dose range from 1 – 50 Gy. We found that a dose of at least 10 Gy is sufficient to inhibit proliferation of KHYG-1 within the first day. A dose of 1 Gy is insufficient to inhibit proliferation. However, a dose of 5 Gy allows the cells to proliferate until day 2 and then inhibits any additional increase in cell numbers. However, only a dose of 10 Gy, but not of 5 Gy, is sufficient to completely abrogate clonogeneic outgrowth of resistant clones. A dose of 50 Gy had the most dramatic inhibitory effect on KHYG-1 proliferation, although this dose also had an apoptotic effect within the first hours after irradiation and dramatically affected viability within the first day. However, even this high dose treatment did not diminish cytotoxicity against K562 or HL60, and had only a minor effect on EM-2 and EM-3 cytolysis. It has been shown that KHYG-1 cells, which are IL-2 dependent, undergo extensive apoptosis (90%) after 48 h (Taguchi et al. 2004). It is conceivable that in an in vivo situation with no additional IL-2 administration, the effect of
IL-2 deprivation will lead to the death of KHYG-1 cells after at least 48 h. Our study shows that irradiating KHYG-1 cells with a dose of at least 10 Gy prevents cell proliferation but does not affect cytotoxicity within the first two days and suggests that the cell line merits further investigation for the adoptive immunotherapy of malignancies.
Acknowledgements AK holds the Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation at the University Health Network and University of Toronto.
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