Leukemia (2005) 19, 835–840 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu CD56dimCD16neg cells are responsible for natural cytotoxicity against tumor targets

O Penack1, C Gentilini1, L Fischer1, AM Asemissen1, C Scheibenbogen1, E Thiel1 and L Uharek1

1Department of Hematology, Oncology, and Transfusion Medicine, Charite´-Campus Benjamin Franklin, Berlin, Germany

The activation of natural killer (NK) cells leads to degranulation lysosomal-associated membrane protein-1 (CD107a). In a series and secretion of cytotoxic granula. During this process, the of experiments, it was shown that CD107a surface mobilization lytic granule membrane protein CD107a becomes detectable at 7 the cell surface. Based on this phenomenon, we have analyzed can be used to isolate and analyze cytolytic T cells ex vivo. by a novel flow cytometry-based assay, the number and Wolint et al used CD107a surface expression to study the phenotype of NK cells responding to tumor targets. Using regulation of cytolytic activity in effector T cells, effector human leukemia and lymphoma cell lines, we observed a close memory T cells and central memory T cells in response to viral correlation between CD107a surface expression and target cell targets. They found that degranulation occur with similar lysis, indicating that NK cell cytotoxicity can be assessed by kinetics in all T-cell subsets. However, degranulation of central this method. The number of degranulating NK cells was closely memory T cells was not followed by target cell lysis due to lack related to the ratio of effector and target cells and showed a 8 maximum at a ratio of 1:1. Moreover, we were able to show that of stored cytotoxic effector molecules. the population of CD56dim/CD16neg NK cells is primarily Target cell killing via the granule-dependent pathway is a responsible for the cytotoxic activity against tumor targets common mechanism of CTLs and NK cells.9,10 Recently, a dim pos bright whereas neither CD56 /CD16 nor CD56 NK cells correlation of NK cell cytotoxicity, CD107a surface expression degranulated in response to the cell lines. Our results indicate and the level of viral replication was found in HIV-1 infected that the CD107a assay represents a promising new method for subjects, suggesting that CD107a expression can serve as a the quantification and characterization of cells exhibiting 11 natural cytotoxicity. marker for NK cell activity in HIV infection. We examined Leukemia (2005) 19, 835–840. doi:10.1038/sj.leu.2403704 whether NK cell activation due to tumor targets can be Published online 3 March 2005 visualized by CD107a surface mobilization and whether Keywords: NK cell; CD107a; flow cytometry; degranulation; activity degranulation correlates with cytolytic activity. For that purpose we determined the surface expression of CD107a on NK cells of healthy donors after coincubation with different leukemia and lymphoma cell lines. We observed a strong correlation between Introduction CD107a surface expression and NK cell cytotoxicity determined in a standard flow cytometry-based cytotoxicity assay. Addi- Natural killer (NK) cells are the predominant cytolytic effector tionally, we were able to determine NK cell activation in cells in innate immunity. They are defined by expression of response to tumor targets on a single-cell level which allows to CD56 and lack of T-cell receptor expression. Different NK cell analyze activation of the distinct NK cell subpopulations compartments can be distinguished by the surface density of separately. CD56 and the presence of the Fc gamma receptor III protein CD16.1,2 More than 90% of peripheral blood NK cells belong to dim the CD 56 subset, which has been shown to express perforin Methods as well as the killer cell Ig-like receptors (KIRs). A subpopulation of CD56dim NK cells expresses the Fc receptor CD16 and is Tumor cell lines involved in antibody-dependent cellular cytotoxicity (ADCC).3 bright Perforin and KIRs are not present on CD56 NK cells, which The following human cell lines were obtained from German are rare in blood but represent the predominant cell type in Collection of Microorganisms and Cell Cultures (Braunschweig, 1 lymph nodes and tissues. It has been hypothesized that the Germany): HEL (ACC11, AML/Erythro-leukemia),12 HL60 bright CD56 subset exhibits immunoregulatory functions through (ACC3, AML),13 K562 (ACC10, CML in blast crisis),14 KASU- the secretion of various cytokines (ie IFN-g, TNF-a, or IL-10), MI-1 (ACC 220, AML),15 ML2 (ACC15, AML),16 EHEB (ACC67, dim whereas CD56 cells are responsible for the cytolytic activity CLL),17 GRANTA-519 (ACC342, mantle cell lymphoma),18 4 against virally infected cells and autologous tumor cells. JEKO-1 (ACC553, mantle cell lymphoma) and 19 KARPAS-422 Target cell killing by cytotoxic T cells (CTLs) and NK cells (ACC 32, diffuse large cell lymphoma).20 mainly relies on degranulation, which leads to the release of The EBV-transformed human lymphoblastoid leukemia lytic granules containing perforin and granzymes. During this L721.22121 cell line was generously provided by Christine S process, lysosomal membranes and plasma membranes merge Falk, Institute of Molecular Immunology, GSF National Research and vesicle membrane proteins become visible on the cell Center for the Environment and Health, Munich, Germany. 5 6 surface. Recently Betts et al reported on the development of a The cell lines were kept at 371C in incubators and supplied pos novel multiparameter flow cytometry assay detecting CD8 with 5% CO2 and air in cell suspension cultures consisting of CTL activity by measuring degranulation via expression of the RPMI medium supplemented with 4 mmol/l L-glutamine (Sigma, Munich, Germany), 100 U/ml streptomycin (Sigma), 100 U/ml Correspondence: Dr O Penack, Department of Hematology, Oncol- penicillin (Sigma) and 10% fetal calf serum (FCS; Kraeber, ogy, and Transfusion Medicine, Charite´-Campus Benjamin Franklin, Wedel, Germany). On the day of testing, cell counting was Hindenburgdamm 30, 12200 Berlin, Germany; performed with trypan blue to assess cell viability. The target Fax: þ 49-30-8445-3395; E-mail: [email protected] This work was supported by a grant of the Deutsche Krebshilfe. cells were washed once in Roswell Park Memorial Institute 1640 6 Received 27 October 2004; accepted 7 December 2004; Published medium (RPMI), resuspended and adjusted to 1 Â 10 viable online 3 March 2005 cells/ml in RPMI. CD56dimCD16neg NK cells mediate cytotoxicity O Penack et al 836 CD107a assay mingen, Hamburg, Germany) in direct immunofluorescence assays: Anti-CD16 FITC (fluorescein isothiocyanate), anti-CD56 Peripheral venous blood from healthy individuals was stored in PE (phycoerythrin), anti-CD107a PE and anti-CD107a PE-Cy5 heparinized tubes and peripheral blood mononuclear cells (phycoerythrin-Cy5). Antibody concentrations were adjusted (PBMCs) were isolated by density gradient centrifugation on a according to the protocol of the manufacturer. Flow cytometric Ficoll-Paque gradient. The mononuclear cells from the interface analysis was performed by a FACScalibur cytometer (BD were washed twice in phosphate-buffered saline (PBS), resus- Pharmingen). In all experiments at least 30 000 events were pended and adjusted to 1 Â 106 cells/ml in RPMI, Sigma, counted and the results were expressed as the percentage of Munich, Germany. All experiments were repeated to assess cells in a gated lymphocyte region. reproducibility. To detect spontaneous degranulation, a control sample without target cells was included in every experiment. An effector/target (E/T) ratio of 1:1 (2 Â 105 effector cells: 2 Â 105 Results target cells in a volume of 200 ml) was used in all experiments. In each tube, containing 200 ml E/T cell suspension, 15 ml of PE- Activated NK cells express CD107a Cy5 conjugated anti-CD107a monoclonal antibody (mAb) was added prior to incubation. PBMCs and target cells were To assess the degranulation of NK cells in response to tumor coincubated (371C) for 3 h in total, after the first 1 h 5 ml of the targets, we examined the surface expression of CD107a after secretion inhibitor 2 mM monensin (Sigma, Munich, Germany) coincubation of PBMC with the NK-sensitive cell line K562 and in 100% ethanol was added. Degranulating effector cells the NK-resistant cell line ML2. No CD107a expression was express CD107a on the cell surface as early as 30 min to 1 h detected on resting NK cells (Figure 1c). After coincubation with following stimulation. As optimal expression of CD107a occurs K562 cells up to 6% of CD56pos cells expressed CD107a, between 3 and 6 h poststimulation, we chose the 3 h coincuba- indicating that a subpopulation of NK cells releases cytotoxic tion period.6,7,11 At the end of coincubation, cells were washed granules after contact with these target cells (Figure 1a). In in PBS and stained with mAbs (CD56, CD16) for flow cytometric contrast, coincubation with NK-resistant ML2 leukemia cells analysis. CD56pos cells, which accounted for 10–30% of the was not followed by an increased surface expression of CD107a gated lymphocytes, were divided into three compartments: (Figure 1b). CD56dimCD16neg, CD56bright and CD56dimCD16pos. Surface expression of CD107a was assessed separately in the NK subsets. Induction of CD107a expression depends on E/T ratios Cytotoxicity assay To determine the influence of different E/T ratios on NK cell NK-cell-mediated cytotoxicity was analyzed as described activation, as indicated by CD107a expression, we performed pos previously.22 Briefly, target cells were stained with 3,30- the assay after immunomagnetic selection of CD56 PBMCs dioctadecyloxacarbocyanine (DiO; Sigma, Munich, Germany) from healthy donors. The NK-sensitive cell lines HL60 and K562 solution for 20 min at 371C. After washing the target cells with were added in E/T ratios ranging from 1:50 to 50:1. As shown in PBS, NK cells and target cells (E:T ratios: 100:1, 50:1, 20:1 and Figure 2 for one example, the highest CD107a surface 10:1) were coincubated for 4 h in RPMI medium. Dead cells expression was detected in the 1:1 E/T ratio. were counterstained using propidium iodide (Sigma) solution and analyzed by flow cytometry. DiO-labeled target cells emit a dim neg green fluorescence and propidium iodide-stained target cells Expression of CD107a is restricted to CD56 CD16 emit a red fluorescence, which where detected by flow cells cytometry. To determine the specific lysis of target cells, the degree of spontaneous cell death was assessed by examining a To further characterize the responding cell population, the sample containing target cells only. mobilization of CD107a to the cell surface was determined in three different NK cell subsets. namely CD56dim/CD16neg, CD56bright and CD56dim/CD16pos cells (gates are shown in Cytotoxicity assay after CD107a positive selection Figure 3a). As demonstrated in Figure 3b, CD56dimCD16neg NK cells degranulated after coincubation with K562 cells. Neither bright dim pos To investigate if CD107apos NK cells rather than CD107aneg NK CD56 nor CD56 CD16 cells showed CD107a surface cells mediate cytotoxicity, we isolated CD56pos cells from expression after leukemia cell stimulation (Figures 3c and d). healthy donors PBMC by positive selection with anti-CD56 Similar results were obtained in experiments with NK cells from magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany). six healthy donors and other target cell lines (Figure 4), dim neg Subsequently we performed the CD107a assay as described suggesting that the CD56 CD16 subset contains the above (CD56pos PBMC: K562 ratio ¼ 1:1). After the 180-min majority of cytotoxic NK cells. incubation period we positively selected CD107apos cells by using anti-CD107a PE conjugated mABs followed by anti-PE magnetic beads (Miltenyi Biotec). Cytotoxicity was assessed CD107a expression predicts NK cell activity as separately in the two cell fractions (CD56pos/ CD107apos and determined by tumor cell lysis CD56pos/ CD107aneg). To investigate whether CD107a surface expression is predictive for cytolytic activity, we performed a flow cytometric cytotoxi- Antibodies and flow cytometry city assay with the myeloid cell lines used for the CD107a assay. As shown in Figure 5, CD107a surface expression and specific Analysis of cell surface markers was performed using the lysis demonstrated a strong positive correlation (r2 ¼ 0.99 for following anti-human fluorescent conjugated mAbs (BD Phar- E/T ratios of 1:1 and 10:1, respectively).

Leukemia CD56dimCD16neg NK cells mediate cytotoxicity O Penack et al 837

Figure 2 CD107a surface expression in CD56pos cells (selected by microbeads) after coincubation with NK-sensitive target cell lines. The x-axis corresponds to the varying effector:target ratios. The y-axis corresponds to the percentage of CD107apos effector cells. One of three similar experiments is shown.

NK cell cytotoxicity after selection of CD56pos/ CD107apos cells

To demonstrate that CD107a expressing cells are responsible for NK-cell-mediated cytotoxicity, CD56pos/CD107apos cells were positively selected by immunomagnetic beads after coincuba- tion with K562 target cells. Only the purified CD56pos/ CD107apos cell fraction exhibited significant cytotoxic activity against leukemia targets, whereas CD56pos/CD107aneg NK cells were not able to lyse K562 cells (Figure 6).

Discussion

Since NK cell-mediated cytotoxicity is a major defense mechanism against tumor cells, methods evaluating NK- mediated target cell killing and NK cell activity are of great interest. Although the chromium (51Cr) release assay has several inherent problems, including hazards associated with radio- activity, cell labeling and high spontaneous release, it has become the standard method for assessing lymphoid, including NK cell, cytotoxicity.23 In recent years flow-cytometric assays, based on the release of fluorescent dyes from target cells or propidium iodide uptake, have been introduced as alternatives for the chromium release assay. They provide several advan- tages such as avoidance of radioactive reagents and detection of cytotoxicity at the single-cell level.24 However, neither the chromium release nor cytometric assays measure NK cell activation directly but rather examine the death of target cells.25 Thus far, methods directly assessing NK cell activation were based on the detection of cytokine production or secretion. Among these methods, ELISpot analysis and ICS (intracellular cytokine staining) are most commonly used.26 They do not allow conclusions regarding the cytotoxic potential of NK cells. Based on the recently described phenomenon that the degranulation of CTLs is followed by expression of the Figure 1 CD107a surface expression in peripheral blood NK cells lysosomal-associated membrane protein-1 (CD107a),6 we as- in response to myeloid leukemia cell lines. The lymphocytic sessed NK cell activity in response to different tumor targets by population was identified by its characteristics in the forward-side flow cytometric detection of CD107a. This new technique has scatter and the shown events are gated for CD56 positivity. NK cells numerous advantages over the existing standard methods. The degranulate in response to NK sensitive K562 target cells indicated by 5.6% CD107a positivity (a). They are not being activated in response main advantage of this simple assay is the opportunity to easily to ML2 cells (b) and in a control sample without target cells (c). identify cytotoxic NK cells on a single-cell level by flow cytometry.

Leukemia CD56dimCD16neg NK cells mediate cytotoxicity O Penack et al 838

Figure 3 CD107a surface expression in peripheral blood lymphocytes of a healthy donor after coincubation with K562 target cells (effector:target ratio 1:1). NK-cell subpopulations are defined by surface expression density of CD56 and CD16 (a). In total, 8.8% CD107a surface expression in the gated [R2] CD56dimCD16neg NK cells indicates degranulation (b). CD56bright NK cells [R3] and CD56dimCD16pos cells [R4] are not activated in response to the K562 cells (c and d).

cells. We showed that CD56pos/CD107pos cells that have been immunomagnetically selected after stimulation with K562 are highly cytotoxic whereas CD56pos/CD107aneg cells are not cytotoxic. The positive correlation between CD107a surface mobilization and the results of a standard flow cytometric cytotoxicity assay suggests that the CD107a assay can be used to assess the individual amount of cytotoxic NK cells at a given time point against a standardized tumor target. Moreover, it allows comparing the capability of different tumor cells to activate cytotoxic NK cells. Its performance is relatively easy and the time required is considerably shorter compared to other assays. Figure 4 Percentage of CD107apos cells in peripheral blood NK Another striking advantage of the novel method is the flow cell subsets after coincubation with myeloid cell lines. The NK cell cytometric acquisition, which not only allows gating out subsets were defined and gated as shown in Figure 3. Only disturbing T cells and monocytes but also permits additional CD56dimCD16neg cells express CD107a. The ML2 Line is generally believed to be NK resistant. Data of six normal donors were pooled. staining of other cell surface molecules, as for example Fc receptors, homing receptors or KIRs. Separate examination of different NK subpopulations will provide new insights into the mechanisms of NK cell activation and cytotoxicity. The finding that degranulation of CD8pos memory T cells As a first example for such an application, we have leads only to low level immediate cytotoxicity due to small investigated the level of CD107a surface mobilization in three amounts of stored cytotoxic effector molecules suggest that ‘classical’ NK cell subsets in response to myeloid leukemia cells. CD107a surface mobilization does not necessarily correlate It is well known that CD56-positive NK cells can be divided into with the cytolytic ability in CTLs.8 We found that degranulation a CD56dim and CD56bright population. While the CD56dim of NK cells in response to tumor targets lead to tumor cell lysis. population dominates in the peripheral blood and contains In this report, we have demonstrated that the CD107a assay mature NK cells with cytotoxic function, the CD56bright reliably predicts NK cell cytotoxicity against different target population is primarily located in the lymph nodes and exhibits

Leukemia CD56dimCD16neg NK cells mediate cytotoxicity O Penack et al 839

Figure 6 Specific lysis against K562 target cells in CD56pos/ CD107apos and CD56pos/CD107aneg PBMC. NK cell populations were selected by microbeads after coincubation with K562 cells. NK-cell- mediated cytotoxicity is restricted to the CD107apos cell fraction, as indicated by a high percentage of specific lysis in contrast to the CD107aneg cell fraction.

CD56dim/CD16neg cells in neoplastic tissues (ovarian and urothelial carcinoma) as well as in normal solid tissues (decidua, intestine) and suggested an involvement in surveillance of solid tissues. Immature NK cell precursors (eg in peripheral blood after allogeneic bone marrow transplantation and in umbilical cord blood) are predominantly CD56pos/CD16neg, during NK cell differentiation CD16 expression increases with impact on NK cell functionality.29–34 In all tests, no considerable expression of CD107a was detected on CD56dim/CD16pos cells, indicating that these cells do not degranulate in response to NK-sensitive leukemia targets and do not exhibit direct cytotoxicity. Since CD16 surface expression is crucial for antibody-dependent cellular cytotoxi- city, it is reasonable that the CD56dim/CD16pos cells are the major effector cells mediating this type of cytotoxic response.3 We are currently investigating whether CD107a expression is induced in the CD56dim/CD16pos cell fraction when the target Figure 5 Correlation of CD107a surface expression and cytotoxi- city. CD107a surface expression (%) in CD56pos PBMC of six normal cell is coated with a relevant antibody, which would extend the donors after coincubation with myeloid leukemia cell lines (a) in application of this assay to antibody-dependent cellular relation to specific lysis (%) determined by flow cytometric measure- cytotoxicity. ment of NK cytotoxicity using propidium iodide uptake to detect cell In summary, we have presented a novel flow cytometric death (b). The correlation of CD107a surface mobilization (E/T ratio of assay, which detects NK cell degranulation as a reliable 1:1) and specific lysis (E/T ratio of 10:1) demonstrates a close indicator of NK cell activation. Using this method we showed relationship (c). that NK-cell-mediated cytotoxicity against tumor targets is restricted to the CD56dim/CD16neg subpopulation. The assay is highly sensitive and its results are strictly correlated with NK cell 1–4 no significant cytotoxic effector function. Our results confirm cytotoxicity. The simple and rapid performance makes it a this concept and demonstrate that surface mobilization of suitable method not only for the scientific analysis of factors dim CD107a is exclusively observed in CD56 cells, indicating influencing NK cell activation on a single cell level but also for that the cytotoxic effector function is restricted to this NK cell broad clinical use to assess NK cell activity in immunocompro- population. mised patients. Functionally, the CD56dim compartment can be further divided into two subsets distinguished by the surface expression References of CD16. Our data suggest that only CD56dim/CD16neg cells are responsible for the natural cytotoxicity against leukemia targets. dim neg 1 Fehniger TA, Cooper MA, Nuovo GJ, Cella M, Facchetti F, In functional terms, the CD56 /CD16 phenotype is poorly Colonna M et al. CD56 bright natural killer cells are present in 27 described in the literature. Nagler et al found that peripheral human lymph nodes and are activated by T cell-derived IL-2: a blood CD56dim/CD16neg cells are preferentially responsive to potential new link between adaptive and innate immunity. Blood very low concentrations of IL-2. These findings were widened by 2003; 101: 3052–3057. Ferlazzo et al, who detected CD56dim/CD16neg cells also in 2 Ferlazzo G, Thomas D, Lin SL, Goodman K, Morandi B, Muller secondary lymphoid organs and showed in vitro that low levels WA et al. The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and of IL-2 render this cell type for target cell killing. They become cytolytic. J Immunol 2004; 172: 1455–1462. hypothesized that in vivo this effect could be initiated by IL-2 3 Cooper MA, Fehniger TA, Caligiuri MA. The biology of human secretion of T-cells.2 Mo¨ller et al28 detected high amounts of natural killer-cell subsets. Trends Immunol 2001; 22: 633–640.

Leukemia CD56dimCD16neg NK cells mediate cytotoxicity O Penack et al 840 4 Ferlazzo G, Munz C. NK cell compartments and their activation by 20 Dyer MJ, Fischer P, Nacheva E, Labastide W, Karpas A. A new dendritic cells. J Immunol 2004; 172: 1333–1339. human B-cell non-Hodgkin’s lymphoma cell line (Karpas 422) 5 Peters PJ, Borst J, Oorschot V, Fukuda M, Krahenbuhl O, Tschopp J exhibiting both t(14;18) and t(4;11) chromosomal translocations. et al. Cytotoxic T lymphocyte granules are secretory lysosomes, Blood 1990; 75: 709–714. containing both perforin and granzymes. J Exp Med 1991; 173: 21 Falk CS, Noessner E, Weiss EH, Schendel DJ. Retaliation 1099–1109. against tumor cells showing aberrant HLA expression using lymp- 6 Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, hokine activated killer-derived T cells. Cancer Res 2002; 62: Roederer M et al. Sensitive and viable identification of antigen- 480–487. specific CD8+ T cells by a flow cytometric assay for degranulation. 22 Mattis AE, Bernhardt G, Lipp M, Forster R. Analyzing cytotoxic T J Immunol Methods 2003; 281: 65–78. lymphocyte activity: a simple and reliable flow cytometry-based 7 Rubio V, Stuge TB, Singh N, Betts MR, Weber JS, Roederer M et al. assay. J Immunol Methods 1997; 204: 135–142. Ex vivo identification, isolation and analysis of tumor-cytolytic T 23 Brunner KT, Mauel J, Cerottini JC, Chapuis B. Quantitative assay of cells. Nat Med 2003; 9: 1377–1382. the lytic action of immune lymphoid cells on 51-Cr-labelled 8 Wolint P, Betts MR, Koup RA, Oxenius A. Immediate cytotoxicity allogeneic target cells in vitro; inhibition by isoantibody and by but not degranulation distinguishes effector and memory subsets of drugs. Immunology 1968; 14: 181–196. CD8+ T cells. J Exp Med 2004; 199: 925–936. 24 Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. 9 Bossi G, Griffiths GM. Degranulation plays an essential part in A novel technique for the fluorometric assessment of T lymphocyte regulating cell surface expression of Fas ligand in T cells and antigen specific lysis. J Immunol Methods 2001; 249: 99–110. natural killer cells. Nat Med 1999; 5: 90–96. 25 Godoy-Ramirez K, Franck K, Gaines H. A novel method for the 10 Kannan K, Stewart RM, Bounds W, Carlsson SR, Fukuda M, simultaneous assessment of natural killer cell conjugate formation Betzing KW et al. Lysosome-associated membrane proteins and cytotoxicity at the single-cell level by multi-parameter flow h-LAMP1 (CD107a) and h-LAMP2 (CD107b) are activation- cytometry. J Immunol Methods 2000; 239: 35–44. dependent cell surface glycoproteins in human peripheral blood 26 Letsch A, Scheibenbogen C. Quantification and characterization of mononuclear cells which mediate cell adhesion to vascular specific T-cells by antigen-specific cytokine production using endothelium. Cell Immunol 1996; 171: 10–19. ELISPOT assay or intracellular cytokine staining. Methods 2003; 11 Alter G, Malenfant JM, Delabre RM, Burgett NC, Yu XG, 31: 143–149. Lichterfeld M et al. Increased natural killer cell activity in viremic 27 Nagler A, Lanier L, Cwirla S, Phillips J. Comparative studies of HIV-1 infection. J Immunol 2004; 173: 5305–5311. human FcRIII-positive and negative natural killer cells. J Immunol 12 Martin P, Papayannopoulou T. HEL cells: a new human 1989; 143: 3183–3191. erythroleukemia cell line with spontaneous and induced globin 28 Mo¨ller M, Kammerer R, Von Kleist S. A distinct distribution of expression. Science 1982; 216: 1233–1235. natural killer cell subgroups in human tissues and blood. Int J 13 Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S Cancer 1998; 78: 533–538. et al. Characterization of the continuous, differentiating myeloid 29 Jacobs R, Stoll M, Stratmann G, Leo R, Link H, Schmidt RE. cell line (HL-60) from a patient with acute promyelocytic CD16ÀCD56+ natural killer cells after bone marrow transplanta- leukemia. Blood 1979; 54: 713–733. tion. Blood 1992; 79: 3239–3244. 14 Lozzio BB, Lozzio CB. Properties of the K562 cell line derived 30 Nagler A, Rabinowitz R, Rosengolts-Rat J, Condiotti R, Schle- from a patient with chronic myeloid leukemia. Int J Cancer 1977; singer M. Natural killer (NK) and T cell-associated surface 19: 136. marker expression following allogeneic and autologous bone 15 Asou H, Tashiro S, Hamamoto K, Otsuji A, Kita K, Kamada N. marrow transplantation (BMT). J Hematother Stem Cell Res 2000; Establishment of a human acute myeloid leukemia cell line 9: 63–75. (Kasumi-1) with 8;21 chromosome translocation. Blood 1991; 77: 31 Schubert J, Heiken H, Jacobs R, Delany P, Witte T, Schmidt RE. 2031–2036. A subset of CD16- natural killer cells without antibody-dependent 16 Ohyashiki K, Ohyashiki JH, Sandberg AA. Cytogenetic character- cellular cytotoxicity function. Nat Immun Cell Growth Regul ization of putative human myeloblastic leukemia cell lines (ML-1, 1990; 9: 103–111. -2, and -3): origin of the cells. Cancer Res 1986; 46: 3642–3647. 32 Tanaka H, Kai S, Yamaguchi M, Misawa M, Fujimori Y, Yamamoto 17 Saltman D, Bansal NS, Ross FM, Ross JA, Turner G, Guy K. M et al. Analysis of natural killer (NK) cell activity and adhesion Establishment of a karyotypically normal B-chronic lymphocytic molecules on NK cells from umbilical cord blood. Eur J Haematol leukemia cell line; evidence of leukemic origin by immunoglobu- 2003; 71: 29–38. lin gene rearrangement. Leuk Res 1990; 14: 381–387. 33 Vitale C, Ciossone L, Morreale G, Lanino E, Cottalasso F, Moretti S 18 Jadayel DM, Lukas J, Nacheva E, Bartkova J, Stranks G, De et al. Analysis of the activating receptors and cytolytic function of Schouwer PJ et al. Potential role for concurrent abnormalities of human natural killer cells undergoing in vivo differentiation after the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B allogeneic bone marrow transplantation. Eur J Immunol 2004; 34: cell non-Hodgkin’s lymphomas. Functional studies in a cell line 455–460. (Granta 519). Leukemia 1997; 11: 64–72. 34 Warren HS, Skipsey LJ. Phenotypic analysis of a resting sub- 19 Jeon HJ, Kim CW, Yoshino T, Akagi T. Establishment and population of human peripheral blood NK cells: the FcR gamma III characterization of a mantle cell lymphoma cell line. Br J (CD16) melecule and NK cell differentiation. Immunology 1991; Haematol 1998; 102: 1323–1326. 72: 150–157.

Leukemia