Leukemia (2002) 16, 736–737  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu KEYNOTE ADDRESS

Immunologic manipulation in AML: from bench to bedside MA Cooper and MA Caligiuri

Department of Internal Medicine and the Comprehensive Cancer Center, The College of Medicine and Public Health, The Ohio State University, Columbus, OH, USA

Leukemia (2002) 16, 736–737. DOI: 10.1038/sj/leu/2402411 subunits with the T cell-derived cytokine IL-2, including the IL-2/15R and common gamma chain (␥c), which together form an intermediate-affinity heterodimeric receptor complex The development of immunotherapeutic strategies for the (IL-2/15R␤␥). However, IL-15 and IL-2 also each utilize priv- treatment of leukemia and other cancers has long been of ate alpha chains (IL-15R␣ and IL-2R␣) that can confer high interest. Natural killer (NK) cells represent one component of affinity binding to their respective heterotrimeric receptors, IL- the innate immune system and have the ability to both lyse 15R␣␤␥ and IL-2R␣␤␥. Although IL-2 is produced primarily target cells and produce immunoregulatory cytokines. Thus, in the periphery and is not required for NK cell development, these large granular lymphocytes have been the subject of as the result of their shared receptor usage, IL-2 can elicit intense investigation for their antitumor effects. Human NK many of the same effects as IL-15 when administered in vivo. cells comprise approximately 10–15% of all peripheral blood In addition, ligands for the receptor tyrosine kinases flt3 and lymphocytes and are defined by the presence of the CD56 c-kit can increase the NK cell precursor frequency of HPCs cell surface antigen and the lack of CD3.1 Two subsets of NK through up-regulation of the IL-15R complex in vitro,6,7 and cells can be distinguished based on their cell surface density in vivo.8 Clinical trials have demonstrated that treatment of of CD56. The majority (ෂ90%) of human NK cells are CD56dim leukemia patients with low-dose IL-2 can safely drive NK cell and express high levels of Fc RIII (CD16), which mediates development and expansion.9 However, administration of antibody- dependent cellular cytotoxicity, while a minority additional NK differentiation factors such as flt3 ligand or c- (ෂ10%) of NK cells are CD56bright CD16dim/neg.2 NK cells have kit ligand in combination with IL-2 has the potential to the unique ability to lyse tumor target cells without prior sensi- enhance NK cell differentiation and expansion in the setting tization, in contrast to T lymphocytes, which depend on acti- of malignancy. vation and presentation of specific antigens associated with Two distinct populations of human NK cells can be ident- HLA molecules to elicit a cytotoxic effect. Because of this fact, ified in the peripheral blood of all healthy donors based upon there has been a great deal of interest in understanding the cell surface density of CD56.10 CD56bright and CD56dim NK mechanisms whereby NK cells mediate their cytotolytic cells differ in many respects including their proliferative effects. It is now clear that NK receptors (NKRs) for MHC class response to IL-2, intrinsic cytotoxic capacity, cytokine pro- I molecules are critical for the recognition of normal vs trans- duction, NKR repertoire, and adhesion molecule expression formed and/or foreign cells. In the setting of therapy for acute (reviewed in Cooper et al2). An important consideration for myeloid leukemia (AML) and other hematologic malignancies, the therapeutic expansion of NK cells in vivo is the fact that there is now good evidence to suggest that mismatch of donor the CD56bright NK cell subset constitutively expresses both NKRs and recipient class I molecules in haplotype- high- and intermediate-affinity IL-2 receptors and efficiently mismatched BMT may have a beneficial effect when that expands in vitro and in vivo in response to low-dose (pM) IL- mismatch is in the donor vs leukemia direction.3 Thus, immu- 2.11 In contrast, resting CD56dim NK cells express only the notherapeutic strategies targeting NK cells have the potential intermediate affinity IL-2/15 receptor and proliferate weakly, to be beneficial for the treatment of leukemia. even in response to high doses of IL-2. Functional studies have It has been known for decades that NK cells develop within demonstrated that the more abundant CD56dim NK cell subset the bone marrow (BM) microenvironment and numerous stud- is highly cytotoxic when compared with the CD56bright ies have definitively identified the cytokine interleukin (IL)-15 subset.2 as the critical factor necessary for the development of human In addition to their cytotoxic effects, NK cells also constitut- and murine NK cells.4,5 IL-15 is produced by a variety of cell ively express receptors for monocyte-derived cytokines types, including normal BM stroma and antigen-presenting (monokines) and produce immunoregulatory cytokines such cells, and can induce NK cell differentation from human hem- as IFN-␥, in response to monokine stimulation. NK cell- atopoietic progenitor cells (HPCs) in the absence of other derived IFN-␥ plays a critical role in the immune response to cytokines in vitro.6 IL-15 shares common signaling receptor infection and possibly in NK cell tumor immunosurveillance. We have recently reported that CD56bright NK cells have the capacity to produce abundant cytokines, including IFN-␥, fol- dim Correspondence: MA Caligiuri, Division of Hematology/Oncology lowing monokine stimulation compared to the CD56 NK and The Comprehensive Cancer Center, The Ohio State University, subset, which produces very low levels of cytokines.12 Collec- A458 Starling Loving Hall, 320 W 10th Avenue, Columbus, Ohio tively, the data support a model whereby CD56bright and 43210, USA; Fax: 617 293 7522 CD56dim NK cells represent functionally distinct subsets of This paper is part of a series of keynote addresses to be published in human NK cells, although very little is known about the devel- Leukemia. They were presented at the Acute Leukemia Forum, San Francisco, 20 April 2001. Supported by an unrestricted educational opmental relationship between these subsets. Therefore, grant from Immunex. knowledge of the distinct functional attributes of human NK Received 2 December 2001; accepted 17 December 2001 cell subsets and the factors involved in their differentiation Immunologic manipulation in AML: from bench to bedside MA Cooper et al 737 and expansion may enable us to design strategies that prefer- absent or expressed at low density in normal tissues, but are entially activate that subset with the greatest therapeutic up-regulated in response to stress, infection, and malignant potential for a particular cancer. transformation.15 While MICA/B were originally thought to be The mechanisms whereby an NK cell recognizes a target restricted to epithelial cells, they have recently been identified cell with subsequent activation or inhibition of killing are on multiple types of tumor cell lines including leukemia, and complex and are just now being elucidated.13 NKRs recogniz- lymphoma lines that were specifically lysed by NK cell clones ing self-MHC class I molecules have been identified in both expressing NKG2D.16 Thus, therapeutic strategies aimed at mouse and man and appear to be critical for the inhibition of up-regulation of activating ligands and/or NKG2D could NK cell lysis of normal ‘self’ cells. NKRs exist in both activat- further potentiate the antileukemic effects of NK cells. ing and inhibitory forms. Inhibitory NKRs are specific for In summary, advances in our understanding of basic human classical (eg HLA-A, B or C) or nonclassical (eg HLA-E or G) NK cell biology, including NK cell differentiation and the class I while activating NKRs also recognize ‘class I-like’ mol- mechanisms of NK cell target recognition have led to the ecules. Ordinarily, when an NK cell encounters a normal host initial design of rational therapeutic strategies for the treatment cell, class I molecules expressed on the surface of that ‘self’ of leukemia. Further investigation of the biology of these cell are engaged by the NK cell’s inhibitory receptors and a potent immune effectors will likely result in additional novel strong inhibitory signal is transduced via an immunoreceptor immune-based strategies for the treatment of cancer. tyrosine-based inhibitor motif in the cytoplasmic domain of the NKR. This signal acts to prevent NK cell activation and References consequent cytolysis. However, when an NK cell’s inhibitory receptors fail to recognize self class I, such as on a foreign or 1 Robertson MJ, Ritz J. Biology and clinical relevance of human transformed cell, the inhibitory signal is absent and NK cell natural killer cells. Blood 1990; 76: 2421–2438. activation can proceed. In contrast, when activating NKRs are 2 Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001; 22: 633–640. engaged by their ligands, NK cell cytolytic and cytokine-pro- 3 Ruggeri L, Capanni M, Casucci M et al. Role of natural killer cell ducing programs are activated to allow attack of target cells. alloreactivity in HLA-mismatched hematopoietic stem cell trans- These receptors do this via an association with membrane- plantation. Blood 1999; 94: 333–339. anchored, activation-inducing adapters such as DAP12 or 4 Williams NS, Klem J, Puzanov IJ, Sivakumar PV, Schatzle JD, DAP10 that activate immunoreceptor tyrosine-based acti- Bennett M, Kumar V. Natural killer cell differentiation: insights 13 from knockout and transgenic mouse models and in vitro systems. vation motif or PI3-kinase pathways respectively. Activation Immunol Rev 1998; 165: 47–61. of NK cytotoxicity is likely mediated by a balance of inhibitor 5 Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance and activating NKR signals as well as various other adhesion to human disease. Blood 2001; 97: 14–32. and/or costimulatory molecules. Three major superfamilies of 6 Mrozek E, Anderson P, Caligiuri MA. Role of interleukin-15 in the NKRs have been described in humans: the killer cell Ig-like development of human CD56+ natural killer cells from CD34+ superfamily, which primarily recognize HLA A, B and C; the hematopoietic progenitor cells. Blood 1996; 87: 2632–2640. 7 Yu H, Fehniger TA, Fuchshuber P, Thiel KS, Vivier E, Carson WE, C-type lectin superfamily, which includes CD94 and NKG2 Caligiuri MA. Flt3 ligand promotes the generation of a distinct receptors recognizing nonclassical HLA-E molecules; and a CD34(+) human natural killer cell progenitor that responds to recently described class of natural cytotoxicity receptors with interleukin-15. Blood 1998; 92: 3647–3657. unknown ligands.14 8 Fehniger TA, Carson WE, Mrozek E, Caligiuri MA. Stem cell factor Regulation of NK cell cytotoxicity by NKRs is relevant in the enhances interleukin-2-mediated expansion of murine natural setting of haplotype-mismatched bone marrow transplantation killer cells in vivo. Blood 1997; 90: 3647–3653. 9 Baer MR, Pixley LA, Schriber JR, Frankel SR, Fehniger TA, Herzig (BMT), where the possibility exists that inhibitory NKRs on GP, Caligiuri MA. Prolonged administration of low-dose interleu- donor-derived NK cells will fail to recognize recipient leu- kin-2 (IL-2) to patients (PTS) with acute myeloid leukemia (AML) in kemic blasts and exert a graft-versus-host effect or vice versa. remission (CR) expands natural killer (NK) cells without significant Elegant work in support of this theory comes from a retrospec- clinical toxicity. Blood 1998; 92 (Suppl. 1): 2534 (Abstr). tive analysis by the Velardi laboratory at the University of Per- 10 Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relation- 3 ship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on ugia. In this study, the investigators examined the role of human peripheral blood NK cells and cytotoxic T lymphocytes. J inhibitory NKRs in mediating a donor NK vs leukemia effect. Immunol 1986; 136: 4480–4486. Donor–recipient pairs where at least one of the haploidentical 11 Caligiuri MA, Zmuidzinas A, Manley TJ, Levine H, Smith KA, Ritz donor’s inhibitory receptors would fail to recognize a class I J. Functional consequences of interleukin 2 receptor expression ligand on the patient’s leukemic blasts had a higher rate of on resting human lymphocytes. Identification of a novel natural engraftment and lower incidence of leukemic relapse, com- killer cell subset with high affinity receptors. J Exp Med 1990; 171: 1509–1526. pared to those without the potential for alloreactive NK cells. 12 Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, This NK vs leukemia effect was borne out in laboratory analy- Ghayur T, Carson WE, Caligiuri MA. Human natural killer cells: a ses showing that donor NK clones were able to lyse patient unique innate immunoregulatory role for the CD56(bright) subset. leukemic blasts. There was no graft-versus-host disease Blood 2001; 97: 3146–3151. observed in these patients, suggesting NK cells may, in the 13 Lanier LL. On guard – activating NK cell receptors. Nat Immunol setting of allogeneic transplantation, mediate a selective NK 2001; 2: 23–27. 3 14 Moretta A, Bottino C, Vitale M et al. Activating receptors and core- vs leukemia reaction. ceptors involved in human natural killer cell-mediated cytolysis. In addition to inhibitory receptors, NK cell activating recep- Annu Rev Immunol 2001; 19: 197–223. tors are thought to play a major role in target cell recognition. 15 Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced One nonclassical activating NKR is the homodimeric C-type MHC molecules by intestinal epithelial gammadelta T cells. lectin receptor, NKG2D, whose ligands are not MHC class I Science 1998; 279: 1737–1740. 16 Pende D, Cantoni C, Rivera P et al. Role of NKG2D in tumor molecules but MHC ‘class I-like’. In humans, these ligands cell lysis mediated by human NK cells: cooperation with natural include MICA and MICB and the RAE-1-like molecules (also cytotoxicity receptors and capability of recognizing tumors of non- known as UL-16 binding proteins, ULBPs).13 These ligands are epithelial origin. Eur J Immunol 2001; 31: 1076–1086.

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