Natural Killer Cells in GvHD and GvL

Chapter 16

Natural Killer Cells in GvHD and GvL Upasana Sunil Arvindam1, Ethan George Aguilar2, Martin Felices1, William Murphy2 and Jeffrey Miller1 1

Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, United States; 2Department of Dermatology,

UC Davis School of Medicine, Sacramento, CA, United States

Chapter Outline Introduction Natural Killer Cell Receptors Natural Killer Cell Signaling Natural Killer Cell Education and Tolerance Natural Killer Cell Memory Role of Natural Killer Cells in Cancer Therapeutics Human Versus Mouse Natural Killer Cells

275 276 279 279 280 281 281

Natural Killer Cells in Humans Adoptive Natural Killer Cell Therapy Expanding Natural Killer Cells Enhancing Natural Killer Cell Function to Eliminate Tumors The Role of Natural Killer Cells in Viral Therapy Concluding Remarks References

282 284 285 285 287 287 288

INTRODUCTION Natural killer (NK) cells were first described for their capacity to reject bone marrow (BM) allografts in lethally irradiated mice without prior sensitization [1]. This rejection ability did not follow the classical laws of transplantation in that parental BM allografts were resisted by F1 hybrid recipients in a phenomenon termed “hybrid resistance” [2] (Fig. 16.1). The radioresistant host effector cells were capable of mediating resistance to BM but not solid tissue allografts suggesting a new cell type existed. NK cell activity was first detected in 1975 in human peripheral blood mononuclear cells by the detection of non-MHCerestricted cytotoxicity toward transformed and virally infected target cells. It was these early experiments that led Karre and colleagues to formulate “the missing self hypothesis” in which NK cell cytotoxicity is triggered by the loss of MHC class I on tumor cells [3]. Following this observation, different families of receptors were identified on NK cells that recognize MHC class I and mediate tolerance in the host. Because of their ability to lyse tumors with aberrant MHC class I expression and produce cytokines and chemokines on activation, NK cells have immense therapeutic potential to treat cancer. In humans, NK cells are identified by the expression of the cell adhesion marker CD56 and lack of the T cell receptor CD3. They are present in many tissues, including the spleen, BM, lymph nodes, and peripheral blood. They are derived from CD34þ progenitor cells in the BM and their maturation is dependent on cytokines and stimuli that occur at distinct stages in the BM, lymphoid tissue and peripheral blood. IL-15 is essential for NK cell development and homeostasis as IL-15 knockout mice lack NK cells. Furthermore, IL-15 activity is enhanced when trans-presented by other cells such as dendritic cells (DCs) [4]. NK cells circulating in the blood can be divided into two functionally distinct groups based on cell surface density of CD56: CD56bright and CD56dim NK cells. Comprising about 10% of circulating NK cells, CD56bright NK cells are more proliferative, have a higher capacity for cytokine production after stimulation with IL-12 and IL-18, and are poor mediators of NK cell cytotoxicity. CD56dim NK cells are more cytotoxic, mediate antibody-dependent cellular cytotoxicity (ADCC) through the Fc receptor CD16 (FCgRIII) and produce cytokines after stimulation with target cells. NK cells produce a wide variety of cytokines and chemokines, including but not limited to, interferon gamma (IFNg), granulocyte colony stimulating factor (G-CSF), tumor necrosis factor alpha (TNFa), transforming growth factor beta, macrophage inflammatory protein 1-beta (MIP-1b), and RANTES. These can modulate the adaptive immune system, recruit and stimulate other immune cells, and stimulate or inhibit hematopoiesis. NK cells also enhance the immune

Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation. https://doi.org/10.1016/B978-0-12-812630-1.00016-5 Copyright © 2019 Elsevier Inc. All rights reserved.

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Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

(A)

(B)

Parental B6 (H2b)

Parental BALB/c (H2d)

Ly49C/I

NK

NK +++

+++

BALB/c BMC

H2d

B6 BMC

Ly49G2/A

H2b -

-

NK

NK

Allogeneic donor BMC transfer

Ly49G2/A

Host NK

Allogeneic donor BMC transfer

Ly49C/I

Host NK

(B6xBALB/c) F1 Hybrid

(C)

Ly49C/I NK -

Parental donor BMC transfer

B6 BMC

+++

H2d

H2b +++

-

BALB/c BMC

Parental donor BMC transfer

NK Ly49G2/A

Host NK FIGURE 16.1 Role of NK cells in parental bone marrow hybrid resistance of B6BALB/c F1 mice. The lack of H-2 recognition by NK cells that express inhibitory receptors for self-MHC class I molecules is involved in allogeneic BM rejection. In parental B6 (A) or BALB/c (B) mice, allogeneic BM rejection is mediated by Ly49C/Iþ and Ly49G2þ NK cells, respectively. In F1 (B6BALB/c) mice (C), NK cells expressing inhibitory receptors to one of the parental MHC are capable of rejecting the BMC of the other parental haplotype. BM, bone marrow; NK, natural killer.

response by the elimination of virally infected or tumor cells through direct killing, mediated by the release of perforin and granzyme, ADCC-mediated killing of antibody-coated cells through CD16, or the induction of apoptosis through Fas ligand (FasL).

NATURAL KILLER CELL RECEPTORS NK cell function is controlled by a balance of signals between activating and inhibitory receptors (Table 16.1). The bestcharacterized family of human NK cell receptors is the inhibitory killer-immunoglobulinelike receptors (KIRs). KIRs are type I transmembrane molecules belonging to the immunoglobulin (Ig) superfamily [5]. KIR mRNA transcripts were discovered through subtractive hybridization in 1995 and the 15 genes encoding KIRs are found on chromosome 19p13.4 in the leukocyte receptor complex (LRC) [6]. They are classified by the number of immunoglobulin-like extracellular domains (two domains [2D] or three domains [3D]) and the length of their cytoplasmic tail (long [L] or short [S]). Inhibitory KIR possess long cytoplasmic tails with immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which get phosphorylated and recruit tyrosine phosphatases, SHP-1 or SHP-2, which in turn dephosphorylate protein substrates associated with activating receptors [7, 7a]. KIRs that have short cytoplasmic tails associate with DAP-12 that contains an immunoreceptor tyrosinebased activation motif (ITAM) leading to cell activation. KIR gene content differs between populations, but can be divided into two broad haplotypes, KIR A and KIR B haplotypes. KIR A haplotypes contain only one activating receptor, KIR2DS4,

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TABLE 16.1 Human Receptor

Mouse Ligand

Inhibitory Receptors

Receptor

Ligand

Inhibitory Receptors

KIR2DL1

HLA-C group 2

Ly49 A-C, E-G, I-O

H-2 class I

KIR2DL2

HLA-C group 1

2B4

CD48

KIR2DL3

HLA-C group 1

CD94/NKG2A

Qa-1b

KIR2DL4

HLA-G

NKG2I

unknown

KIR2DL5

unknown

NKR-P1B

m12, Clr-b (Ocil)

KIR3DL1

HLA-Bw4

NKR-P1D

Clr-b (Ocil)

KIR3DL2

HLA-A

KLRG1

Cadherins

KIR3DL3

unknown

LAIR-1

Collagen

NKG2A

HLA-E

SICLEC-E

Sialic acid

LIR-1

HLA class I

PILR-a, b

CD99

Activating Receptors

Activating Receptors

KIR2DS1

HLA-C group 2

Ly49D

H-2Dd

KIR2DS2

unknown

Ly49P

MCMV

KIR2DS3

unknown

Ly49H

m157 MCMV

KIR2DS4

HLA-A*11

NKR-P1A

m12 MCMV

KIR2DS5

unknown

NKR-P1C (NK1.1)

m12 MCMV

KIR3DS1

HLA-F

NKR-P1F

Clr-g

NKG2C

HLA-E

CD94/NKG2C, E

Qa-1b

NKG2D

MICA, MICB, ULBP1-4

NKG2D

Rae-1s, H60, MULT-1

CD16

IgG 1, 3, 4

CD16

IgG

NKp30

B7-H6, BAT3, CMV pp65

NKp46

Viral HA

NKp44

Viral HA

DNAM-1

CD112, CD155

NKp46

Viral HA

2B4

CD48

NKp80

AICL

CRACC

CRACC

CD160

HLA-C

LFA-1, CD11a

ICAM-1, 2, 3

DNAM-1

CD112, CD155

CD96

CD155, CD111

2B4

CD48

CRACC

CRACC

NTB-A

NTB-A

whereas KIR B haplotypes are far more varied consisting of two or more activating receptors [8]. KIR genes are highly polymorphic, with several alleles described for each KIR gene. Inhibitory KIR recognize allelic epitopes present on human leukocyte antigens (HLA) class I molecules, HLA-A, HLA-B, and HLA-C allotypes [9]. In mice, Ly49 receptors are type II membrane-anchored glycoproteins that bind different MHC class I H-2 alleles. Although structurally different, mouse Ly49s functionally resemble human KIRs in that both are involved in the acquisition of tolerance to cells expressing self-MHC class I alleles. The structural organization of Ly49s is divided into three regions, a ligand-binding extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extensive

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allelic polymorphism observed in the Ly49 genes can be explained by the different regions being encoded in unique exons of the NK gene complex region in chromosome 6 that allows for multiple possible rearrangements, provides high receptor diversification, and accounts for the many Ly49 receptors that have been identified [10,11]. Currently, 23 different Ly49 transcripts have been identified; among them, 13 Ly49 genes are considered inhibitory receptors based on the presence of ITIMs in their cytoplasmic domains (Ly49A, B, C, E, F, G, I, J, O, Q, S, T, and V) and 8 activating because of the lack of ITIMs (Ly49D, H, L, M, P, R, U and W) [11]. Several studies have suggested that Ly49 receptors can interact with multiple H-2 alleles, although each may show a stronger affinity for a particular H-2 allele [12]. For example, Ly49D interacts with H-2Dd and this has been demonstrated through cytotoxicity assays in vitro and through its implication on H2Dd BM allografts rejection in vivo [13]. In H-2Dd strains, the coexpression of self-specific inhibitory receptors was sufficient to allow self-tolerance of Ly49Dþ NK cells. The frequency and amount of Ly49 expression in an NK cell seems to be partially influenced by H-2 expression in the host. However, because of the distinctive mechanism of gene regulation that results in randomized Ly49 expression, NK cells from a single host can express inhibitory receptors that bind different H-2 alleles and, therefore, there are NK cells with inhibitory receptors able to recognize self-MHC and NK cells with inhibitory receptors that do not. To explain how NK activation with such a differential expression of Ly49s is regulated, a process known as NK licensing [14], education [15], or tuning [16] has been proposed. This will be discussed later. Both humans and mice encode a family of C-type lectin-like receptors, known as the CD94/NKG2 receptor family. They are disulfide-linked heterodimers that are composed of an invariant common subunit, CD94. This subunit is linked to a glycoprotein encoded by a gene of the NKG2 family [17]. The NKG2 family consists of four genes: NKG2A/B, NKG2C/D, NKG2E, and NKG2F (only present in humans). CD94/NKG2A is an inhibitory receptor that binds to HLA-E in humans and Q1-ab in mice. As the expression of HLA-E is promoted by binding of peptides clipped from leader-sequence of classical HLA class I molecules, it is thought that HLA-E expression acts as a barometer of classical class I expression. The purpose of CD94/NKG2A may, therefore, be to monitor class I expression in a quantitative way whereas KIR receptors monitor each allele individually. CD94/NKG2B appears to be a splice variant of NKG2A and binds HLA-E. CD94/NKG2C and NKG2D are activating receptors. CD94/NKG2C also recognizes HLA-E, but like Ly49D affinity for H2d, this recognition is weaker than for the inhibitory CD94/NKG2A receptor [18]. NKG2D is a homodimer and does not associate with CD94 like the other members of this family. NKG2D recognizes the nonclassical MHC molecules: MICA and MICB and other non-MHC molecules ULBP1, ULBP2, and ULBP3 that are upregulated by the cell in times of stress, such as viral infection and tumorigenic transformation. In the mouse NKG2D ligands are retinoic acid early inducible-1 (Rae-1), minor histocompatibility antigen H60 and the murine UL16-binding protein-like transcript-1 [12]. Another family of immunoglobulin receptors, the leukocyte immunoglobulin-like receptors (LILR), is also expressed by NK cells, as well as other immune cells [19], and the genes encoding these receptors are in the LRC. The LILR deliver an inhibitory signal to NK cells. LILRB1 (LIR-1) expressed on NK cells binds to an array of classical and nonclassical HLA class I molecules, including HLA-F and HLA-G. It has also been shown to bind to the cytomegalovirus (CMV) encoded glycoprotein UL-18 [20]. The natural cytotoxicity receptors (NCRs) are a family of immunoglobulin-activating receptors and include the receptors NKp30 and NKp44 in humans and NKp46 in both human and mouse [21]. They have been shown to mediate NK cell cytotoxicity involved in lysing of tumor cells. NKp30 and NKp46 are constitutively expressed on both resting and activated NK cells, whereas NKp44 is only expressed on activated NK cells. Their ligands are varied. NKp30 recognizes the tumor ligand B7-H6, the HLA-B-associated transcript 3 (BAT3) (a nuclear protein released on heat shock treatment), and the CMV protein pp65. NKp80 binds to AICL, which is expressed on myeloid cells and may be involved in triggering NK cell lysis of AML [21]. Influenza virus hemagglutinin (HA) has been identified as a ligand for NKp46, but tumor ligands for this receptor remain unknown [22,23]. Mouse and human NK cells also express other activating receptors. CD16, a member of the Fc family of receptors, binds to the Fc portion of IgG antibodies. On engagement CD16 mediates ADCC to lyse antibody-coated targets. NK cells also express a variety of activating receptors that recognize a wide range of diverse ligands [24]. DNAM-1 and CD96, also known as Tactile, have been shown to bind to CD112 and CD155, both poliovirus receptors that mediate cell adhesion and increase NK cellemediated killing. More recently, these receptors have been shown to oppose each other during NK cell antitumor responses, with CD96 outcompeting DNAM-1 for CD155 binding [25]. The glycoprotein CD160 interacts with HLA-C triggering NK cell cytotoxicity. CD2 serves as a costimulatory molecule through binding of LFA-3. Finally, SLAM family heterophilic receptor 2B4 (CD244) yields activation through binding to CD48, whereas NTB-A and CRACC are homophilic and can also promote NK cell activation [26].

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NATURAL KILLER CELL SIGNALING When an NK cell encounters a potential target, it must first adhere to the target and form an immune synapse [27]. In the absence of this adherence, NK cells are unable to lyse target cells. NK cell receptors such as LFA-1, CD2, CD16, and 2B4 enhance NK cell adherence. LFA-1 binds to ICAM-1 on the target cell and initiates signal transduction. After adhesion to the target cell, activating receptors can now bind to their ligands. The ITAM-bearing activating receptors on NK cells use transmembrane adaptors with intracellular signaling domains to begin the activation cascade. CD16, NKp30, and NKp46 can couple with FcεRIg, CD3z, or both, whereas NKp44 uses DAP-12. The activating KIR, activating Ly49, NKG2C, and NKG2E all couple with DAP-12. On ligation, protein tyrosine kinases of the Src family phosphorylate ITAM-bearing subunits. This leads to recruitment of ZAP-70 and/or Syk initiating further downstream signaling cascades ultimately leading to polarization of perforin and granzyme containing granules and release of these cytotoxic granules to lyse the target cell. The ITAM-dependent NKG2D receptor couples with DAP-10 in humans, whereas in mice it can couple to both DAP-12 and DAP-10. On ligation, DAP-10 signaling recruits either p85 or Grb2, leading to the phosphorylation of PI3K. The SLAM families of receptors, which include 2B4, contain a signaling motif called an immunoreceptor tyrosine-based switch motif (ITSM). On ligation ITSM is recognized by three cytoplasmic SH2 domains containing adaptor molecules: SAP, EAT, and ERT. Mutated SAP results in X-linked lymphoproliferative disease. 2B4 signaling recruitment of SAP is thought to activate the NK cells, whereas recruitment of EAT or ERT is thought to inhibit NK cell activation [28]. As NK cells express a wide range of activating receptors that interact with different adaptor molecules and signal through different pathways, defects in one signaling pathway do not ultimately lead to dysfunction. Mice lacking the tyrosine kinases ZAP-70 and Syk can still lyse tumor targets by presumably upregulating ITAM-independent signaling pathways [28]. It is also apparent that separate pathways are involved in cytotoxicity and cytokine signaling. Colucci and colleagues [29] found that Vav1/ mice develop NK cells normally, but these NK cells are only capable of target celleinduced IFNg production and are unable to lyse target cells. Vav1 was shown to control ERK activation and exocytosis of cytotoxic granules. CD45/ mice could lyse targets but were unable to produce IFNg. CD45 was required for the full activation of Syk, Vav1, and phosphorylation of JNK and p38 after ITAM activation. However, CD45 has no effect on ITAM-mediated P13K activity required for signaling of cytotoxicity [30]. As most activating receptors can signal for both cytotoxicity and cytokine production, it is unclear if activating receptors trigger both pathways simultaneously or if cytotoxicity and cytokine production are triggered at different signally thresholds.

NATURAL KILLER CELL EDUCATION AND TOLERANCE NK cells can express inhibitory receptors for both self- and nonself-MHC class I molecules. To explain how NK cells acquire tolerance to self, several groups proposed what is known as NK licensing [14], NK arming [31], NK tuning [16], or NK education [15]. NK cells that express inhibitory receptors for self-MHC gain functional properties and become educated. Vivier and colleagues [32] have shown that activating receptors are confined to nanodomains in the plasma membrane of educated NK cells but remain in the actin meshwork of uneducated cells. This process is not permanent because mature, uneducated NK cells placed in an environment with self-ligands become educated, whereas educated NK cells placed in an environment devoid of self-ligands lose education [33,34]. A ligand-instructed model of KIR acquisition has been proposed, where initial education of an NK cell by a self-ligand through a KIR can influence subsequent acquisition of other KIR specific for the same HLA ligand [35]. Recently, Parham and colleagues showed that HLA class I educates NK cells through interactions with KIRs and by providing peptides that bind HLA-E to form ligands for CD94/NKG2A receptors. They identified that dimorphism in the leader peptide of HLA-B modulates this latter function: 21methionine (21M) delivers functional peptides, whereas 21threonine (21T) does not. Therefore, there are two forms of HLA haplotype: one preferentially supplying CD94/NKG2A ligands and the other preferentially supplying KIR ligands. This HLA-B dimorphism divides the human population into three groups: M/M, M/T, and T/T. The M/M and M/T individuals have NK cells that are better educated than the T/T individuals [36]. NK cells that lack inhibitory KIRs and NKG2A receptors for self-MHC remain relatively hyporesponsive. Uneducated NK cells, although hyporesponsive in resting stages, can become functional on activation [14]. These activated cells have an important role during mouse CMV infection [37,38]. It has been shown that educated cells are more active and show augmented NK cell function. In a mouse model of CMV infection, educated NK cells provided the primary antiviral defense after hematopoietic stem cell transplantation (HSCT) [39]. In humans, NK cell education makes cells alloreactive against tumor targets missing self-HLA ligand. Uneducated or hyporesponsive NK cells do not seem to contribute to the clinical benefits of alloreactive NK cells [40]. Our understanding of the rules governing the maintenance of self-tolerance and breaking of tolerance is incomplete. After

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HSCT, the education and function of engrafting alloreactive cells is affected by interactions with class I HLA and transplanted NK cells may be educated by hematopoietic cells transferred by the graft itself. One study has demonstrated that while infusion of a low marrow cell dose is not adequate to prevent host elimination of donor NK cells, a high marrow cell dose can induce recipient tolerance to the donor NK cells. The high exposure to donor MHC results in reprogramming of NK cell tolerance from the recipient to the donor [40]. Additional studies have demonstrated that donor instruction by donor MHC can direct the recipient NK cells to tolerate the allogeneic stem cells [41]. After HSCT, donor NK cell tolerance to recipient leukemic cells can be broken by increased expression of activating ligands to NKG2D, DNAM-1, and NCRs on the leukemic cells [42,43]. Alternatively, loss of inhibitory signaling in the setting of donor KIR recipient KIR ligand (HLA) mismatch can also break tolerance. Multiple mechanisms regulate the activating potential of NK cells. Owing to the stochastic expression of Ly49s, one NK cell can display one or multiple receptors. The rheostat tuning model postulates that the presence of multiple inhibitory receptors within one NK cell allows for a stronger regulation of activation and those cells exhibit higher cytotoxic function when engagement of those inhibitory receptors does not occur [44,45]. It is likely that KIR and Ly49 receptors work in concert with NKG2A, 2B4, and other inhibitory receptors in regulating NK cell subset function and is in fact the total NK receptor expression of a subset that may generate a net positive (through binding activating receptors such as Ly49D or H, KIRDS, as well as NCRs) or negative effect (through binding inhibitory Ly49 or KIRDL receptors and NKG2A) on overall function after binding the appropriate ligands.

NATURAL KILLER CELL MEMORY The capacity for immune memory has long been considered a property of T and B cells; however, there is growing evidence that NK cells demonstrate memory responses. NK cells have been shown to have hapten-specific memory in mice [46]. Mice that lacked both T and B cells still mounted a strong contact hypersensitivity response and depletion of NK cells abolished the response, suggesting that NK cells are capable of adaptive immune responses. Furthermore, hapten-specific memory was transferrable. Combination of IL-12, IL-18, and low-dose IL-15 has been shown to induce a population of cytokine-induced memory-like (CIML) NK cells in mice and humans [47]. CIML NK cells were transferred into naïve hosts and on restimulation produced significantly more IFNg than naïve NK cells and showed more cytotoxicity and proliferation. Combined cytokine activation of both CD56bright and CD56dim NK cells with IL-15 plus IL-18 or IL-12 plus IL-18 resulted in robust CD25 expression and increased STAT5 signaling [48]. Romee and colleagues demonstrated that these cells show enhanced cytotoxicity against leukemia cell lines and primary human AML blasts in vitro regardless of KIReKIR-ligand interactions. Human CIML NK cells xenografted into mice reduced AML burden in vivo and improved overall survival. These data led to a phase 1 human clinical trial where adoptively transferred CIML NK cells expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions [49]. Sun and colleagues [50] were the first to demonstrate NK cellemediated viral immune memory. During cytomegalovirus (CMV) infection in mice, NK cells expressing the activating receptor Ly49H preferentially expand and persist in high numbers after CMV infection and this response is driven through interaction of Ly49H with the viral protein m157. These NK cells could mount a robust response to subsequent CMV infections after adoptive transfer and had higher IFNg transcripts than naïve Ly49HþNK cells. Since then, these observations have been extended in several other murine viral models [51,52] as well as a rhesus macaque model of SIV immunization [53]. In addition, these memory NK cells could be isolated from mice several months after the initial infection and were still capable of mounting robust responses. In humans, there is a population of long-lived CD56dim CD57 þ NKG2Cþ NK cells that expand after acute human cytomegalovirus (HCMV) reactivation in transplant recipients [54,55] and coculture with CMV-infected fibroblasts [56]. These cells are thought to be analogous to mouse Ly49Hþ memory NK cells. However, no receptor has been identified that mediates the interaction with HCMV. In humans, they have been termed adaptive NK cells. The genome-wide DNA methylation patterns of adaptive NK cells are strikingly similar to cytotoxic effector CD8 T cells but differ from those of canonical NK cells. Adaptive NK cells have been shown to have subsets that do not express FcεR1g, Syk, and EAT-2 and have reduced PLZF expression that affects cytokine production [57,58]. In vitro experiments have demonstrated that, relative to other NK cell subsets, adaptive NK cells exhibit stronger CD16-mediated responses and greater TNF-a and IFN-g production in response to K562 myeloid leukemia cells. Cancer cells can downregulate classical class I HLA molecules while retaining the expression of HLA-E. The switch in receptor usage for HLA-E recognition from predominantly inhibitory NKG2A to activating NKG2C may be a key mechanism by which adaptive NK cells mediate graft-versus-leukemia (GvL) effects.

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CMV causes asymptomatic or mild illness in healthy individuals. However, for patients immunosuppressed due to HIV infection or solid organ or hematopoietic stem cell transplantation (HSCT), CMV is a potentially life-threatening complication. CMV remains latent in the host and latent CMV reservoirs have been found in cells of the myeloid lineage and endothelial cells [59]. CMV reactivation after HSCT has been shown to be beneficial. Several groups have reported an association between CMV reactivation and relapse protection after HSCT. Elmaagacli and colleagues [59] reported that early CMV reactivation is associated with a reduced risk of relapse in AML patients undergoing allogeneic HSCT from HLA-matched sibling or unrelated donors. The risk of leukemic relapse was 9% at 10 years after HSCT compared with 42% in patients who did not reactivate CMV. In recipients of umbilical cord blood (UCB) HSCT, CMV reactivation has been shown to induce a more rapid reconstitution of fully functional, educated NK cells with increased survival capacity and the ability to respond rapidly with cytokines [60]. In the absence of CMV reactivation, NK cells remain immature and recovery of full effector functions takes at least 1 year. Cichocki and colleagues demonstrated that in 674 allogeneic HSCT recipients, those with reactivated CMV had lower leukemia relapse and superior disease-free survival 1 year after reduced intensity conditioning compared with CMV seronegative recipients. Furthermore, expansion of these cells 6 months after transplant independently trended toward a lower 2-year relapse risk. This protective effect was independent of age and graft-versus-host disease (GvHD) but highly correlated with the expansion of adaptive NK cells [61]. Therefore, adaptive NK cells have the potential to be therapeutically beneficial in both controlling HCMV infections and treating patients with hematological malignancies. The same group has also developed an adaptive NK cell product. They demonstrated that addition of a small molecule inhibitor of glycogen synthase kinase 3 to peripheral blood NK cells from a CMVþ donor expanded ex vivo with IL-15 markedly enhanced CD57þ NKG2Cþ cells. This was associated with the expression of several transcription factors associated with late-stage NK cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting cell viability or proliferation. These cells were highly cytotoxic expressing high levels of TNF and IFN-g in response to K562 target cells. They also showed high ADCC responses [62]. This discovery has been translated into a phase I clinical trial for AML that is currently recruiting patients at the University of Minnesota.

ROLE OF NATURAL KILLER CELLS IN CANCER THERAPEUTICS Human Versus Mouse Natural Killer Cells Studies in mouse models have allowed for the analysis of several parameters involved in NK maturation, activation, and function that provide essential data to understand NK cell biology and have a strong potential for clinical/human translation. Mouse models have been invaluable in identifying molecules relevant for NK activation or suppression with the discovery of the Ly49 family members [13]. IL-15 was first demonstrated to be required for NK maturation and activation as IL-15deficient mice showed profound NK defects compared with IL-2-deficient mice [63]. The concept of NK licensing was also postulated after the study of mouse NK cells displaying differential inhibitory receptors for MHC [14]. The use of NK cells in cancer immunotherapy was initially described using mouse models of allogeneic HSCT. Mouse NK studies demonstrated efficacy of using NK cells to promote graft-versus-tumor (GvT) effects while inhibiting GvHD in allogeneic hematopoietic transplantation [64]. As NK cells do not initiate the rejection of solid tissue allografts, it was reasonable to hypothesize that the solid organ tissues often targeted in GvHD (skin, gut, liver) would not be targets of donor NK cells. However, NK cells can be found in GvHD lesions suggesting that they may contribute to the pathology once initiated. In nonmyeloablative conditioning, donor NK cells may suppress host hematopoiesis. Indeed, it has been suggested that donor NK cells may allow for less conditioning to be given to the recipient and promote donor myeloid engraftment [65]. Mouse models also have demonstrated that donor-type NK cells can act as “veto” cells and inhibit host effector cells (T and NK cells) capable of mediating graft rejection [66]. Interestingly, as opposed to licensed host NK cells that can reject allogeneic HSCs, unlicensed host NK cells were found to produce GM-CSF after allogeneic MHC-I recognition and promote hematopoietic engraftment [67]. Thus, NK cells, both donor and radioresistant host NK subsets, can promote allogeneic donor engraftment through multiple mechanisms. NK cells have also been shown to mediate resistance to metastatic spread in numerous mouse models, suggesting that solid tumors can also be potentially targeted. The receptors NK cells use to recognize tumors have focused on NKG2D and the Ly49 receptor family. Inhibitory Ly49 molecules have been thought to inhibit NK cell antitumor responses. Blockade of these receptors using Fab fragments have been shown to promote antitumor effects without inducing myelosuppression in mouse models [68], suggesting that these approaches may be of use in the clinic. The implication of activating NK receptors, such as NKG2D or DNAM-1, in tumor surveillance has also been demonstrated in mouse models. Furthermore, downregulation of NKG2D expression has been detected in cancer patients and correlated with poor cytotoxic NK function. Finally, there is increasing evidence that NK cells play important roles in immunoregulation. There is ample

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evidence in mouse models that NK cells can both promote and inhibit T cell responses, either directly or indirectly. Unlicensed NK cells were found to indirectly promote antigen-specific T cell responses after viral infection through production of GM-CSF [38]. These data would suggest that while direct antitumor effects can occur when using activated NK cells in cancer therapy, care must be exercised as these same cells can also promote or suppress T cell responses that may be necessary for long-term efficacy. Mouse models are a valuable tool for the study of NK biology. However, there are critical differences between human and mouse NK cells that should be taken into account when attempting to translate mouse data into human research. One of the main differences between human and mouse NK cells is the lack of CD56 expression in the latter, which is the primary marker used to identify human NK cells. This means that there are no CD56bright and CD56dim subsets that are present in humans. There have been multiple attempts to find a common marker that can be used in both species. Hayakawa and colleagues were able to differentiate mouse NK maturation stages through the expression of the TNF receptor family member CD27, also expressed in humans, and CD11b [69]. Four maturation stages have been identified: CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and CD11bhighCD27low. In humans, a CD27high subset is found within the CD56bright NK population while CD27low is mainly expressed on peripheral blood CD56dim NK cells. Similar to CD56bright, CD11blowCD27high NK cells produce higher levels of cytokines than CD11bhighCD27low. However, CD11blowCD27high NK cells also display strong cytotoxic function differently than CD56bright. In addition, there is a paucity of NK cells present in the mouse LN, whereas human CD56bright NK cells are relatively abundant in human LN. As CD56bright NK cells are poorly lytic and thought to predominantly mediate effector functions through production of cytokines such as IFN-g, this suggests a significant divergence between the species regarding possible immunoregulatory roles of NK cells. Resting mouse NK cells have much lower cytotoxic functions. The generation of inbred mice as well as the housing conditions (specific pathogen-free) may account for the poor lytic function of resting mouse NK cells. In addition, mouse NK cells cultured in vitro survive for a short period compared with human NK cells that can be maintained longer with stable KIR expression [66]. These results would suggest that similar effects might occur in vivo in which mouse NK cell therapies may underestimate potential efficacy due to their short-lived nature.

NATURAL KILLER CELLS IN HUMANS The first trials to harness the antitumor properties of NK cells focused on the use of IL-2 to activate autologous NK cells both ex vivo and in vivo. Ex vivo IL-2 stimulated autologous PBMCs were infused into patients with melanoma, lymphoma, and renal cell carcinoma with additional high dose IL-2 [70]. Despite inducing NK cell cytotoxicity, the treatments were unsuccessful. High-dose IL-2 infusions to stimulate NK cells in vivo were also clinically disappointing and were associated with high toxicities. Ultimately, infusion of IL-2 or adoptive transfer of IL-2eactivated autologous NK cells failed for four main reasons: competition with the recipient’s lymphocytes for cytokines and “space”, autologous NK cells were inhibited by self-HLA class I, immunosuppression and stimulation of regulatory T cells (Tregs). As inhibitory KIR and their ligands were further characterized, the next approach to utilizing NK cells as immunotherapy focused on allogeneic NK cells. These NK cells would be alloreactive against a target that lacked the ligand for their inhibitory receptors. Allogeneic NK cells with the potential to mediate antitumor effects can be delivered in the context of an HSCT or adoptively transferred after ex vivo stimulation. Efficacy of the first approach was highlighted in the 2002 study from the Perugia group who provided evidence that donor alloreactive NK cells decreased graft rejection, enhanced engraftment, and mediated the GvL effect in the absence of GvHD after mismatched HSCT [65]. The potential for NK alloreactivity in the GvHD direction was determined using what would become known as the KIR ligand incompatibility or KIR ligand mismatch model. For example, the donor expresses all three ligands for the inhibitory KIR (C1, C2, and Bw4), whereas the recipient only expresses C2 and Bw4. Therefore, it is predicted that the donor will have C1-specific alloreactive NK cells that will not be inhibited in the recipient and can potentiate the GvL effect. This model assumes that the donor expresses the inhibitory KIR that recognizes each self-HLA ligand. Of the 92 AML and ALL patients who received HLA mismatched grafts, 34 patients were KIR ligand incompatible in the GvH direction. Transplanted donor alloreactive NK cells protected against graft rejection, GvHD, and against AML relapse. Without NK cell mismatch, the probability of event-free survival at 5 years was 5%, and with an NK cell mismatch, the probability was 65%. This only occurred in patients with AML. That there were no beneficial effects seen for patients with ALL is speculated to be due to lack of the adhesion molecule, LFA-1, on the surface of ALL blasts [71]. Donor alloreactive NK clones could also be identified in the recipient up to 12 months after transplant. Based on these findings, several retrospective analyses were conducted with conflicting results. Giebel and colleagues [72] demonstrated a beneficial effect of KIR ligand mismatching studying 130 unrelated donor transplants, of which 20 had

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predicted NK cell alloreactivity. Event-free survival was markedly increased (87%e46%, P ¼ .006). As in the Perugia study, NK cell alloreactivity was beneficial predominately for myeloid malignancies. In perhaps one of the largest retrospective analysis of KIR ligand mismatching, a study that examined over 1500 unrelated transplants found no beneficial effect and reported that KIR ligand incompatibility was associated with increased GvHD, treatment-related mortality, and shorter leukemia-free survival compared with HLA-matched transplants [73]. Therefore, in human trials, the role in GvHD is still controversial. Improved survival was observed after HLA-matched sibling transplant in myeloid patients where the recipient lacked a KIR ligand [65] and lower relapse after unrelated HSCT was also associated when the recipient lacked a KIR ligand. There are several factors that may explain the disparities between the Perugia findings and subsequent retrospective analyses and studies. In the Perugia study, donor allografts were extensively T celledepleted using a combination of CD34þ selection, soybean agglutination, and E-rosetting. Differing pretransplant conditioning regimes and posttransplant GvHD prophylaxis may also play a role in the efficacy of NK cell alloreactivity. KIR ligand mismatching has also been studied after UCB HSCT. Willemze and colleagues [74] reported on the outcomes of 218 single-unit UCB transplants in AML and ALL patients. Donorerecipient pairs with KIR ligand incompatibilities showed improved leukemia-free survival and decreased relapse for patients with AML. Patients with ALL also showed a trend toward better outcomes when there was predicted KIR ligand incompatibility. By contrast, Brunstein and colleagues [75] reported a negative effect of KIR ligand incompatibilities in a cohort of 257 UCB HSCT recipients. Two other models for predicting NK alloreactivity have also been studied. The KIR-ligand absence model uses recipient HLA typing to predict the potential for NK alloreactivity. This model assumes that the respective inhibitory KIR that would recognize the missing ligand in the recipient is present in the donor. The receptor-ligand model uses donor KIR genotyping and recipient HLA typing to predict NK cell alloreactivity. This model allows for the potential of NK cells expressing a known inhibitory receptor to eliminate recipient cells that lack the receptor for that KIR. Even with all these models, donorerecipient pairs do not behave as predicted. This is most apparent with Bw4 and KIR3DL1. Differing levels of surface expression of KIR3DL1 alleles have been described that may influence generation of Bw4-specific clones. For example, KIR3DL1*01502 is a stronger allele for Bw4 than KIR3DL1*007 [68]. The Bw4 epitope, which spans amino acids 77e83, can have either an isoleucine (Ile) at position 80 or a threonine (Thr). In HIV, Bw4 alleles with Ile80 have been associated with delayed progression to AIDS [7] and it could be predicted that Bw4 alleles with an Ile80 may have stronger interactions with KIR3DL1 and result in higher numbers of Bw4-specific clones. Disparities with HLA-C mismatching predictions may also occur. Yawata and colleagues [76] examined NK cell receptor repertoire expression and function in 58 healthy individuals. They found that various combinations of HLA-C alleles and KIR genes resulted in differing levels of NK cell function. KIR2DL3 expressing NK cells from donors who were homozygous for the C1 allele HLA-C*07 produced high levels of IFNy, those expressing the C1 alleles HLA-C*01, *03, *08, or *1403 produced moderate levels of IFNy and those donors homozygous for HLA-C*1402 were poor producers of IFNy. KIReHLA combinations that result in lower NK cell function may be due to weak interaction between the inhibitory KIR and its cognate ligand. Indeed, studies looking at NK cell education and acquisition of function suggest that inhibitory receptor interaction with its cognate ligand must reach a threshold to gain function and that this threshold is much higher for cytokine-producing functions than for cytotoxicity [45]. Another approach is to examine the immunogenetics of donor and recipient KIR genes. As described earlier, KIR genes can be divided into two main haplotypes based on gene content: haplotype A and haplotype B. KIR A haplotypes have a fixed gene content of the main inhibitory KIR and only one activating KIR, whereas B haplotypes have variable gene content comprising up to five activating KIR. The genes on each KIR haplotype can be divided into either centromeric (2DS2, 2DS3/5) or telomeric (2DS1, 3DS1) regions or those that can be on either or both (2DL5 and 2DS3/5). These are denoted as Tel-A or Cen-A for the A haplotype and Tel-B and Cen-B for the B haplotype. In 2010, 1086 AML and 323 ALL transplant pairs were evaluated for the relative contributions of centromeric and telomeric KIR B genes [77]. Donors with 2 centromeric or telomeric KIR B motifs were associated with better outcome. Predicting NK cell alloreactivity is further complicated by the presence of other inhibitory and activating receptors present on donor NK cells. The inhibitory receptors NKG2A and LIR-1 may suppress NK cell function after transplantation. HLA-E, the ligand for NKG2A, is expressed on hematopoietic cells and could inhibit NK cell function even if there is a KIR mismatch, especially considering that reconstituting NK cells have high NKG2A expression. Furthermore, while some tumors may downregulate class I, HLA-E usually remains intact. Pende and colleagues [78] demonstrated KIR2DS1-mediated lysis of C2 homozygous leukemic blasts and confirmed that NKG2A inhibitory signaling could be overcome. A large study of 1409 unrelated transplants examined the role of donor and recipient KIR haplotypes and found that transplants from a KIR B haplotype donor resulted in lower relapse and improved survival for patients with AML [79]. The effect was not seen for patients with ALL. As described earlier, KIR A haplotypes consist

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predominately of inhibitory KIR and only one activating KIR2DS4. KIR B haplotypes, however, have varying activating KIR gene content. Even if donorerecipient pairs are carefully selected based on predicted NK alloreactivity and confirmation with in vitro lysing of recipient leukemic blasts, the transplant environment still needs to be considered. NK cells are the first lymphocyte to reconstitute after HSCT, and NK cell function varies depending on the transplant source. Reconstituting NK cells have different receptor profiles than the donor, which includes higher expression of NKG2A, altered KIR and NCR expression, and lower than normal overall function [80]. Recovery of normal receptor repertoires and function may take up to 1 year or more. Graft source also influences NK cell recovery after HSCT. Cooley and colleagues [81] compared KIR receptor repertoires between unmanipulated BM transplants and T celledepleted transplants. T cell depletion was mediated through ATG, elutriation, or campath. They found that KIR expression was increased in the patients who received T celledepleted grafts. Foley and colleagues [82] compared NK cell function (cytotoxicity and cytokine production) after HCST with three different graft preps, unmanipulated (T cell replete), CD34þ selected (T cell deplete) without posttransplant immune suppression and UCB. Overall NK cell function was lower after HCST compared with donor NK cells before transplantation. Surprisingly, NK cell function was highest after T cellereplete HCST, and this was associated with higher KIR expression. In recipients of T cellereplete HCST, it was demonstrated that target-induced IFNy production was associated with KIR and not NKG2A as NK cells expressing only NKG2A were poor producers of IFNy. Furthermore, IFNy-producing NK cells expressed a KIR that recognized itself. As NKG2A-expressing NK cells were cytotoxic, it was postulated that in the posttransplant setting, the interaction between NKG2A and HLA-E supports the education of NK cells to degranulate but that the signal is too weak to meet the threshold to educate for cytokine production. Therefore, these studies suggest that T cells may be beneficial in promoting NK cell function rather than hindering it. NK cells may persist in the recipient for longer than originally reported and infused mature donor NK cells may contribute to the increased function seen after T cellereplete HSCT.

ADOPTIVE NATURAL KILLER CELL THERAPY The second method to deliver alloreactive NK cells to the patient involves adoptive transfer of donor NK cells purified ex vivo and infused into the recipient. These NK cells are presumed to be mature, fully functional, and educated in the donor. The first trial of this approach was conducted at the University of Minnesota [83]. Forty-three patients with metastatic melanoma, metastatic renal cell carcinoma, Hodgkin’s disease, or poor prognosis AML were enrolled in the trial. PBMCs were collected from haploidentical related donors and CD3 depleted before being incubated overnight in IL-2. Before NK cell infusion, patients underwent a preparative regimen that involved three different chemotherapy preps: high cyclophosphamide and fludarabine (Hi-Cy/Flu), low cyclophosphamide and methylprednisone or fludarabine (Flu). After infusion, patients received IL-2 daily for 14 days. NK cell expansion was only observed for patients receiving the preparatory regimen of Hi-Cy/Flu. Successful expansion of NK cells was determined by the detection of greater than 100 NK cells/mL of blood 12e14 days after infusion. On this protocol, 30% of poor prognosis AML patients achieved a complete remission, whereas no beneficial effects were observed for the other diseases. However, this remission was not durable and patients ultimately relapsed. Because NK cells only expanded after high-dose preparative regimens, it was proposed that the addition of 400 cGy of total body irradiation to further deplete host immune cells and create space for donor NK cells to expand. This addition also meant that patients needed to be “rescued” with a CD34þ selected stem cell infusion. On this protocol, NK cell expansion was much more successful and no acute GvHD was observed, although death due to infection was common (unpublished data). It should also be noted that while there was induction of remission, ultimately this therapy did not result in lasting remissions. In addition, there was no influence of KIR-ligand incompatibilities. The current strategy at the University of Minnesota is to use NK cells, cytokines, and lymphodepleting chemotherapy (Hi-Cy/Flu) as therapy to achieve remission in patients with refractory AML. These trials use donor NK cell persistence and in vivo expansion as measures to improve clinical efficacy given the correlation between leukemia clearance and donor-derived NK cells 7 and 14 days after adoptive transfer. The use of adoptive transfer of NK cells to treat various malignancies has resulted in mixed results. Shi and colleagues [84] infused haploidentical KIR mismatched NK cells into 10 patients with relapsed multiple myeloma followed 14 days later with an autologous stem cell graft. Five patients achieved near complete remission. Bachanova and colleagues [85] treated six patients with non-Hodgkin’s lymphoma with infusion of haploidentical NK cells and found that NK cells poorly expanded in vivo and host Tregs were significantly increased after NK cell infusion and IL-2 administration. In an experimental tumor model, pretreatment with Ontak (denileukin diftitox), to deplete host T regulatory cells, allowed the adoptively transferred NK cells to eliminate tumors more efficiently compared with mice not treated with Ontak [86]. A similar strategy was used in humans in a clinical trial where Ontak treatment in conjunction with haploidentical NK cells

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induced improved clearance of AML [87]. Therefore, depleting host T regulatory cells has the potential for improving NK cell expansion and function in humans. Recently, IL-15 has been developed for use in NK cellebased therapies. An interesting aspect of IL-15 is that it uses trans-presentation where IL-15eproducing cells present the cytokine in the context of IL-15 receptor a (IL-15Ra) chain on the cell surface. DCs are one cell type that can trans-present IL-15 to NK cells [19]. IL-15R is a heterotrimeric receptor consisting of a unique a chain, a shared b subunit with IL-2 (CD122), and a common g subunit (CD132) shared with several cytokines [88], implying similar biological activities of IL-2 and IL-15. IL-15 activates NK cells without activating Tregs. Engagement of IL-15 receptor on NK cells causes the autophosphorylation and activation of JAK1 or JAK3, which induce at least three parallel signaling cascades: RaseRafeMAPK, activation of transcription factor STAT5, and PI3KeAKTemTOR pathways [88]. IL-15 controls NK cell proliferation and survival, and maintains stocks of effector proteins granzyme B and perforin. Huntington and colleagues recently showed that cytokine-inducible SH2-containing protein (CIS) and inhibitor of DNA binding 2 (id2) regulate IL-15 signaling. CIS, encoded by the CISH gene, is being described as an intracellular checkpoint because deletion of CISH rendered NK cells hypersensitive to IL-15 that led to enhanced proliferation, survival, IFN-g production, and cytotoxicity toward tumor cells [89]. id2 regulates NK cell responsiveness to IL-15 and knockouts have impaired IL-15 signaling and metabolic function [90]. ALT-803 (Altor BioScience) is an IL-15/IL-15Ra-Fc superagonist complex. In preclinical mouse models, ALT-803 has shown potent antitumor activity [91e93]. At the University of Minnesota, a clinical trial with 16 patients was performed in patients with relapsed hematologic malignancy after allogeneic hematopoietic cell transplantation (>60 days) with no active GvHD and having >10% residual donor chimerism. Treatment increased NK cell proliferation and in vitro analysis showed an increased frequency of NK cells that degranulated against K562 targets and produced IFNg after IL-12/IL-18 stimulation [94]. Another trial used a recombinant human IL-15 administered as a daily intravenous bolus infusion for 12 consecutive days in patients with malignant melanoma or metastatic renal cell cancer. They observed up to 10-fold expansion of NK cells in the circulating blood [95]. Currently, there are a number of open clinical trials investigating if IL-15, monomeric or in complexes, can be used to activate and expand NK cells to improve this modality of immunotherapy.

EXPANDING NATURAL KILLER CELLS In the initial report describing the clinical efficacy of adoptively transferring NK cells, PBMCs were CD3 depleted and activated with IL-2 overnight. After infusion, IL-2 was added daily for 14 days to expand these donor NK cells in vivo. Platforms currently used also involve the depletion of CD19 cells. The alternative is to expand NK cells ex vivo and then infuse into patients. Several centers are working on this approach. Imai and colleagues engineered the class I negative K562 cell line to express 4-1BBL and membrane-bound IL-15 [96]. Under GMP conditions, they described on average a 1000-fold expansion of NK cells after 3 weeks in culture with retained cytotoxic capabilities. K562 cells expressing membrane-bound IL-21 and 4-1BBL (K562-mb21-41BBL) expanded NK cells significantly more than the ones with IL-15 [97]. Another approach is using particles derived from plasma membranes of the K562-mb21-41BBL cells that express 4-1BBL and membrane-bound IL-21 (PM21 particles). Ex vivo, PM21 particles caused specific NK cell expansion from PBMCs from healthy donors and AML patients. They also stimulated in vivo NK cell expansion in NSG mice. Ex vivo preactivation of PBMCs with PM21 particles (PM21-PBMC) before intraperitoneal (i.p.) injection resulted in 66-fold more NK cells in peripheral blood of mice and these cells were found in the BM, spleen, lung, liver, and brain even 16 days after injection of the PM21 particles [98]. Another GMP-compatible lymphoblastoid cell line and IL-2 expand NK cells on average 500-fold in 30 days. These expanded NK cells were shown to upregulate CD25 (IL-2R), CD48, NKG2D, and TRAIL compared with resting NK cells. They also increased secretion of many cytokines and chemokines, including IFNy, TNFa, GM-CSF, and MIP-1b [99]. Removal of IL-2 resulted in decrease expression of both NKG2D and TRAIL and multiple cytokines including IFNg. Studies have also explored the use of NK cell lines for potential adoptive therapy. Both irradiated NK-92 and KHYG-1 could prove to be an unlimited source of cytotoxic NK cells for adoptive transfer; however, their survival in vivo is still unclear [100]. Irradiated NK-92 cells were used in a phase I clinical trial for renal cell cancer and melanoma with only mild infusion toxicities [101]. Another method involves the generation of NK cells from human embryonic stem cells or induced pluripotent stem cells using a spin-embryoid body differentiation system that allows for human NK cells development completely in vitro [102].

ENHANCING NATURAL KILLER CELL FUNCTION TO ELIMINATE TUMORS In addition to expansion and adoptive transfer, genetic engineering has enabled NK cells to express certain receptors that increase their ability to recognize and eliminate tumors. Chimeric antigen receptors (CARs) typically involve fusions

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of single-chain variable fragments (scFv) derived from monoclonal antibodies such as anti-CD19 fused to the CD3z transmembrane endodomain. Addition of costimulatory molecules such as CD28, CD137 (4-1BB), and/or CD134 (OX40) increases the efficacy of CARs. NK-92 cells have been genetically modified with CARs that consist of CD20 or Her-2/neu ectodomains, both fused to CD3z endodomain, and have shown increased NK cell killing of CD20-positive lymphoma or breast cancer cells, respectively [103]. Pegram and colleagues [104] genetically engineered mouse NK cells with a CAR consisting of anti-erbB2 (Her-2/neu) and CD28 fused to CD3z. These NK cells were adoptively transferred into Rag-1/ mice and inhibited growth of erbB1þ lymphoma by directly lysing the tumors. Several tumor-targeted monoclonal antibodies have been developed and have been used clinically to treat various malignancies. Because inhibitory KIR bind to self-HLA class I and inhibit NK cell function, monoclonal antibodies directed against the inhibitory KIR have been developed. Romagne and colleagues [105] generated a human monoclonal antibody called 1e7F9 that recognizes inhibitory KIRs KIR2DL1, KIR2DL2, and KIR2DL3. It does not cross-react with KIR3DL1. During preclinical characterization, it was demonstrated that 1-7F9 did not stimulate peripheral blood NK cells to degranulate or produce IFNy and blocking KIR with 1-7F9 increased lysis of primary AML blasts. Although preclinical studies yielded excitement for this reagent, clinical results have been lackluster. Carlsten and colleagues described one mechanism that might explain limited efficacy of the humanized version of this blocking antibody, IPH2101, tested in a small phase II clinical trial for smoldering multiple myeloma. Immune-monitoring studies showed that treatment with IPH2101 led to an unexpected depletion of KIR2D þ NK cells resulting from monocyte- or neutrophil-mediated trogocytosis, which is dependent on binding of FcgR1 to the Fc portion of the IgG4 IPH2101 antibody. This reduces NK cell education and the NK cells become hyporesponsive [106]. Unlike other activating receptors present in human NK cells, CD16 can robustly mediate activation without the need for coengagement of other receptors [107]. NK cells recognize antibody-coated targets through CD16 and mediate antibody dependent cellular cytotoxicity (ADCC). Allelic polymorphisms within the CD16 gene have been shown to influence NK cellemediated ADCC. One such polymorphism is at position 158, a region of the receptor that interacts with the hinge region of IgG antibodies, has either a phenylalanine (F) or valine (V) at this position, and alters NK cell binding [108]. The 158V polymorphism results in higher CD16 binding to IgG. Some of the monoclonal antibodies that mediate NK cell ADCC include trastuzumab (Her2 on breast cancer), alemtuzumab (CD52 on CLL), and cetuximab (EGFR on colorectal cancer) [109]. Recently, a mechanism that inhibits CD16 signaling has been identified. A disintegrin and metalloprotease17 (ADAM17), also known as TACE, is expressed by NK cells and cleaves CD16 on NK cell activation. Its selective inhibition can prevent CD16 shedding and lead to enhanced IFNg production, especially when triggering was delivered through CD16. Fc-induced production of cytokines by NK cells exposed to rituximab-coated B-cell targets was further enhanced by ADAM17 inhibition [110]. While individual monoclonal antibodies can trigger NK cell function, combinations of antibodies may enhance NK cell responses. Blockade of inhibitory KIR in combination with tumor-targeted monoclonal antibodies may increase CD16-mediated ADCC. CD137 or 4-1BB is a costimulatory molecule of the TNF receptor family. It is expressed on activated NK cells, T cells, B cells, dendritic cells, monocytes, and neutrophils. On resting NK cells, its expression is low but CD16 activation can induce its expression [111]. CD137 can be activated by binding to its natural ligand or triggered with a monoclonal antibody against it. Anti-CD137 antibodies have been used in combination with other monoclonal antibodies to increase NK cell activation. Anti-CD137 antibodies in combination with rituximab have been shown to increase degranulation and IFNy production [112]. On engagement of CD16 with rituximab-coated lymphoma cells, CD137 is upregulated on the NK cell and addition of an agonist against CD137 increased NK cellemediated ADCC. Clinical trials with anti-CD137 antibodies have been conducted. One such antibody, urelumab was tested in a multidose phase IeII trial in patients with locally advanced or metastatic solid tumors. Three partial response and four stable disease cases occurred at all three doses tested in expansion cohorts [113]. Since then, many combinations of trials with chemotherapy and checkpoint blockade are ongoing. In addition to monoclonal antibodies, bispecific killer engagers (BiKEs) and trispecific killer engagers (TriKEs) have been used to direct NK cell killing of tumor targets. BiKEs and TriKEs are small molecules containing a single variable portion (VH and VL) of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. These molecules facilitate synapse formation between the NK cell and the tumor target. All of these molecules contain an anti-CD16 scFv that activates the NK cell. BiKEs and TriKEs specific for CD16 and CD19/CD22 not only targeted Raji tumor cells, but they also resulted in increased secretion of IFN-g, TNF, MIP-1a, GM-CSF, RANTES, and IL-8, indicating that these reagents elicit both direct killing and NK cellemediated inflammatory responses. This TriKE could elicit better CD107a responses to primary ALL and CLL targets than rituximab [110]. Newer TriKEs now contain an IL-15 moiety, in place of the third scFv, which enhances proliferation, survival and priming of NK cells. A CD16IL-15CD33 TriKE has shown efficacy in vitro and in vivo against AML targets. The TriKE showed cytotoxicity

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against HL-60 targets and primary AML targets. These results were confirmed in an immunodeficient mouse HL-60 tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks [114]. Anti-EpCAM, anti-CD133, and anti-EpCAM/CD133 have also been described highlighting the versatility of this platform in different tumor settings [115,116]. Histone deacetylase and proteasome inhibitors have also been shown to sensitize tumors to NK cell killing. Bortezomib, a proteasome inhibitor, has been shown to upregulate death receptors such as Fas and TRAIL-R2/DR5 [117] and induce apoptosis through Fas/FasL and TRAIL/DR5 interactions, both expressed by NK cells. Bortezomib has been shown to be effective in inducing apoptosis of quiescent CD34þ cells in CML patients and increasing the efficacy of ex vivo expanded adoptively transferred autologous NK cells. Finally, NK cells have been shown to enhance activity against tumor cells with a cancer stem cell (CSC) phenotype [118]. In addition, radiation has been shown to enhance targeting of these CSCs by NK cells through both CSC enrichment and stress ligand/death receptor upregulation [119]. Other reagents such as Lenalidomide have also shown enhancing roles by decreasing the threshold for activation of NK cells. Combinational approaches, using a number of these drugs at the same time, will likely be the best route to maximize NK cell immunotherapy [120].

THE ROLE OF NATURAL KILLER CELLS IN VIRAL THERAPY Another potential therapeutic approach to eliminate tumors is the use of oncolytic viruses. An oncolytic virus is a virus that preferentially infects, replicates, and lyses malignant cells. In most instances, the virus is engineered to increase tumor specificity. The first report of an engineered oncolytic virus described the ability of a thymidine kinaseenegative mutant herpes simplex virus-1 (HSV-1) that was attenuated for neurovirulence to treat gliomas [121]. The virus can lyse human glioma cell lines in vitro, inhibit growth of human glioma in nude mice, and prolong survival. As discussed earlier, while NK cells have antitumor properties, NK cells also have potent antiviral properties. Individuals with NK cell deficiencies are particularly susceptible to viral infections [122]. Because NK cells are critical components of the host response to viral infections, they may be both beneficial and a hindrance when using oncolytic viruses as a cancer therapy. For effective use of oncolytic viruses, they must be able to enter the cell, infect, and replicate before the immune response targets the virus. HSV-1e and vaccinia viruseinfected cells, commonly used as oncolytic viruses, can be rapidly lysed by NK cells preventing virus dissemination, thereby potentially preventing the efficacy of these oncolytic viruses [123]. On activation, NK cells will also recruit other innate immune cells such as monocytes and dendritic and cells of the adaptive immune response, T cells and B cells, that may further impede efficacy of the virus. To counteract the host’s antiviral immune response, the addition of chemotherapy to suppress the immune system coupled with oncolytic viruses may increase the potential for this line of therapy. Alternative approaches involve harnessing the potent antiviral properties of NK cells. This may involve viruses that upregulate ligands for NK cell activating receptors and downregulate ligands for the inhibitory receptor, for example, HLA-class I. Bergmann and colleagues [124] engineered influenza A virus to be used as a potential oncolytic virus. NKp46 expressed by all NK cells recognizes influenza A HA on infected cells. An alternative approach involves immune suppression to ensure total viral infection of the tumors, particularly in metastatic tumors, followed by adoptive transfer of activated NK cells that will be able to eradicate the infected tumor cells.

CONCLUDING REMARKS NK cells have been considered important effector cells in eliminating aberrant cells ever since they were initially identified by their ability to spontaneously reject BM allografts and lyse cells with low MHC class I expression without prior sensitization. In the clinical setting, NK cells both in the context of HSCT and adoptively transferred have been shown to have remarkable therapeutic properties, including the ability to enhance engraftment, decrease both rejections, decrease GvHD in some studies, and eliminate leukemic cells. The use of animal models and in vitro assays to demonstrate NK cell effectiveness in eliminating cancer cells has been fundamental. Further characterization and identification of NK cell receptors and their ligands on tumor cells will enable a clearer understanding of how NK cells recognize tumors and how this can be exploited in the clinical setting. Because not all tumors respond to NK cells in the same manner, certain NK cell therapies may be more applicable to particular cancers. In addition, combinations of different therapies may be essential to enhance the efficacy of NK cell immunotherapy in vivo. Since their discovery over 40 years ago, new and exciting areas of NK cell biology continue to emerge. The effective use of NK cells to treat cancer can be expected to increase as we advance our understanding of how NK cells gain function, how the multitude of different receptors expressed by NK cells control function, and how this knowledge can be exploited to eliminate tumors.

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