(PTEN) Regulates Natural Killer Cell Function

Elucidation of the Mechanism by which Phosphatase and Tensin Homologue Deleted on Chromosome Ten (PTEN) Regulates Natural Killer Cell Function

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Edward Lloyd Briercheck

Graduate Program in Integrated Biomedical Science Program

The Ohio State University

2013

Dissertation Committee:

Professor Michael A. Caligiuri, MD (Advisor)

Professor William E. Carson, III, MD

Professor Gregory B. Lesinski, Ph.D.

Professor Guido Marcucci, MD

Edward Lloyd Briercheck

2013

Abstract

Human natural killer (NK) cells are CD56+CD3- large granular lymphocytes of the innate immune system which are characterized by the ability to both directly kill and initiate an immune response to virally infected or malignantly transformed cells. Human NK cells in peripheral blood can be divided into two developmentally and functionally distinct subsets based upon surface expression of CD56. In contrast to the more mature CD56dim

NK cell, the less mature CD56bright NK cell is unable to kill malignant cells at rest. We sought to determine the mechanism of this difference in cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We observed that CD56bright NK cells showed a ~5 fold increase in PTEN protein expression over CD56dim NK cells. Human and murine NK cells overexpressing PTEN demonstrated decreased cytolytic activity and IFN-γ secretion, with concurrent decreases in their downstream (MAPK and AKT) targets that are critical for cytolysis. Paradoxically, human NK cells with near complete PTEN knockdown also showed decreased cytolytic activity despite elevations in AKT and MAPK. Confocal microscopy revealed that near complete PTEN knockdown results in a disruption of the NK cell’s ability to organize immunological synapse components including decreased adhesion, decreased polarization of the microtubule organizing center toward the target cell and decrease in coalescence of cytolytic granules. Thus, PTEN is differentially expressed in mature human NK cell subsets and our studies suggest it must be expressed at an optimum level to maximize NK cytolytic activity. ii

Dedication

This document is dedicated to Ani and my family.

iii

Acknowledgments

I would like to thank my advisor Dr. Michael A. Caligiuri for his mentorship both in and outside the laboratory throughout my training. I have learned an incredible amount and am continually inspired by your passion, drive and belief in those around you. I have been truly lucky to have a mentor whose door, text messaging, email, phone is always open. Thank you for this opportunity, and your continued support.

To my friends and colleagues in the Caligiuri lab. I have learned so much from all of you these past five years. I simply could not have completed this journey without you. I would especially like to thank Dr. Rosanna Trotta for her scientific expertise, willingness to teach, and providing me with a foundation of learning to do truly well designed science. I would also like to thank my students Jordan Cole, Tyler Cole, and Alex

Hartlage for their contributions to this work. You all have tremendous potential and I look forward to having you as colleagues.

I would like to thank my committee members Dr. Guido Marcucci, Dr. William E.

Carson III, and Dr. Gregory B. Lesinski. I am very fortunate to have such an accomplished and willing committee during my graduate studies. Thank you all once again.

I would also like to extend my appreciation the Medical Scientist Training Program

(M.S.T.P.) and the Biomedical Sciences Graduate program at The Ohio State University

iv for the opportunity to be a part of such a special group of faculty and students. I would like to thank Dr. Larry Schlesinger, Dr. Lawrence Kirschner, and Ashley Bertran for their tireless work in creating a world-class training program. I would also like to express my gratitude to the late Dr. Alan Yates who first welcomed me to the program and laid the foundation for the M.S.T.P.

I would like to acknowledge the Pelotonia Graduate Fellowship program, the Medical

Scientist Training Program and the National Cancer Institute for their support of the research presented in this thesis.

I would like to thank my mother for her commitment to creativity and education and my father who has never wavered in his belief in hard work and his son. Thank you both and thank you to the rest of my family including Brennen Cocklin for their tremendous support. Finally, Ani there are no words to describe what you mean to me and how you have supported me through this process, most often from 800 miles away. I am constantly amazed at your talent, determination, and grace. You make me want to be a better person.

With love, thank you.

Vita

May 2002 ...... Jackson High School

May 2006 ...... B.S. Biology, University of Toledo

2006 to present ...... MSTP Program The Ohio State University

Publications

Trotta R, Chen L, Costinean S, Josyula S, Mundy-Bosse BL, Ciarlariello D, Mao C, Briercheck EL, McConnell KK, Mishra A, Yu L, Croce CM, Caligiuri MA. Blood. 2013 Feb 19 Overexpression of miR-155 cause expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells. Blood. 2013 Feb 19 [Epub ahead of print] McClory S, Hughes T, Freud AG, Briercheck EL, Martin C, Trimboli AJ, Yu J, Zhang X, Leone G, Nuovo G, Caligiuri MA. Evidence for a stepwise program of extrathymic T cell development within the human tonsil. J Clin Invest. 2012 Apr 2;122(4):1403-15.

Trotta R, Ciarlariello D, Dal Col J, Mao H, Chen L, Briercheck EL, Yu J, Zhang J, Perrotti D, Caligiuri MA. The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells. Blood. 2011 Feb 24;117(8):2378-84. doi: 10.1182/blood-2010- 05-285130. Epub 2010 Dec 14.

Hughes T, Becknell B, McClory S, Briercheck EL, Yu J, Mao C, Giovenzana C, Nuovo G, Wei L, Zhang X, Gavrilin M, Wewers M, Caligiuri MA. IL-1β selectively expands and sustains IL-22(+) immature natural killer cells in secondary lymphoid tissue. Immunity. 2010 Jun 25;32(6):803-14

Briercheck EL, Freud A, Caligiuri MA. Natural Killer Cells: Basic Science and Clinical Application Nov 2009 Human Natural Killer cell development, Elsevier.

Hughes T, Becknell B, McClory S, Briercheck EL, Freud AG, Zhang X, Mao C, Nuovo GJ, Yu J, Caligiuri MA. Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the Th17 cytokine interleukin-22. Blood 113(17): 4008-10, 2009. vi

Chang JS, Santhanam R, Trotta R, Neviani P, Eiring AM, Briercheck EL, Rochetti M, Roy DC, Calabretta B, Caligiuri MA. High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2 dependent suppression of C/EBPalpha-driven myeloid differentiation. Blood 110(2):994-1003, 2007.

Fields of Study

Major Field: Integrated Biomedical Science Program

vii

Table of Contents Abstract ...... ii

Dedication ...... iii

Acknowledgments...... iv

Vita ...... vi

List of Figures ...... xi

CHAPTER 1: BACKGROUND ...... 1

1.1 NK Cell Biology...... 1

1.1a The role of NK cells in immunity ...... 1

2 1.1b NK cell development...... 2

4 1.1c NK signaling through activating and inhibitory receptors ...... 4

9 1.1d Phosphoinositide-3 kinase pathway in NK cell function ...... 6

11 1.1e Mechanism of the NK cell immunological synapse ...... 8

1.2 Phosphatase and tensin homologue deleted on chromosome ten (PTEN) ...... 12

1.2a PTEN as a tumor suppressor ...... 12

1.2b The regulation of PTEN ...... 14

1.2c PTEN in immunity ...... 15

viii

1.3 Summary and Significance ...... 16

CHAPTER 2. PHOSPHATASE AND TENSIN HOMOLOGUE DELETED ON

CHROMOSOME TEN (PTEN): A CRITICAL BALANCE OF EXPRESSION IS

REQUIRED FOR MAXIMUM NATURAL KILLER CELL CYTOLYSIS OF TUMOR

CELLS ...... 17

2.1 Introduction ...... 17

2.2a Results ...... 19

2.3 Discussion and Summary ...... 26

2.4 Experimental Procedures ...... 32

2.5 Figures and Tables ...... 40

3. Future studies and extended discussion ...... 56

3.1 Introduction ...... 56

3.1a ...... 56

3.2b ...... 57

3.2 Results ...... 58

3.2 Discussion and Summary ...... 61

3.2a ...... 61

3.2b ...... 63

3.4 Experimental Procedures...... 64

3.6 Concluding Remarks ...... 70

Bibliography ...... 71

List of Figures

Figure 1. PTEN is differentially expressed in human CD56bright vs. CD56dim NK cells

...... 41

Figure 2 PTEN overexpression negatively regulates NK cell cytolytic activity...... 43

Figure 3 PTEN overexpression negatively regulates NK cell Interferon-ϒ (IFN- ϒ) production and secretion...... 45

Figure 4 PTEN overexpression does not affect receptor expression or cell NK development...... 47

Figure 5 PTEN overexpression negatively regulates the activation of the AKT and

MAPK pathways...... 49

Figure 6 PTEN loss has a dose dependent effect on NK cell cytolytic activity...... 51

Figure 7 Loss of PTEN in NK-92 cells leads to a deficiency in target cell adhesion ...... 53

Figure 8 Loss of PTEN leads to a disruption of polarization and cytolytic granule coalescence...... 55

Figure 9 PTEN mRNA among NK cell developmental subsets in human tonsil, peripheral blood and miR-26b-3P expression in CD56bright and CD56dim NK cells...... 67

Figure 10 PKCθ expression and Ca 2+ flux in stimulated shPTEN-NK-92 cells and Over-

PTEN-NK-92 cells...... 69

CHAPTER 1: BACKGROUND 1.1 NK Cell Biology

1.1a The role of NK cells in immunity

1 The immune system is comprised of diverse populations of cells that work in concert

to mediate the body’s response to invading or infectious pathogens, eliminate

damaged cells, and protect against malignant transformation. The immune system is

broadly divided into two arms termed innate and adaptive immunity. Cells of innate

immunity are characterized by their ability to rapidly respond to and protect the host

from invading pathogens without the need for prior antigen priming. Conversely, the

adaptive immune system requires priming by a specific antigen and is comprised of

B and T lymphocytes. In 1975 a third lymphocyte was identified that had

morphology similar to B and T cells, but responded with cytolytic activity to

leukemic target cells without prior to antigen stimulation [1, 2]. This immediate

response led these cells to be identified as Natural Killer (NK) cells. NK cells have

since been shown to be critical components of the body’s immune defense against

viral infection as well as malignant transformation [3, 4]. More recent evidence

indicates that developing NK cells may also participate in immune defense against

bacterial pathogens through the secretion of IL-22 [5, 6]. Additionally, the NK cell

response can be made specific through engagement of the FcϒRIIIA (CD16) cell

surface receptor binding to the Fc portion of target bound antibodies [7]. Further

blurring the label of innate or adaptive to NK cells is their clonal expansion during

infection and the generation of long lived “memory cells” [8, 9]. These

characteristics have made NK cells an alluring possibility for immune based cancer

therapeutics including cytokine mediated activation, monoclonal antibodies, and

adoptive transfer of autologous and allogeneic NK cells [10]. Maximizing NK cell

based therapy requires an understanding of the development and regulation of NK

cell functions.

2 1.1b NK cell development

3 While human NK cells have been broadly defined as CD3-CD56+CD16+/- and mouse

NK cells as CD3-NKp46+NK1.1+/-DX5+/-, there is great diversity in the phenotypic

and functional developmental intermediates leading to a mature NK cell. NK cells

like their lymphocyte counterparts B and T cells are initially derived from a common

lymphoid progenitor (CLP) [11, 12]. NK cells may then transition through six

additional stages of maturation during their lifetime: 1) pre-NK precursor (pre-NKP),

NK precursors (NKP), immature NK (iNK), mature NK1 (mNK1), mature NK2

(mNK2), and memory NK (ref JHY/Ronnie rvw). Lymphocytes in the pre-NKP stage

retain the ability become T or NK cells, but the transition to the NKP stage coincides

with the acquisition of the IL-15 receptor (IL-15R) beta chain (CD122) [13]. The

importance of IL-15 for committed NK development is further highlighted by mice

which are deficient in IL-15 exhibiting defects in NK cell development, while IL-15

transgenic mice have increased NK cell numbers and can develop NK cell or NKT

2 cell leukemias [14-17]. Until recently the iNK cell was only a phenotypically distinct developmental intermediate before reaching the mNK1 stage. However, recent evidence indicates that these cells or at least subpopulations of iNK cells have distinct function. Our lab showed that human iNK cells from secondary lymphoid tissue (SLT) uniquely produce IL-22, and further that this stage of NK cells can be maintained by culturing the cells in vitro with IL-1β [6, 18]. Other labs have identified IL-22-producing cells in mouse that show some striking similarities to the phenotype of mouse and human iNK cells, however some discrepancies between these populations exist and more work will be necessary to determine if these cells represent true NK cell developmental intermediates whose differences are influenced by a dynamic microenvironment or if they are separate lineages from a heterogeneous population [5, 19-21]. The mNK1 population is the first to exhibit classical NK cell functions including IFNϒ secretion and perforin mediated cytolytic activity. This stage also highlights an important difference in functional development between mouse and human NK cells. In mouse mNK1 cells are most easily defined by expressing both CD27 and CD11b, the latter at a lower density than in mNK2 cells. Importantly, these double positive cells exhibit the highest level of both IFNϒ secretion and cytolytic activity [22]. CD27 is also expressed on the human NK cells.

However, in humans CD27+ cells have higher cytokine secretion, but lower cytolytic activity [23]. This finding correlates with the previously characterized roles of human

CD56bright and CD56dim NK cells. Human CD56bright NK cells secrete high levels of cytokine by monokine stimulation, but demonstrate relatively weak cytolytic activity

compared to CD56dim NK cells [24]. It is likely that CD56bright, mNK1 will

eventually become CD56dim, mNK2 in humans, as this transition from a CD56bright to

CD56dimNK cell, but not the reverse, has been shown to occur both in vitro and in

vivo[25, 26]. CD56bright NK cells also display longer telomeres than CD56dim NK

cells, indicating a less mature cell [27]. In addition, CD94 expression can further

dissect the CD56dim NK cell population, with the CD94highCD56dim NK cell showing

intermediate functional characteristics between the least mature CD94highCD56dim

and the most mature CD94lowCD56dim subsets, indicating that the CD94highCD56dim

population represents a transitional stage of NK cell development [28].

Accompanying the decreased cytokine production and increased cytolytic activity in

the transition from human mNK1 to mNK2 is the acquisition of FcϒRIIIA (CD16),

which binds the Fc portion of antibodies and mediates antibody-dependent cellular

cytotoxicity (ADCC) [29]. The relative abundance of mNK2 cells in peripheral blood

and their ability to immediately respond to antibody tagged tumor cells allow NK

cells to be important players in monoclonal antibody based cancer therapeutics.

Further, combining these therapies in conjunction with cytokines and/or adoptive

NK-cell immunotherapy is a logical next step [30]. In order to maximize these

treatment strategies it will be important to clearly define the complex signaling

pathways regulating NK cell activation.

4 1.1c NK signaling through activating and inhibitory receptors

5 Activation receptor signaling

6 Elucidating NK cell signaling is difficult due to both the redundancy of signaling

proteins and the lack of dominant activation and inhibitory receptors. Nowhere is this

more evident than the Src-family kinases. Loss of individual members of this family

including Lck, Fyn, Src, Lyn, Yes, and Fgr results in normal NK cell function, yet

global inhibition of this family blocks NK cell activation [31]. Activating natural

cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46 signal through induction

of Src-family kinase-mediated tyrosine phosphorylation of the immunoreceptor-

based activation motif (ITAM), which induces binding of tyrosine kinases Syk and

Zap70 leading to activation of signaling complexes including phosphatidyl-inositol-

3-OH (PI3K), phospholipase C (PLC-ϒ1, PLC-ϒ2), and Vav-1, 2, 3. Conversely,

NKG2D signals through DAP10, with a tyrosine-based signaling motif that (YINM),

that will then require phosphorylation by the Src-family kinases to induce the

formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3 ) and downstream

NK cell activation pathways. The slam-related receptor (SRR) family member 2B4

similarly requires the Src-family kinases to recruit SLAM-associated protein (SAP)

and induce NK cell activation [32]. Finally NK cells are able to mediate ADCC

through the binding of the Fc portion of the antibody to the FCRϒRIIIa on NK cells,

which also signals through ITAM phosphorylation by Src-family kinases. In addition

to the Src-family kinases downstream molecules PLC-ϒ and Vav are essential for

NK cell killing [31]. PLC-ϒ2 is critical to calcium mobilization and granule release

[33, 34]. NKG2D-DAP10 signaling requires Vav1, while ITAM-coupled receptors

require Vav2 and Vav3 [35].

7 Inhibitory receptor signaling

8 The key to the NK cell’s ability to survey the body and induce an immediate

response, while maintaining tolerance to self relies on the expression of cell surface

inhibitory receptors that bind to MHC class I molecules [36]. MHC class I molecules

are expressed on normal healthy cells, but are often downregulated in the context of

viral infection and malignant transformation. Therefore NK cell surface receptors

that can bind to MHC Class I molecules allow the NK cell to recognize a healthy cell

and prevent activation [37]. These receptors are the Killer cell Ig-like receptors (KIR)

in humans and the Ly49 family in mice, which signal through immunoreceptor

tyrosine-based inhibition motif (ITIM). Upon ligand binding ITIMs will recruit the

tyrosine phosphatases SHP-1 and SHP-2, which leads to dephosphorylation of

VAV1, thereby inhibiting several possible pathways of NK cell activation [38]. In

addition to Ly49 and KIR, mouse and human NK cells express inhibitory receptors

of the C-type lectin-like family consisting of NKG2A or NKG2C complexed to

CD94. This inhibitory receptor complex binds the non-classical MHC class I

molecule Qa-1 and HLA-E in mouse and human respectively [39, 40]. Both KIR and

CD94-NKG2A leads to phosphorylation of Crk, which results in disassembly of the

Crk, c-CBl, and C3G complex that are involved in actin remodeling and LFA-1

mediated adhesion, which are necessary for cytolytic activity [41].

9 1.1d Phosphoinositide-3 kinase pathway in NK cell function

10 The PI3K pathway has been well studied for its role in both normal and pathologic

cellular activity. Activation of the PI3K pathway results in the generation of

6 phosphatidylinositol (3,4,5)-tripsphosphate (PI(3,4,5)P3 ) at the plasma membrane.

PI(3,4,5)P3 acts as a docking site for plasma membrane proteins with pleckstring homology (PH) domains including Akt/PKB, PDK1, BTK, VAV, and PLCϒ.

Pharmacologic inhibition of the PI3K pathway results in loss of the NK cell’s ability to polarize the microtubule organizing center (MTOC) and cytolytic granules towards the target cell by blocking the MAPK pathway [42, 43]. Genetic approaches to studying the PI3K pathway in NK cells have shown both redundancy and specificity for PI3K isoforms. Loss of p110δ leads to decreased IFN-, TNF-alpha, and GM-CSF secretion, while p110γ loss has no effect [44]. Cytolytic activity is not disrupted by either p110δ or p110γ loss individually, but loss of both results in significantly impaired lysis of target cells [44]. Additionally, loss of both p110δ and p110γ inhibited NK cell development. Physiologic inhibition of this pathway occurs in part through the dephophosphorylation of PI(3,4,5)P3 by lipid phosphatases including SH2-containing inositol phosphatase 1/2 (SHIP1/2) and the tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN).

The role of PTEN in NK cells has yet to be explored and will be a focus of this work discussed in detail below. SHIP-2 is ubiquitously expressed, while SHIP-1 is restricted to cells of the hematopoietic lineage. The importance of PI3K signaling for

NK cell activation, and SHIP-1’s inhibition of PI(3,4,5)P3 formation suggests SHIP-

1 would function as a negative regulator of NK cell activation. Indeed, in human NK cells SHIP-1 was shown to colocalize with CD16 and through localization to CD16 negatively regulated ADCC and IFN-γ production [45, 46]. Additionally, our lab

showed that SHIP-1 protein is expressed at lower levels in the CD56brightNK cell

subset, that produces high levels of IFN-γ and that overexpression of SHIP-1

negatively regulated IL-12, IL-15, and/or IL-18 induced IFN-γ secretion. Similarly,

miR-155 and adapter SAP have been shown to activate NK cell function by

inhibiting SHIP-1 activity [47-49]. Mouse knockout models of SHIP-1 have been

less clear. SHIP-/- mice showed increased numbers of NK cells with an altered

inhibitory receptor profile, that led to a decrease in NK cell cytolytic activity and

cytokine production in response to allogeneic transplant [50-52]. Further

complicating the role of SHIP-1 was the finding that differences in haplotype can

alter the result in SHIP-1-/- NK cells. While SHIP-1-/- H2b haplotypes show the

aforementioned deficiency in cytolytic activity, H2d haplotypes show an elevation

that may be due to the elevation in expression of Ly49A, a key licensing receptor for

H2d NK cells [53]. Finally, SHIP-1-/- stimulated with IL-12 showed an elevation in

ADCC and IFN-γ [46]. These findings suggest that in most physiologic contexts

SHIP-1 acts a negative regulator of NK cell activity, but also has an important role in

murine NK cell development.

11 1.1e Mechanism of the NK cell immunological synapse

12 Of critical importance to unlocking the therapeutic potential of NK cells is

understanding how the complex molecular machinery integrates at the moment an

NK cell encounters a potential target cell. The concept of NK activation being

regulated by a delicate balance of signaling between activating and inhibitory

receptors was introduced previously and becomes even more dynamic when one

8 imagines that NK mediated killing relies not only on activation, but rather a focused site of activation occurring in 3-dimensions throughout the NK cell. Understanding this process has led to investigations regarding the nature of the NK cell immunologic synapse, which has been defined as “the intentional arrangement of molecules in an immune cell at the interface with another cell,” and includes diverse cellular molecules including receptors, signaling molecules, cytoskeletal elements and cellular organelles [54]. The NK cell’s initial contact and transition to firm adhesion followed by signaling is a gradual process and setting a defined line regarding these transitions is difficult. L-selectin and L-selectin ligand models have suggested a role for selectins in the initial NK cell recruitment to tumor infiltrates in lymph nodes, though whether this occurs by direct binding of NK cells to tumor or cells in the microenvironment was not clear [55]. CD2 binding to sialyl-Lewis X is also likely important in the initiation of binding as CD2 on the NK cell surface accumulates at the immune synapse and activation of CD2 leads to aggregation of

NK activating receptors and downstream activation pathway signaling [54, 56]. The next step, firm adhesion is mediated at least in part by binding of integrin molecules including lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) and

MAC1 (CD11b/CD18), which upon binding can themselves initiate NK cell activation [54, 57-60]. Throughout this process the NK cell is also interacting with potential inhibitors of synapse progression mediated through inhibitory receptor signaling. Once NK cells have progressed to the effector stage reorganization of the immune synapse begins. Early studies of the immune synapse recognized the

9 polarization of F-Actin at the immune synapse, and subsequent studies have shown this event to be dependent on VAV1 activity and Wiskott-Aldrich syndrome protein

(WASP). While many molecules accumulate at the synapse, and have been shown to be important for NK cell function, their ability to aggregate and ultimately produce a mature NK cell immune synapse is dependent on the accumulation of F-Actin [54,

61, 62]. Upon the accumulation of activation signals within the immune synapse, the

NK cell must mobilize its lytic granules towards the target cell. The lytic granules will move along microtubules and accumulate at the microtubule organizing center

(MTOC), which itself polarizes towards the active immune synapses, thereby delivering the lytic granules in a directed manner. The mechanisms underlying lytic granule movement to the MTOC are incompletely understood, but the subsequent movement of the MTOC to the immune synapse in NK cells requires ERK, VAV1 and PYK2 activation [63-65]. Once the MTOC has delivered lytic granules to the immune synapse a small conduit is cleared in the accumulated F-Actin likely to allow centralized movement of the lytic granules, which is assisted at least in part by the myosin-II motor protein [58, 62, 66, 67]. The final steps necessary for the release of lytic granule contents have yet to be fully elucidated in NK cells, but are known to include a final priming step before lytic granule fusion to the inner NK cell membrane. MUNC13-4, syntaxin-11, and MUNC18-2 deficiency in patients with familial hemophagocytic lymphohistiocytosis (FHL) leads to deficiencies in NK cell degranulation and has given clues to the important players in this final step [68].

MUNC13-4 bound to Rab27a associates with the lytic granule, and can bind SNARE

(soluble N-ethylmaleimide-sensitive-factor accessory proteins receptor) family proteins which act to help fuse the vesicular membrane (v-snares) and target membrane (t-snares) together. Among the SNARE proteins syntaxin-11, and VAMP7

(vesicle-associated membrane protein 7) have been shown to be required for NK cell lytic function [69-71]. Finally, at the termination of the NK synapse NK cells detach from their target. This process is not fully understood, but the downregulation and degradation of NK cell activating receptors such as NKG2D, 2B4, and NKp46 may be one component allowing the NK cell to release [72-74]. It is additionally possible that other “active” detachment pathways have yet to be shown in NK cells or the eventual integration of components from the inhibitory complex, that may have initially been overridden by stronger, but shorter acting activation signals, ultimately mediate detachment. An additional question regarding how the cell mediates activity at the immune synapse is determining what signals are being processed in the remaining parts of the cell. Is creating an “inactive” portion of the NK cell also an active process and if so what molecules are responsible for these pathways?

Elucidating this mechanism will be important to further our understanding of NK cell activation.

1.2 Phosphatase and tensin homologue deleted on chromosome ten

(PTEN)

1.2a PTEN as a tumor suppressor

The observation that chromosome 10q23 was frequently deleted in human cancer led to the discovery of a new phosphatase, termed phosphatase and tensin homologue deleted on chromosome ten (PTEN) [75, 76]. Subsequently it was demonstrated that PTEN acts as a lipid phosphatase, which regulates PI(3,4,5)P3 levels [77]. Since these seminal discoveries the importance PTEN has been demonstrated by the presence of PTEN mutations in a wide variety of cancers including 38% of endometrial, 20% of central nervous system (CNS), 17% of skin, and 14% of prostate cancers[78]. Additionally, inherited mutations of PTEN encompass a clinical spectrum of disorders referred to as

PTEN hamartoma tumor syndromes, including Cowden syndrome (CS), Bannayan-Riley-

Ruvalcalba syndrome, Proteus syndrome and Proteus-like syndrome. CS patients present with multiple hamartomas and a highly elevated risk of breast, thyroid, and endometrial carcinomas due to germline haploinsufficiency [79]. Interestingly, haploinsufficiency may at times be enough to drive malignant transformation, and in some contexts may even provide a growth advantage over homozygous deletions. In a mouse model of prostate cancer, step-wise decreases in Pten expression led to increases in tumor severity

[80]. Intriguingly, this same group was able to show that complete loss of PTEN can induce a p53 mediated senescence pathway, termed PTEN induced cellular senescence

(PICS) whereas a heterozygous loss of PTEN does not. This senescence will then continue until concomitant loss of p53 [81]. This infers that in some tumor environments 12 partial PTEN loss may offer a growth advantage, and explains why at presentation prostate malignancies do not select for complete loss of PTEN [81]. In addition to increased proliferation upon PTEN loss, PTEN has shown a role in regulating chemotaxis in some cell types and therefore may have function in regulating tumor metastases through both PI3K/AKT dependent and independent mechanisms [82-86]. In addition,

PTEN loss in the tumor microenvironment, can have dramatic consequences. When

PTEN was deleted in mammary fibroblasts, epithelial tumors were larger and showed increased metastases [87]. Further, the loss of PTEN within the mammary stromal fibroblasts initiates a secretome that reprograms other cells in the tumor microenvironment to down regulate PTEN in a Pten-miR-320-Ets2 pathway [88]. While many of the consequences of PTEN loss both within the tumor and the tumor microenvironment have focused on its cytoplasmic role, emerging evidence indicates a role for PTEN within the nucleus. Nuclear PTEN positively regulates RAD51, which is a key protein in double-strand break (DSB) repair and this occurs independently of PTEN’s lipid phosphatase activity, though negative regulation of cell cycle regulator checkpoint kinase 1 (CHEK1) which also promotes accumulation of double strand breaks (DSBs) is mediated through activated AKT [89, 90]. Nuclear PTEN also has been shown to inhibit oncoproteins polo-like kinase (PLK1) and Aurora kinases (AURKS) through a phosphatase independent enhancement of APC/C binding to its activator CDH1. Within the cytoplasm and the nucleus the role of PTEN’s protein phosphatase activity is yet to be fully understood. Protein substrates including focal adhesion kinase (FAK), cAMP responsive-element-binding protein (CREB), and nonreceptor tyrosine kinase c-SRC

(SRC) suggests that this is in an important area for understanding the oncogenic consequences and potential therapeutic approaches to PTEN loss.

1.2b The regulation of PTEN

PTEN is highly regulated with mechanisms described at the genetic, epigenetic, transcriptional, translational, and post-translational levels. This diversity in regulation mechanisms provides a repertoire of options for the cell to alter PTEN activity in both the physiologic and pathologic setting. Epigenetic silencing of PTEN has been observed in melanoma, and splice variants of PTEN have been observed in sporadic breast cancer

[91, 92]. Transcription factors mediating PTEN expression include p53, PPARΥ, and early growth response protein 1 (EGR1), while several including SNAIL, oncogenic factor inhibitor of DNA-binding 1 (ID1) (both of which compete with the p53 binding site), ecotropic virus integration site 1 protein (EV1), BMI1, and c-JUN negatively regulate PTEN transcription [93]. Post-transcriptionally several microRNAs can downregulate PTEN including the miRs-19, -21, -22, -26a, -29b, -106b~25, -216a, -217, -

221, -222, -301, -486, and -494 [93]. In addition to standard miR regulation PTEN was shown to be regulated both in vitro and in the disease setting by competing endogenous

RNA (ceRNA) through PTEN’s pseudogene PTENP1 [94]. Post-translational phosphorylation at Ser370, Thr382, Thr383 or Ser385 stabilizes PTEN, but leads to a closed conformation, thereby inhibiting PTEN’s lipid phosphatase activity [95].

Conversely, in leukocytes phosphorylation at Ser229 and Thr321 activates and induces

PTEN to translocate to the membrane [96]. Acetylation inhibits PTEN’s catalytic activity while excluding PTEN from nucleus. Conversely, deacetylation by sirtuin 1(SIRT1) 14 allows for PTEN nuclear localization [97, 98]. Monoubiquitination of PTEN is important for nuclear localization, while polyubiqutination leads to PTEN degradation [99-101].

Finally PTEN activity is regulated by various protein-protein interactions. Importantly, proteins which reduce PTEN’s lipid phosphatase activity PREX2A (breast), SIPL1

(cervical), and MAN2C1 (prostate) are highly expressed in various cancers [93].

1.2c PTEN in immunity

Along with its well studied function as a tumor suppressor, an emerging role for PTEN in governing immune homeostasis has been shown, with much of this work being done in T cells. Initial studies showed that murine Pten haploinsufficiency led to a lethal autoimmunity and impaired activation induced apoptosis of T and B lymphocytes [102].

Mice with a complete T cell specific loss of Pten showed lymphadenopathy, splenomegaly, enlarged thymi and premature death due to CD4+ T cell lymphomas. In agreement with the haploinsufficient model, Pten-/- T cells showed impaired negative selection, elevations in cell-mediated autoreactivity and increases in serum levels of anti- ssDNA antibody [103]. In contrast to these findings, OX40Cre inducible deletion of Pten in CD4+ T cells does not result in lymphoma or autoimmunity, but instead enhanced response to listeria and tumor clearance [104]. This difference is significant when considering the potential of targeting PTEN in immunotherapy. Targeting PTEN in mature T cells may lead to the enhanced effector function desired, without the dire consequences observed in early thymocyte deletions. Regulatory T cells (Treg) showed no difference in development and tolerance in the absence of Pten, though constitutively active AKT does inhibit the development of Treg cells [105-107]. Mature Treg cells are 15 able to proliferate in response to IL-2 in the absence of Pten, while maintaining their suppressive function. Interestingly, murine Vα14iNKT cells depleted of Pten show impaired development and deficiency in tumor immunity [108]. Like T cells, macrophages show diverse and context dependent responses to PTEN modulation. PTEN-

/- macrophages display enhanced FcΥR signaling, but in the same study showed reduced

TLR4 response. Similarly, PTEN mediates prostaglandin E2 (PGE2) inhibition of alveolar macrophage FcΥR phagocytosis, while loss of PTEN decreases macrophage clearance of Leishmania major [109-111].

1.3 Summary and Significance

NK cell activation is a dynamic and balanced integration of signals, which allow the immune system to immediately respond to virally infected and malignantly transformed cells. The importance of the lipid phosphatase PTEN as a tumor suppressor has been clearly demonstrated, and further understanding its emerging role in immune cells will be an important consideration in the development of immune based therapies. Based on the importance of PTEN as a tumor suppressor, and previous work showing molecules similar to PTEN can regulate NK cell function, we sought to investigate if the important tumor suppressor gene PTEN plays a role in one our immune system’s most important tumor suppressing cells, the NK cell.

CHAPTER 2. PHOSPHATASE AND TENSIN HOMOLOGUE DELETED

ON CHROMOSOME TEN (PTEN): A CRITICAL BALANCE OF

EXPRESSION IS REQUIRED FOR MAXIMUM NATURAL KILLER CELL

CYTOLYSIS OF TUMOR CELLS

2.1 Introduction

In human peripheral blood NK cells can be divided into two developmentally and functionally distinct subsets based upon cell surface expression of CD56. In contrast to the more mature CD56dim NK cell the less mature CD56bright NK cell is unable to kill malignant cells at rest. Conversely, the CD56bright NK cell secretes cytokines including

IFN-γ with significantly higher efficiency compared to the CD56dim NK cell. The molecular mechanisms underlying these differences remain incompletely defined.

In order for NK cells to carry out an effective, yet controlled response, NK cell activation is mediated by a dynamic balance of signaling through activating and inhibitory receptors. Previous reports have demonstrated the importance of the PI3K/AKT and

MAPK pathways for regulating NK cell cytolytic activity and IFN-γ secretion [42, 43,

112, 113]. Our lab and others have shown that in human NK cells the 3’-lipid phosphatase SHIP-1 is a negative regulator of PI3K/AKT and MAPK, and that this event correlates with decreased NK cell function including IFN-γ production, natural cytotoxicity, and antibody-dependent cell-mediated cytotoxicity [45, 46, 114].

Conversely, SHIP-1-/- mice have shown decreases in NK cell function [51, 115, 116].

Despite these differences the ability of changes in SHIP-1 expression to alter NK cell activity is clear, and we sought to determine if other lipid phosphatases might also be involved in the regulation of NK cell activity and thus account for the functional differences between CD56bright and CD56dim NK cells.

The importance of PTEN as a tumor suppressor was described in detail in chapter 1.

Briefly, the importance of PTEN has been demonstrated by the presence of PTEN mutations and/or deletions in a wide variety of cancers including, 38% of endometrial,

20% of central nervous system (CNS), 17% of skin, and 14% of prostate cancers [78].

Additionally, inherited mutations of PTEN encompass a clinical spectrum of disorders referred to as PTEN hamartoma tumor syndromes, including Cowden syndrome (CS),

Bannayan-Riley-Ruvalcalba syndrome, Proteus syndrome and Proteus-like syndrome

[79]. A major mechanism of PTEN mediated tumor suppression has been attributed to its function as a lipid phosphatase inhibitor of the PI3K/AKT pathway, however PTEN has proven to be a complex regulator of cellular homeostasis with both lipid phosphatase dependent and independent roles in proliferation, senescence, motility, and chromosomal stability [81-90]. PTEN has also emerged as an important regulator of immune function.

Loss of Pten in T cells can lead to variable consequences ranging from a lethal

18 autoimmunity to enhancement or deficiency tumor immunity depending upon the developmental timing and the subset specificity of Pten deletion [103-108]. Despite these findings a role for PTEN in natural killer cells has yet to be investigated.

Therefore, we initially investigated PTEN’s protein expression level in functional, mature human NK cells. We show that PTEN is more highly expressed in CD56bright NK cells than CD56dimNK cells. In both human and murine NK cells high expression levels of

PTEN can negatively regulate IFN-γ production and cytolytic activity through inhibition of the PI3K/AKT and MAPK pathways. Conversely, complete loss of PTEN also leads to a decrease in cytolytic activity, despite elevated levels of PI3K/AKT and MAPK.

Alternatively, we demonstrate that in the absence of PTEN human NK cells show decreased adhesion to target cells, decreased coalescence of granules to the MTOC, and decreased polarization of the MTOC towards the immune synapse.

2.2a Results

PTEN is differentially expressed between human CD56brightNK and CD56dimNK cells

In contrast to the more mature CD56dim NK cell, the CD56bright NK cell is unable to kill malignant targets at rest. We sought to determine the mechanism behind this profoundly different cytolytic activity by exploring changes in gene expression. We isolated

CD56bright and CD56dim NK cells by fluorescence activated cell sorting (FACS) and assessed for PTEN protein expression. Western blots showed that expression of PTEN protein is increased 5 fold in CD56brightNK cells compared to CD56dimNK cells (Figure

1A and 1B average increase 5.29; p<.04, n=4). In order to confirm these differences we 19 fixed enriched NK cells on poly-L-lysine coated slides and stained for expression of both

CD56 and PTEN protein. In agreement with our western blot data NK cells which were brightest for CD56 expression also showed the highest levels of PTEN protein expression

(Figure 1C and 1D; Average 2.2 fold increase in mean fluorescent intensity x area, p<.0001).

Effect of PTEN overexpression on NK-cell lytic function

In order determine whether PTEN regulates NK cell cytolytic activity we isolated

NKp46+CD3- NK cells from the previously described Super-PTEN transgenic mice

[117]. We first confirmed by western blot that PTEN is indeed upregulated in the NK cells of Super-PTEN mice (Figure 2A) and then tested the ability of their NK cells to kill the prototypic murine NK cell tumor cell target YAC-1. Freshly isolated Super-PTEN

NK cells lysed YAC-1 tumor cells with lower efficiency compared to WT littermate controls (Figure 2B). Following 8 days of culture in IL-2 both Super-PTEN and WT NK cells showed enhanced spontaneous cytotoxic function. Despite this increased activation

Super-PTEN NK cells had a significantly decreased efficiency of killing (Figure 3C, p<.0001). In addition, following IL-2 activtion, Super-PTEN NK cells demonstrated an even greater suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) against P815 antibody-coated target cells when compared with WT NK cells (Figure 2C, p<.03).

To validate these findings in human NK cells we infected the human NK cell line NK-92 with a lentiviral vector encoding GFP (pCDH-EF1-MCS-T2A-copGFP, empty vector), or a lentiviral vector encoding GFP and PTEN (pCDH-EF1-MCS-T2A-copGFP-PTEN,

Over-PTEN) and confirmed PTEN protein overexpression by western blot (Figure 2E).

Similar to Super-PTEN murine NK cells, Over-PTEN NK-92 cells showed a significantly decreased efficiency in natural cytotoxicity against the NK sensitive leukemic cell line

K562 (Figure 2F, n=3, p<.02). In order to confirm these findings we infected primary human NK cells isolated from healthy donors with either empty vector or Over-PTEN lentiviral vectors. These cells were then FACS sorted for GFP expression, co-cultured with K562 target cells and CD56+GFP+ cells were assessed for CD107A (a marker of degranulation). Over-PTEN cells showed a significant reduction in the mean fluorescent intensity (M.F.I.) of CD107A+ cells correlating to decreased cytolytic activity (n=6, p=.003).

Effect of PTEN overexpression on NK-cell IFN-γ secretion

NK cell IFN-γ secretion is a critical component of the NK cell’s immunomodulatory role in the clearance of infectious pathogens and tumor surveillance [29]. Previous work in our lab, and others has shown that the lipid phosphatase SHIP-1 can negatively regulate

NK cell IFN-γ secretion [46, 114]. NK cell IFN-γ secretion is highly induced by synergistic stimulation with IL-12 and IL-18 [118]. Therefore, to determine whether

PTEN overexpression also regulates IFN-γ secretion we treated WT and Super-PTEN murine NK cells for 24 hours with IL-12 and IL-18 and assessed for IFN-γ secretion by enzyme-linked immunosorbent assay (ELISA) on cell free supernatants. Super-PTEN NK cells showed a decrease in IFN-γ secretion compared to WT littermate controls.

Similarly, Over-PTEN NK-92 cells showed a significant decrease in IFN-γ secretion

(n=5, p<.04). In order to determine if PTEN overexpression induced decrease in IFN-γ

21 secretion was due to decreased intracellular levels of IFN-γ or inhibition of IFN-γ release we assessed IFN-γ by intracellular flow cytometry. Both Super-PTEN NK cells and

Over-PTEN NK-92 showed decreased intracellular levels of IFN-γ compared to controls

(Figure 3A, n=3, p<.02, and 3C) indicating that decreased IFN-γ secretion is due to a decrease in IFN-γ production.

Effect of PTEN overexpression on NK cell development and receptor expression

In order to determine if the decreased efficiency of cytolytic activity and IFN-γ secretion of Super-PTEN NK cells were due to differences in NK cell development or receptor expression we analyzed the phenotype of the Super-PTEN NK cells and WT controls by flow cytometry. CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and

CD11bhighCD27low represent 4 sequential stages of terminal mouse NK cell maturation

[22, 119]. After gating on NKp46+CD3- NK cells we saw no significant change in any of the aforementioned populations, thus indicating that PTEN overexpression does not disrupt NK cell development (Figure 4A). In addition there were no significant differences in the expression of NK1.1, 2B4, DX5, Ly49H, Ly49D, Ly49A, CD117,

CD94, NKG2D, CD27, KLRG1, CD69, CD62L, NK2ACE, and CD16 (Figure 4B).

PTEN overexpression negatively regulates AKT and ERK

The PI-3K/AKT pathway has been previously shown to be important for NK cell mediated cytotoxicity and IFN-γ secretion [42, 112]. Further, PTEN has been demonstrated to regulate the PI-3K/AKT pathway; therefore we sought to determine if negative regulation of PI-3K/AKT might account for the decrease in cytolytic activity

22 and IFN-γ observed in PTEN overexpressing cells [120]. In order to test this we stimulated Super-PTEN and WT murine NK cells with fixed YAC-1 targets cells for 0, 1,

3 and 10 minutes and assessed their expression of P-AKT. Super-PTEN NK cells showed a marked reduction in P-AKT (Figure 5A). Similarly Over-PTEN NK-92 cells stimulated with K562 target cells also showed a marked reduction P-AKT. An additional downstream target of PI-3K is ERK, which is a known positive regulator of NK cell cytotoxicity (Figure 5B). Sustained P-ERK was significantly decreased in both Super-

PTEN NK cells and Over-PTEN NK-92 cells compared to respective controls (Figure 5A and 5B). In order to determine if the negative regulation of cytolytic activity by PTEN was due to activity at PTEN’s lipid phosphatase catalytic site we generated an NK-92 cell line that overexpressed PTEN with a glycine to glutamate substitution in position 129

(Over-PTEN-G129E), a mutation previously shown to disrupt PTEN’s lipid phosphatase activity (Figure 5C) [121]. This mutation led to a rescue of cytoxicity to levels near empty vector and recovery of P-AKT and P-ERK levels upon stimulation with fixed

K562 target cells (Figure 5D and 5E).

Effect of PTEN loss on NK cell cytolytic activity

Considering the suppressed NK cell function observed in the context of PTEN overexpression we next sought to determine if the absence of PTEN would lead to an increase in NK cell function. Murine studies have shown that homozygous loss of Pten is embryonic lethal at day 7.5 [122]. We therefore isolated NK cells from previously described Pten heterozygous mice (Pten+/-) [87]. Pten+/- NK cells show ~50% PTEN protein expression compared to WT littermate controls (Figure 6A). Freshly isolated

Pten+/- NK cells did not show a significant increase in cytolytic activity over WT littermate controls (data not shown). However, following 8-day cultures with IL-2 Pten+/-

NK cells lysed YAC-1 target cells with significantly higher efficiency than WT littermate controls (Figure 6B, n=4, p<.0001). Further, in agreement with our data in PTEN overexpression, Pten+/- NK cells show an elevation in P-AKT and P-ERK upon stimulation with fixed YAC-1 target cells (Figure 6C). In order test the effect of PTEN loss in human NK cells we infected NK-92 cells with a lentiviral vector carrying either cDNA for GFP alone (pSIH1-H1-copGFP, empty vector) or GFP plus a short hairpin

RNA (shRNA) targeting PTEN (pSIH1-H1-copGFP-shPTEN, shPTEN). FACS sorted shPTEN NK-92 cells showed nearly undetectable levels of PTEN protein by western blot compared to empty vector (Figure 6D). Intriguingly, shPTEN NK-92 cells lysed K562 target cells (Figure 6E, n=5, p<.003) with significantly lower efficiency compared to empty vector. Despite this deficiency in cytolytic activity shPTEN cells stimulated with fixed K562 target cells demonstrated highly elevated levels of P-AKT and P-ERK

(Figure 6F). This data suggested that the role of PTEN in NK cells is more complex than a linear negative regulation of cytolytic activity.

Complete loss of PTEN leads to a deficiency in NK cell-target cell adhesion

A critical initial step in NK cell mediated cytolytic activity is the binding of the NK cell to its target cell at the immunologic synapse [54]. We were interested in determining if

PTEN had any effect on the ability of the NK cell to form conjugates with target cells. shPTEN NK-92 cells and the corresponding empty vector control both express GFP. We took advantage of this by staining K562 leukemic target cells with the red fluorescent dye

PKH26. NK-92 cells and K562 leukemic target cells were then co-cultured at multiple time points and assessed by flow cytometry. GFP+PKH26Red+ events were counted as immune conjugates. shPTEN NK-92 cells exhibited a significantly decreased level of adhesion to target cells compared to empty vector control (Figure 7A and Figure 7B, n=3, p<.01). Importantly we saw no significant difference in adhesion of Over-PTEN cells compared to empty vector (Figure 7A top and Figure 7C, n=3, p=.94). Firm adhesion of

NK cells to their target cells is mediated through multiple receptors including the integrins lymphocyte function associated antigen-1 (LFA-1; CD11a-CD18) and macrophage receptor 1 (MAC1; CD11b-CD18). Therefore, in order to determine if alterations in adhesion molecule or activation receptor binding was the cause of decreased binding by shPTEN-NK-92 cells we assessed surface expression of these markers via flow cytometry. However, flow analyses revealed no difference in CD11a,

CD18, or CD11b between shPTEN NK-92 and empty vector control (Figure 7D). In addition no difference was observed in the activating receptors NKG2D or NKp44, while shPTEN-NK-92 showed elevated levels of NKp46, CD11c and NKp30 indicating expression of adhesion and activating receptors was not responsible for the decreased binding in shPTEN-NK92 cells (Figure 7D). Rather, shPTEN-NK92 cells appeared to be more activated than empty vector control. The NK cell must polarize in response to target binding, to kill and PI-3K is crucial in generating directional polarization of NK cells.

Evidence in other cell types which demonstrate the necessity of PTEN organized polarization led us to hypothesize that a complete absence of PTEN may be disrupting the coordinated steps necessary for NK cell polarization and cytolytic activity, which would

25 also result in the observed decrease in adhesion. To test this hypothesis we compared NK cell-target cell conjugates between shPTEN-NK-92 cells and NK-92 cells infected with empty vector. shPTEN-NK-92 cells exhibited diffuse F-actin, decreased polarization of cytolytic mediators, and decreased coalescence of lytic granules to the microtubule organizing center (MTOC) (Figure 8).

2.3 Discussion and Summary

NK cells are critical components of the innate immune system’s defense against malignantly transformed as well as virally infected cells. The NK cell’s ability to both directly kill target cells, as well as, produce immunomodulatory molecules such as IFN-γ is tightly regulated, yet incompletely understood. Deciphering the signaling pathways regulating NK cell functional activation and how the signaling mediators involved are modified within the immune microenvironment will allow for the discovery of new therapeutic targets. Lipid phosphatases are key regulators of NK cell activity against malignant targets [123]. However, the important tumor suppressor and lipid phosphatase

PTEN had yet to be investigated in NK cells. In this chapter we have provided evidence that PTEN is more highly expressed in CD56dim NK cells subset than in CD56bright NK cells and that maximum cytolytic activity of NK cells requires an optimal level of PTEN expression. Though, high PTEN expression inhibited cytolytic activity by blocking the

PI-3K/AKT and MAPK pathways (Figure 2 and Figure 5), we were intrigued to find that while a partial loss of Pten in murine NK cells led to an increase in cytolytic activity a complete knockdown of PTEN in NK-92 cells also led to decreased cytolytic activity despite elevated activation of the PI-3K/AKT and MAPK pathways (Figure 6). We then 26 showed that the complete loss of PTEN leads to a significant decrease in NK cell-target cell adhesion, which is unaffected by PTEN overexpression. Further, confocal microscopy revealed that shPTEN-NK-92 cells that were in conjugation with K562 target cells failed to polarize the microtubule organizing center (MTOC) and showed a significant decrease in granule coalescence to the MTOC, together indicating that complete PTEN loss leads to an activated yet disorganized phenotype (Figure 8).

Collectively, the data presented here are the first to describe a role for PTEN in the regulation of NK cell function and further that PTEN regulation of NK cell function exists at an optimized balance such that high levels of PTEN and complete loss of PTEN will both lead to decreases in cytolytic activity.

The higher levels of expression of the 3’-lipid phosphatase PTEN we observed in human

CD56bright NK cells as compared to CD56dim NK cells, differs from previous finding for the 5’ lipid phosphatase SHIP-1. SHIP-1 was shown to be a negative regulator of IFN-γ and the lower expression of SHIP-1 in CD56brightNK cells likely in part accounts for their relatively higher production of IFN-γ [114]. We found that overexpression of PTEN decreases both cytolytic activity and IFN-γ secretion (Figure 2 and Figure 3). In agreement with PTEN’s role as negative regulator of NK cell cytolytic activity NK cells from Pten+/- mice show an elevation in cytolytic activity compared to littermate controls, though we did not observe a significant difference in IFN- γ expression (data not shown).

CD56brightNK cells exhibit relatively weak cytolytic activity at rest and have high levels of PTEN expression (compared to the strongly cytolytic CD56dimNK cells that have lower PTEN expression) may, in part, provide a molecular explanation for the difference

27 in cytolytic function [29]. Developmentally, CD56brightNK cells have yet to express a large number of inhibitory killer-cell immunoglobulin-like receptors (KIR) and one possibility is that PTEN may provide a necessary obstacle to cytolytic activation prior to

KIR acquisition [124]. This may be especially important in the activating cytokine rich environment of secondary lymphoid tissue (SLT) in which 90% of NK cells are of the

CD56brightNK cell subset. Our finding that PTEN mediated suppression of the PI-

3K/AKT and MAPK pathways, directly correlated with cytolytic activity is in agreement with previous reports [42, 43, 112, 113]. Surprisingly, with complete loss of PTEN this correlation was lost indicating that an alternative mechanism for decreased killing efficiency must exist. We found that complete loss of PTEN leads to a significant decrease in the percentage of NK cells adhering to target cells, despite the fact that we concurrently observed normal or elevated levels of adhesion molecule expression. Upon encountering a target cell NK cells must initially adhere to the target cell and then undergo cytoskeletal changes that allow further binding of activating receptors followed by complex protein rearrangements that polarize the cell for a focused release of cytolytic mediators in the direction of the target cell [54]. Initial studies in Dictyostelium cells and subsequently in neutrophils, and neuronal cells, have shown that PI(3,4,5)P3 gradients are generated by both stimulation at the anterior of the cell and localization of PTEN at the posterior [125]. In addition to decreases in adhesion our data shows that NK cells depleted of PTEN, which do form conjugates have significantly decreased polarization of the MTOC as well as decreased granule coalescence to the MTOC (Figure 8).

The “distracted” phenotype of PTEN depleted NK cells is analogous to early studies of

PTEN in neutrophils which demonstrated that PTEN helps to “prioritize” responses to chemokines by regulating PI(3,4,5)P3 gradients within the cell [126]. Interestingly, another group demonstrated that SHIP-1, is critical for neutrophil chemotaxis upon integrin adhesion. This group described a model in which PTEN accumulates at the posterior end of the cell facilitating accumulation of PI(3,4,5)P3 at the anterior end, while SHIP1 works at the cell substratum interface to abolish integrin mediated

PI(3,4,5)P3 gradients thereby allowing the cell to disengage at its current position, while binding and activating PI(3,4,5)P3 at the leading edge [127]. It is possible that a similar interplay also exists between PTEN and SHIP-1 in NK cells and future studies should investigate concomitant knockdowns. Further our data’s suggestion that an optimum level of PTEN exists to mediate cytolytic activity may give clues to seemingly contradictory studies in SHIP-1. Our group and others have previously shown that SHIP-

1 can act as a negative regulator of natural cytotoxicity, ADCC, and IFN ϒ production

[45, 46, 114]. In contrast, groups using SHIP1-/- murine models have found that loss of

SHIP1 leads to decreased NK function by a variety of mechanisms including disruption of intracellular of inhibitory signaling mediated through abnormal association between

2B4 and SHP-1, inappropriate expression of Ly49B, and arrested development [51, 115,

116]. The former studies focused primarily on overexpression of SHIP-1, cytokine induced downmodulation, or inhibition at receptor specific sites (i.e. CD16, IL-12). The later, experiments while they describe a complete SHIP1 KO are all conducted in mice that demonstrate some developmental abnormalities making inferences about the role of

SHIP-1 in mature human NK cells speculative. However, as described in our studies of

PTEN, some functional deficits seen in the SHIP-1-/- mice may be due to disruptions in

NK cell polarity, an effect that may not be apparent upon overexpression, downmodulation, or signaling through a single receptor such as CD16 in ADCC. Indeed, overexpression of PTEN in human NK-92 cells and Super-PTEN murine NK cells led to a significant decrease in natural cytotoxicity, and an even greater loss of cytolytic activity in the context of ADCC. While the role of PTEN has yet to be explored in NK cells our findings would seem to contrast with a previously published study of PTEN in

Vα14iNKT cells, which showed both a heterozygous and complete loss of PTEN resulted in decreased IFN-γ production and decreased antitumor immunity. However, similar to investigations of complete SHIP-1 knockout both heterozygous and complete PTEN loss in Vα14iNKT cells result in arrested development, reduced proliferation, and elevated expression of the Ly49 inhibitory receptor, again making inferences about the role of

PTEN in mature Vα14iNKT cells difficult [108]. In contrast, Pten+/- murine NK cells showed no apparent changes in receptor expression and seemed to develop normally indicating that the elevated level of cytotoxicity was the result of Pten function in fully developed NK cells. Additionally, our approach of depleting PTEN in the human NK cell line NK-92 which exhibits characteristics of mature NK cells including natural cytotoxicity and IFN-γ secretion allowed us to investigate PTEN’s role after NK cell functional maturation. Considering the marked effect of SHIP1 knockout on murine NK cell development, and the embryonic lethality of homozygous deletion, it will be important in future studies to generate an NK specific Pten KO.

The role of PTEN in NK neoplasms is still unclear. An initial screen of anaplastic large cell lymphomas (ALCL) showed a 67% rate of complete PTEN deletion [128]. A more recent analysis of miR expression in NK-cell lymphomas showed that miR-21 and miR-

155 were up regulated compared to normal NK cells and that miR-21 and miR-155 targeted PTEN and SHIP-1 respectively. Recent evidence from our lab confirms miR-

155’s role as a negative regulator of SHIP-1 and that this offers a proliferative and survival advantage to NK cells, though NK neoplasms were not observed [49]. We did not observe a notable change in NK cell proliferation in our models of PTEN expression and to our knowledge NK neoplasms have not been reported in patients with PTEN hamartoma syndromes. Therefore it is likely that PTEN loss in NK cell neoplasms exists as either a secondary event to transformation or a primary event followed by additional undetermined mutations. The later seems likely considering PTEN’s recently described role in chromosomal stability [89, 90].

While, our data describing an optimum balance of PTEN expression infers that PTEN may be a difficult target to activate NK cells towards tumor cells, it may be the ideal target for factors secreted by immune evasive tumor cells. Recent evidence indicates that circulating exogenous miRs can modify NK cell function [129] and several miRs including miR-21, miR-26a, miR-29b, miR-214, miR-221, and miR-222 have all been shown to negatively regulate PTEN and therefore secretion of these miRs into the tumor microenvironment could disrupt optimum PTEN levels within NK cells. Perhaps most intriguing is recent evidence that PTEN can be secreted in exosomes, and that the exported PTEN retains its lipid phosphatase ability upon entering the recipient cell [130].

This raises the possibility that PTEN could traffic from the target cells, possibly at the immune synapse thereby immediately inhibiting NK cell activity. A similar mechanism could be potentially applied to other phosphatases including SHIP-1. While not at the protein level preliminary evidence has shown the presence of SHIP-1 mRNA within the exosomes of acute myeloid leukemia (AML) cell lines and primary AML patient samples

[131]. Whether at the mRNA or protein level, export of such proliferation inhibiting molecules may offer a duel immune evasion strategy where tumors can increase proliferation while inhibiting immune effector cells.

Collectively, to our knowledge we have demonstrated for the first time that the tumor suppressor PTEN can regulate normal NK cell activation. PTEN is expressed at higher levels in the human CD56brightNK cell subset compared to CD56dimNK cells, and at high expression levels negatively regulates NK cell cytolytic activity against tumor targets and the secretion of IFN-γ in response to monokine stimulation. Further, we show in the NK-

92 cell line that a near complete loss of PTEN disrupts cytolytic activity through loss of adhesion and organization of the NK cell immune synapse indicating that an optimum level of PTEN likely exists for maximum NK cell cytolytic activity.

2.4 Experimental Procedures

Cells lines and NK cell preparations. The human IL-2 dependent NK cell line NK-92

(gift of Dr. H. Klingemann, Rush Cancer Center, Chicago, IL) was maintained in culture in RPMI-1640 medium (Invitrogen, Carlsbad, CA), supplemented with 20% heat- inactivated FBS (Invitrogen), 2 mM L-glutamine and 150 IU/ml rhIL-2 (Hoffman-

LaRoche Inc., Nutley, NJ). The YAC-1 mouse T-lymphoma cell line, and human erythroleukemia cell line K562 were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS (Invitrogen), 2 mM L-glutamine , and the murine mastocytoma

P815 cells were maintained in DMEM medium, both with 10% FBS (Invitrogen) and 2 mM L-glutamine (Gibco).The amphotropic-packaging cell line 293 T was maintained in culture in DMEM (Invitrogen)/10% FBS medium and grown for 16-18 h to 80% confluence prior to transfection by calcium phosphate-DNA precipitation (Profection system, Promega, Madison, WI). Human NK cells were isolated from peripheral blood leukopacks of healthy individuals (American Red Cross, Columbus, OH) by incubation for 30 min with RosetteSep NK cell antibody cocktail (StemCell Technologies Inc.,

Vancouver, Canada), followed by Ficoll-Hypaque density gradient centrifugation. NK cell preparations containing >99% CD56+ NK cells were obtained by positive selection using CD56 MicroBeads and MACS Separation Columns from Miltenyi (Miltenyi

Biotech Inc, Auburn, CA), as determined by direct immunofluorescence using an anti-

CD56 phycoerythrin (PE)-conjugated monoclonal Ab (Beckman Coulter). All murine experiments were performed with purified NK cells via microbead selection (Miltenyi

Biotech Inc.) followed by FACS, selecting on NKp46+CD3- cells giving >99.5% purity.

Cultured murine NK were in RPMI-1640 (Invitrogen), supplemented with 20% heat- inactivated FBS (Invitrogen), 2 mM L-glutamine containing 900 IU/ml of human IL-2

(Hoffman-LaRoche Inc.) and 55M -mercaptoethanol (Gibco). All work with human materials was approved by the Institutional Review Board of The Ohio State University

Comprehensive Cancer Center.

Mice. Wild-type and Super-PTEN transgenic mice (C57BL/6J) [117] were a kind gift from the laboratory of Dr. Pierre-Paolo Pandolfi (Beth Israel Deaconess Hosptial, Boston,

MA) Wild-type and Pten+/∆4–5 (FVB/N) [132] were a kind gift from the laboratory of Dr.

Gustavo Leone (The Ohio State University, Columbus, OH). Animal work was approved by The Ohio State University Animal Care and Use Committee.

Lentivirus infection of NK-92 cell line and primary human NK cells. A short hairpin

RNA targeting PTEN (shPTEN) was cloned into a lentiviral vector PSIH-H1-copGFP

(System Biosciences), and cDNA encoding PTEN (Over-PTEN) (Origene), or PTEN-

G129E (Over-PTEN-G129E) (Addgene plasmid 30377) [121] was cloned into pCDH-

MCS-T2A-copGFP-MSCV (System biosciences). NK-92 cells and primary NK cells were infected following a protocol similar to previously published standards [133]

Briefly, infectious supernatant from Empty Vectors, shPTEN, Over-PTEN, and Over-

PTEN-G129E transfected 293 T cells were concentrated with Polethylene Glycol after 48 hours and used for one to three cycles of infections at multiplicity of infection of 10. All vectors contain the gene for green fluorescent protein (GFP). Upon infection, NK-92 cells and CD56+ NK cells were sorted for GFP expression on a FACSAria II (BD Biosciences,

San Jose, CA). GFP+CD56+ primary NK cells were used for experimentation immediately after sorting. Expression and knockdown of PTEN was confirmed by western blot.

Real-time RT-PCR. RNA was extracted using the Total RNA Purification Kit (Norgen).

Reverse transcription was performed with Taqman MicroRNA Reverse Transcription Kit and RT primers specific for PTEN or 18S as control (Applied Biosystems). Real-time

RT-PCR reactions were performed as described[48]. Data were analyzed with the comparative CT method using internal control 18S RNA levels to normalize differences in sample loading. Results (mean ± SEM of triplicate reaction wells) represent the n-fold difference of transcript levels in a particular tonsil sample for previously published NK cell developmental stages 1-4 [124] and CD56brightCD94high, CD56dimCD94high,

CD56dimCD94low NK cells from peripheral blood.

Western blot analysis. Cells were harvested, washed once with ice-cold PBS and lysed

(108 cells/ml RIPA buffer: 0.15 M NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 8.0), supplemented with protease and phosphatase inhibitors, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, 50 mM NaF, 10 mM -glycerol- phosphate, 1 mM EDTA and a protease inhibitor cocktail tablet from Roche Applied

Science, Indianapolis, IN, as described.[134] Alternatively, cells were directly lysed in

Laemmli buffer (2x105 cells /20 l). Western blotting was performed according to previously published protocols [135] and Ab-reactive proteins were detected with horseradish peroxidase-labeled sheep anti-rabbit, mouse and/or goat Ig sera and enhanced chemiluminescence (ECL; Amersham Corp., Arlington Heights, IL). Proteins were analyzed in 4-15% SDS-PAGE (BIO-RAD Laboratories, Hercules, CA) using reducing conditions. Abs used were: rabbit monoclonal anti-PTEN, anti-phospho-AKTSer473, and anti-phospho-ERKThr202/Tyr204 (Cell Signaling Technology, Boston, MA); monoclonal mouse anti-GRB2 Ab (BD Pharminogen San Diego, CA).

Flow cytometry analysis. Murine splenocytes were stained with Abs (clones) from BD

Biosciences reactive against: NK1.1 (PK136), CD3 (145-2C11), CD117 (ACK45),

CD27 (LG.3A10), CD11b (M1/70), CD49b (DX5), CD122 (TM-beta1), Ly-49C/I (5E6),

Ly-49I (YLI-90), Ly-49A (A1), CD16/32 (2.4G2), Ly-49D (4E5), Ly-49G2 (4D11). Rat

Abs to mouse CD94 (18d3), CD69 (H1.2F3), NKG2D (CX5), NKG2ACE (20d5), 2B4

(eBio244F4), NKp46 (29A1.4), Ly-49C/I/F/H (14B11) were from eBioscience.

NKp46+CD3- cells were gated for FACS analysis of these antigens and analyzed with

FlowJo v7.6.1 (TreeStar). Human NK-92 cells were stained with Abs (clones) from BD

Biosciences reactive against: CD11a, CD117, CD18, NKp44, CD11b, NKG2D, NKp46,

CD11c, and NKp30 and analyzed with FlowJo v7.6.1 (TreeStar).

IFN-γ Detection. ELISA: Quantification of human IFN- γ was performed using commercially available mAbs pairs (Endogen Inc.). Cell free supernatants were collected after 18 or 24 h of incubation at 37oC with the indicated stimuli. Wild-type and Super-

PTEN purified mouse NKp46+CD3- NK cells were left untreated or stimulated with monokines and/or immobilized 18 to 24 h at 37oC. Results are shown as the mean of triplicate wells +/- SEM. Intracellular (IC) staining for IFN- γ was performed after 18-24 hours of stimulation with murine or human IL-12 and IL18, adding 2 M GolgiPlug during the final 4 hours of incubation. Cells were then permeabilized and fixed using the

Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) and stained with anti- mouse IFN-γ-PE mAb or anti-human IFN-γ-APC mAb (BD Biosciences). Cells were analyzed immediately as previously described [136] using an LSR II cytometer (BD

Biosciences) and FlowJo software (Tree Star, Inc.). Non-specific staining was detected with an appropriately labeled isotype antibody.

CD107A Degranulation Assay. Lentiviral infected human NK cells were sorted for

CD56+CD3-GFP+ and cultured overnight in RPMI-1640 medium (Invitrogen, Carlsbad,

CA), supplemented with 20% heat-inactivated FBS (Invitrogen), 2 mM L-glutamine and

150 IU/ml rhIL-2 (Hoffman-LaRoche Inc., Nutley, NJ). NK cells were then co-cultured with K562 target cells at a 5:1 ratio for 4 hours in the presence of monensin

(ebiosciences), and CD107A-APC antibody or Isotype control (BD Biosciences). Cells were then washed, gated on GFP+ and assessed for CD107A expression.

Cell stimulation. WT, Super-PTEN, Pten+/- NK cells were stimulated with IL-2 (90 ng/ml), or IL-12 (20 ng/ml; Genetics Institute Inc.) plus IL-18 (10 ng/ml; R&D Systems) for indicated times. For stimulation with YAC-1 tumor cells, murine NK cells were expanded in IL-2 for > 8 days, starved from IL-2 for 2 hours on ice and then mixed with paraformaldehyde-fixed YAC-1 cells at 5:1 ratio and stimulated for the indicated times.

For stimulation with K562 leukemic cells NK-92 cells starved from IL-2 for 2 hours on ice and then mixed with paraformaldehyde-fixed K562 cells at a 5:1 ratio and stimulated for the indicated times.

Cytotoxicity assays. Mouse: YAC-1, or P815 cells coated with an anti-mouse lymphocyte rabbit Ab (Accurate Chemical) were used as targets in a 4-hour 51Cr release assay[113] with fresh NK cells or NK cells cultured in IL-2 90 ng/ml for 8 days. Human:

K562 were used as targets in a 4-hour 51Cr release assay [113] with empty vector, Over-

PTEN-, Over-PTEN-G129E-, and shPTEN-NK92 cells that were cultured in 150 u/ml of

IL-2.

Conjugate formation. Immune complex formation among GFP+ NK-92 cell lines and

K562 leukemic cells were examined by flow cytometry. K562 leukemic cells were stained with PKH26 Red Fluorescent Dye (Sigma-Aldrich). For the conjugate assay,

2x105 NK-92 cells were mixed with 2x105 stained K562 target cells in 200 l of cold

RPMI-1640 with 10% FBS. To induce the formation of immune-complexes, cells were incubated at 37oC at indicated. Cells were immediately run on LSR II flow cytometer and conjugates were detected by FACS analysis as previously described [47].

Immune Conjugates by Confocal Microscopy Empty vector, Over-PTEN or shPTEN

NK-92 effector cells were allowed to form conjugates with K562 erythroleukemic cells for 25 minutes at 370C. Conjugates were adhered in Poly-L-lysine coated slide (Sigma)

0 for another 15 mints at 37 C, and in the presence of 5% CO2. Adhered cells were rinsed in PBS and fixation-permeabilization was performed using Permafix buffer (BD

Biosciences). Fixed cells were rinsed in 1% BSA in PBS with 0.1% saponin (Permwash buffer) and stained for PTEN as described above. After a blocking step with mouse IgG at 10ug/ml concentration cells were rinsed and incubated with anti-alpaha tubulin- biotynlated Ab. Cells were rinsed and further incubated with streptavidin-Alexaflour 568

(Molecular Probes). Perforin FITC (BD) and AF-568 Phalloidin were used at 1:3 and

1:100 dilution respective to visualize the effect of PTEN on F-actin polymerization, granule convergence towards MTOC, and localization of the MTOC at the immune synapse. Cells were visualized using a Spinning disck confocal laser scanning microscope (Zeiss, Observer 1), and data was collected and analyzed by Volocity software (Perkin Elmer).

For analysis, a fixed intensity threshold was set up to obtain perforin or alpha tubulin staining above the background, alpha tubulin defined MTOC was located as described previously [137]. To measure MTOC polarization, a straight line connecting the MTOC to the center of the center of immune synapse was obtained using Volocity software. To measure granule convergence to the MTOC, x and y coordinates of the MTOC and all lytic granule regions in the plane of the MTOC were obtained. The length of the shortest line connecting the MTOC and each granule region was calculated as if it were the hypotenuse of the triangular region defined by the individual object coordinates, thus representing the MTOC-to-granule distance (MGD). The MGD was determined for each lytic granule region present in an individual image from a single time point, and mean

MGD was determined. Thus, the MGD was calculated using the following equation.where x and y were the coordinates of the MTOC centroid and xi and yi were the coordinates of the centroid of an individual lytic granule region. Distance of MGD in

PTEN ko cells were found to be significantly higher (p< 0.05)than the MGD of empty vector transduced cells as determined by paired two tailed TTEST.

2.5 Figures and Tables

Figure 1. PTEN is differentially expressed in human CD56bright vs. CD56dim NK cells: NK cells isolated from the peripheral blood of healthy donors by positive bead selection were separated by FACS based upon CD56 surface expression. (A) Two representative donor western blots showing PTEN protein expression. GRB2 is shown a loading control. (B) Average PTEN protein expression in CD56brightNK cells (blue) relative to CD56dimNK (white bar) in 7 healthy controls. (C) A representative 3- dimensional confocal image of a CD56brightNK cell (left) and CD56dimNK cell (right) showing stained with PTEN (blue) and F-Actin (green). (D) Mean fluorescence intensity

(MFI) of PTEN expression in CD56bright and CD56dim NK cells.

Figure 1 PTEN is differentially expressed in human CD56bright vs. CD56dim NK cells

Figure 2. PTEN overexpression negatively regulates NK cell cytolytic activity. (A)

Western blot for PTEN protein expression in NK cells isolated from wild-type (WT) littermate controls and Super-PTEN transgenic mice. Grb2 was used as a loading control.

(B) Cytolytic activity of freshly isolated NK cells in Super-PTEN mice (solid line, triangles) vs. WT littermate controls against YAC-1 lymphoma cells. (C) Cytolytic activity of NK cell cultured for at least 8 days in IL-2 from Super-PTEN mice (solid line, triangles) vs. WT littermate controls (dashed line, squares) against YAC-1 lymphoma cells. (D) Antibody-dependent cell-mediated cytotoxicity (ADCC) of NK cell cultured for at least 8 days in IL-2 from Super-PTEN mice (solid line, triangles) vs. WT littermate controls (dashed line, squares) against antibody labled P815 target cells. (E) Western blot of PTEN protein expression in NK cells infected with lentivirus containing cDNA encoding GFP alone (empty vector) or GFP and PTEN (Over-PTEN). Grb2 was used as loading control. (F) Cytolytic activity of over-PTEN NK-92 cells (solid line, triangles) vs. empty vector (square, dashed line) against K562 erythroleukemia cells. (G) CD107A expression (degranulation) in primary human NK cells infected with either empty vector

(dashed line) or Over-PTEN co-cultured with K562 target cells. (H) Average Mean fluorescent intensity (M.F.I.) of CD107a+ NK cells from 6 healthy donors infected with either empty vector or Over-PTEN virus.

Figure 2 PTEN overexpression negatively regulates NK cell cytolytic activity.

Figure 3. PTEN overexpression negatively regulates NK cell Interferon-ϒ (IFN- ϒ) production and secretion. (A) Intracellular IFN-ϒ production of NK cells cultured for at least 8 days in IL-2 from WT littermate control (solid) vs. Super-PTEN NK cells (white) followed by stimulation with IL-12 and IL-18 for and assessed by flow cytometry. (B)

The secretion of IFN-ϒ production in IL-12 and IL-18 stimulated WT littermate control

(solid) vs. Super-PTEN mice (white) measured by ELISA. (C) Intracellular IFN-ϒ production of NK-92 cells from Over-PTEN (solid) vs. empty vector infected NK cells

(white) stimulated with IL-12 and IL-18 and assessed by flow cytometry. (D) The secretion of IFN-ϒ production in IL-12 and IL-18 stimulated Empty vector (solid) vs.

Over-PTEN (white) measured by ELISA.

Figure 3 PTEN overexpression negatively regulates NK cell Interferon-ϒ (IFN- ϒ) production and secretion.

Figure 4. PTEN overexpression does not affect receptor expression or cell NK development. (A) Freshly isolated splenocytes from WT littermate controls (solid) and

Super-PTEN NK cells were gated on NKp46+CD3- cells and assessed for expression of

NK cell activating and inhibitory receptors by flow cytometry. (B) Mean percentage of

CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and CD11bhighCD27low subsets in splenocytes gated on NKp46+CD3- cells between WT littermate controls

(solid) and Super-PTEN NK cells (white).

Figure 4 PTEN overexpression does not affect receptor expression or cell NK development.

Figure 5. PTEN overexpression negatively regulates cytolytic activity through inihibition of the AKT and MAPK pathways. (A) Western blot of NK lysates of P-

AKT and P-ERK in WT and Super-PTEN mice after stimulation with fixed YAC-1 target cells for 0, 1, 3, and 10 minutes. (B) Western blot of NK lysates of P-AKT and P-ERK in empty vector (left) and Over-PTEN NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes. (C) Western blot of PTEN protein expression in NK cells infected with lentivirus containing cDNA encoding GFP alone (empty vector), cDNA encoding GFP and WT PTEN (Over-PTEN) or GFP and PTEN with a glycine to glutamate substitution at position 129 (Over-PTEN-G129E). (D) Western blot of NK lysates of P-AKT and P-ERK in empty vector (left) and Over-PTEN NK-92 (middle) and

Over-PTEN-G129E NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes. (E) Cytolytic activity of Over-PTEN-G129E NK-92 cells (solid line, open square) vs. empty vector (dashed line, filled square) and Over-PTEN-G129E (solid line, triangle) against K562 erythroleukemia. (E) Western blot of NK lysates for P-AKT and P-ERK in empty vector (left) and Over-PTEN NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes. Grb2 was used as a loading control for all western blots, which are representative of at least 3 independent experiments.

Figure 5 PTEN overexpression negatively regulates the activation of the

AKT and MAPK pathways.

Figure 6. PTEN loss has a dose dependent effect on NK cell cytolytic activity. (A)

Western blot for PTEN protein expression in NK cells isolated from wild-type (WT) littermate controls and mice with a heterozygous deletion of Pten (Pten+/-). (B) Cytolytic activity of freshly isolated NK cells in Super-PTEN mice (solid line, triangles) vs. WT littermate controls against YAC-1 lymphoma cells. (C) Western blot of NK lysates for P-

AKT and P-ERK in empty WT (left) and Pten+/- NK cells after stimulation with fixed

Yac-1 target cells for 0 and 2 minutes. (D) Western blot of PTEN protein expression in

NK cells infected with lentivirus containing cDNA encoding GFP alone (empty vector) or GFP and a short hairpin RNA targeting PTEN (shPTEN). (E) Cytolytic activity of shPTEN NK-92 cells (solid line, triangles) vs. empty vector (square, dashed line) against

K562 erythroleukemia cells. (F) Western blot of NK lysates for P-AKT and P-ERK in empty vector (left) and shPTEN NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes.

Figure 6 PTEN loss has a dose dependent effect on NK cell cytolytic activity.

Figure 7. Loss of PTEN in NK-92 cells leads to a deficiency in target cell adhesion.

(A) One representative experiment of empty vector (top) and shPTEN NK-92 cells

(bottom) cells were co-cultured for 0, 5, 20, 40 and 60 minutes with stained pKH26 Red

K562 target cells and analyzed by flow cytometry. Boxes indicate double positive events representing the percent of NK cells forming conjugates with target cells. (B) Average % of max adhesion for three independent experiments with empty vector (solid) vs. shPTEN and (C) empty vector (solid) vs Over-PTEN (white). (D) Flow cytometric analyses of adhesion and activating receptors in empty vector (dashed line) and shPTEN NK-92.

Figure 7 Loss of PTEN in NK-92 cells leads to a deficiency in target cell adhesion

Figure 8. Loss of PTEN leads to a disruption of polarization and cytolytic granule coalescence. Confocal representative images of (A) empty vector and (B) shPTEN NK-

92 (top double sided arrow) in immune conjugation with K562 target cells (bottom double sided arrow). Conjugates are stained with anti-tubulin (yellow), anti-perforin

(red), anti-F-Actin (green) and anti-PTEN (blue). Empty vector and shPTEN NK-92 cells were assessed for their ability to (C) polarize to the target cell (measured by distance from the microtubule organizing center (MTOC)) to the immune synapse and (D) the coalescence of granules to the MTOC (measured the average distance of perforin containing granules to the MTOC.

Figure 8 Loss of PTEN leads to a disruption of polarization and cytolytic granule coalescence.

3. FUTURE STUDIES AND EXTENDED DISCUSSION

3.1 Introduction

In the previous chapter we presented data which suggested that an optimum level of

PTEN expression exists for maximum NK cell cytolytic activity. Following these findings our current investigations are focused on three areas of inquiry regarding PTEN in NK cells: a.) What is PTEN’s role in NK cell development and how is differential

PTEN protein expression regulated between CD56bright and CD56dim NK cells? b.) How does loss or gain of PTEN modify the balance of secondary lipid phosphatase signals including PI(3,4)P2 and PI(4,5)P2? c.) Are tumors able to evade NK cell lytic activity by modifying NK cell PTEN or similar negative regulators of NK cell activation?

3.1a

While this was the first investigation of PTEN in NK cells, previous work in T cell specific Pten-/- mice suggests lipid phosphatase signaling has an important role in development. Further supporting this are several previously described studies in SHIP-1-/- mice that show NK cells have arrested development and decreased function. Not surprisingly deficiency in PI kinases and their subunits can also lead to defective NK cell development and function [44, 112, 138, 139]. Previously our lab defined five stages of

NK development in human secondary lymphoid tissue (hSLT). The first two CD34+

56 stages retain the ex vivo ability to develop into T cells, dendritic cells, and NK cells. The

CD34-CD117+CD94- stage 3 cell initially thought to be only a committed NK progenitor has recently been discovered to have an expanded role in immunity including the production of the IL-22 cytokine. The final two stages correspond to the CD56brightNK cell and the CD56dimNK cell discussed previously. In hSLT approximately ~90% of

CD56+ cells are of the CD56bright phenotype, with ~10% characteristic of CD56dimNK cells. The converse is true in peripheral blood where ~10% of NK cells are CD56brightNK cells and ~90% are CD56dimNK cells. In addition CD94 expression can divide CD56dim

NK cells into two populations with CD56dimCD94highNK cells showing intermediate functional characteristics of CD56bright NK cells and CD56dim NK cells. In these preliminary studies we show that PTEN transcript is highly expressed in the early NK cell developmental stages and that the differential protein expression in CD56bright NK cells and CD56dim NK cells may be due to expression of miR-26b-3p.

3.2b

An important distinction between SHIP-1 and PTEN is the differences in the end product of PI(3,4,5)P3 metabolism. SHIP-1 is a 5’-lipid phosphatase and therefore produces

PI(3,4)P2. Conversely, PTEN, a 3’-lipid phosphatase results in the production of

PI(4,5)P22. PI(3,4)P2 binds AKT with a greater affinity than PI(3,4,5)P3 so it is conceivable that upon stimulation SHIP-1 could amplify AKT signals. In agreement it has been shown that PI(3,4)P2 levels govern activation of protein kinase B (PKB) in mast cells and B cells [140-142]. PTEN metabolism of PI(3,4,5)P3 to PI(4,5)P2 also has important downstream consequences. PLCΥ metabolizes PI(4,5)P2 into diacylglycerol 57

(DAG) which activates Protein Kinase C (PKC), and inositol 1,4,5-triphosphate

2+ [I(1,4,5)P3] thereby releasing intracellular Ca stores. While PI(4,5)P2 is relatively abundant compared to PI(3,4,5)P3 it appears that regions containing high concentration of

PI(4,5)P2 exist in the cell and therefore PTEN may help to produce these changes. This seems especially pertinent to our findings presented in Chapter 2 in which complete loss of PTEN led to disorganization of the immunological synapse. It has been demonstrated that an increase in PI(4,5)P2 at the plasma membrane is required, albeit not necessarily driven by PTEN, for effective immune synapse formation [58, 143-146]. In order to build upon our previous findings we are investigating how PTEN modulation can affect pathways driven by the presence of PI(4,5)P2.

3.2 Results a. PTEN is mRNA is differentially expressed during human NK cell development

Our finding in chapter 2 showing that CD56bright NK cells express higher levels of PTEN protein than CD56dim NK cells and the developmental abnormalities observed in SHIP-1-

/- NK cells and PTEN-/- T cells led us to ask if gene expression of PTEN differs between these stages in hSLT. We observed that after peaking in stage 2 PTEN mRNA levels decreased six fold by stage 4 (Figure 9A, n=2). Considering that we saw high PTEN protein expression in the stage 4 CD56bright NK cells, relative to CD56dimNK cells we next sought to determine if this difference was reflected at the transcript level. In contrast the 5-fold decrease in protein expression from CD56brightNK cells to CD56dimNK cells,

PTEN mRNA increased modestly from CD56brightCD94highNK cells to

CD56dimCD94highNK cells to CD56dimCD94low NK cells indicating that PTEN protein 58 levels must be post-transcriptionally regulated during the transition from CD56bright to

CD56dim NK cells. We next explored the possibility of microRNA (miR) regulation of

PTEN by running a nanostring array looking at differences in miR expression between the CD56brightCD94high, CD56dimCD94highNK, and CD56dimCD94low NK cell subset in four healthy donors. Among the top 2 differentially expressed miRs is miR-26b-3p of which

PTEN is a predicted target (Figure 9B, n=4, p<.04). b. PTEN modulation in NK-92 cells disrupts activation of PI(4,5)P2 driven pathways

PI(4,5)P2 is a phosphatidylinositol molecule that acts as a substrate for PLCΥ. PLCΥ metabolizes PI(4,5)P2 into DAG which activates protein kinase C (PKC) and inositol

2+ 1,4,5-trisphosphate (I(1,4,5)P3) which triggers the release of intracellular Ca . Since both

PKCθ and Ca2+ are essential for NK cell mediated cytotoxicity we sought to determine if loss of PTEN effects the activation of these pathways. In order to test this we stimulated empty vector and shPTEN NK-92 cells with fixed K562 targets cells for 0, 1, 3 and 10 minutes and assessed their expression of P-PKCθ. We observed a marked decreased in

PKCθ expression in shPTEN NK-92 cells compared empty vector (Figure 10A). We also assessed Over-PTEN NK-92 cells and also found a minimal decrease in p-PKCθ expression following stimulation with K562 target cells (Figure 10B). In order to detect differences in calcium flux upon activation we stained shPTEN NK-92 and Over-PTEN

NK-92 cells with an indo-1 AM, a UV light – excitable, ratiometric Ca2+ indicator and then stimulated with a cocktail of biotinylated antibodies to activating receptors (2B4,

NKG2D, and NKp44) of NK cells, followed by streptavidin. We found that in both

59 shPTEN and Over-PTEN cells we observe a modest decrease in Ca2+ flux compared to empty vector control, which correlated with our previously observed cytolytic activity.

3.2 Discussion and Summary

3.2a

Determining how the common lymphoid progenitor becomes a mature cytolytic NK cell is an important area of investigation. This is especially true in pathologic conditions in which NK development may be inhibited or where an elevation in mature NK cells may be an avenue of immunotherapy. However, recent evidence in our lab and others indicate that developing stage 3 NK cells have specific role in immunity not observed in other stages of NK development [6, 18, 19, 21, 147]. Therefore, understanding the factors that govern how an NK cell develops may lead to further discoveries about other immune roles a developing NK cells may undertake, either permanently or transiently.

Considering our initial findings showing high PTEN protein expression in the less mature

CD56brightNK cell than the more mature CD56dimNK cells and mouse models demonstrating the importance of SHIP-1 and PI3K in normal NK development we investigated mRNA expression differences among the four stages of human NK development found in tonsil. We observed a dramatically higher expression of PTEN in the CD34+ stage 1 and stage 2 NK cell precursors suggesting that PTEN may have an important role in maintaining these pluripotent lymphoid progenitors (Figure 9A). These findings suggests that a NK cell specific PTEN knockout model may uncover important an important role for PTEN in the development of NK cells.

The CD56bright to CD56dim is the final developmental transition for NK cells and we have described in chapter 2 that CD56bright NK ~5 fold higher levels of PTEN protein expression than CD56dim NK cells. Interestingly, when exploring differences among the 61

CD56brightCD94high, CD56dimCD94highNK, and CD56dimCD94low NK cells we saw minimal differences in mRNA transcript that did not reflect our observations for protein expression presented in chapter 2, suggesting that PTEN is regulated at least in part through post-transcriptional mechanisms in mature NK cells subsets (Figure 9B).

Concurrently, our recent results from a nanostring array of CD56brightCD94high, and

CD56dimCD94low NK cells showed that mir-26b-5p, of which PTEN is a predicted target

(microRNA.org, mirDB, and targetscan), is expressed at significantly higher levels in the low PTEN expressing CD56dimCD94low NK cell than the high PTEN expressing

CD56brightCD94highNK cell. Intriguingly, Fehninger et. al., showed mir-26b as one of the top three miRs down-regulated upon IL-15 stimulation of murine NK cells and miR-26b was found to be one of the most highly expressed miRs in both human and murine NK cells [148, 149]. While miR-21 was previously shown to negatively regulate PTEN in the context of NK cell malignancy we did not observe an appreciable difference in our preliminary findings, suggesting that high level expression of miR-21 may reflect a pathologic rather than physiologic mechanism of PTEN regulation [150]. miR regulation of NK cells is an emerging, but new area. miR-150 and miR-181 have been shown to drive NK development in mouse and human respectively, while mir-223 negatively regulates granzyme B in murine NK cells. Only miR-155 has been shown to be differentially expressed between between CD56bright and CD56dim NK cells and was demonstrated to positively regulate IFN- γ production by downregulating SHIP-1[48]. It is likely that a collection miRs regulate NK cell development and function and our preliminary data suggests that miR-26b through regulation of PTEN may be among them.

3.2b

The work presented here and previous studies from our lab and others regarding SHIP-1 in NK cells make dissecting the interplay of these similar, but different, lipid phosphatases an important area for understanding NK cell signaling and activation. While we have shown that SHIP-1 and PTEN are differentially expressed among CD56bright

(higher PTEN) and CD56dim NK cells (higher SHIP-1) each is still expressed in each subset and how they metabolize components of the NK activating and inhibitor machinery may finely tune NK cell responses. Our data showing that PTEN loss leads to a decrease in cytolytic activity and that this results from disruption of necessary cytoskeletal dynamics led us to explore pathways that might be dysregulated in the absence of PTEN. Among the important pathways of NK cell activation is the metabolism of PI(4,5)P2 by PLCΥ into DAG which activates protein kinase C (PKC) and

2+ inositol 1,4,5-trisphosphate (I(1,4,5)P3), which mediates intracellular Ca release. We found that depletion of PTEN in NK-92 cells leads to a notable decrease in p-PKCθ, while increased levels of PTEN lead to marginal differences. This data infers that PTEN metabolism of PI(3,4,5)P3 may help to provide the NK cells with adequate levels of

PI(4,5)P2, and may in part explain the decreased cytolytic activity and disorganized phenotype of the shPTEN NK-92 cells described in chapter 2. The importance of PKCθ for NK cell function likely depends on the stimulus and target as PKCθ knockout studies have shown results ranging from no effect, to dramatically increased tumor burden [151-

153]. In agreement with our findings presented in chapter 2, PKCθ has been directly linked to cytoskeletal arrangement of the NK cell immune synapse by mediating the

63 phosphorylation of WASp-interacting protein (WIP), loss of which leads to severely decreased cytolytic activity [154]. It would be of interest in future studies to determine if constitutively active WIP can rescue the deficiency in cytolytic activity observed in

PTEN depleted NK cells.

As mentioned directly above, the second half of PLCΥ’s metabolism of PI(4,5)P2 is

2+ production of I(1,4,5)P3 and subsequent release of intracellular calcium stores. Ca flux is a required for NK cell cytolytic activity as evidenced by patients loss of the calcium release-activated calcium channel ORAI1 displaying nearly absent cytolytic activity against NK sensitive targets [155]. We observed minimal decreases in calcium flux in the shPTEN-NK-92 cells as well as in the Over-PTEN-NK-92 cells compared to their respective controls. This finding is not surprising considering that both shPTEN-NK-92 cells and Over-PTEN-NK-92 cells have reduced, but not absent cytolytic activity. The decrease in Over-PTEN-NK-92 may be mediated by a reduction in both PI3K/AKT and

MAPK pathways which stimulate calcium release, while decreased PI(4,5)P2 availability in the absence of PTEN may account for the decrease in calcium flux in shPTEN-NK-92 cells.

3.4 Experimental Procedures

Real-time RT-PCR. RNA was extracted using the Total RNA Plus Purification Kit

(Norgen). Reverse transcription was performed with Taqman MicroRNA Reverse

Transcription Kit and RT primers specific for PTEN and Sybr green for 18S as control

(Applied Biosystems). Real-time RT-PCR reactions were performed as described[48].

Data were analyzed with the comparative CT method using internal control 18S RNA 64 levels to normalize differences in sample loading. Results (mean ± SEM of triplicate reaction wells) represent the n-fold difference of transcript levels in a particular tonsil sample for previously published NK cell developmental stages 1-4 [124] and

CD56brightCD94high, CD56dimCD94high, CD56dimCD94low NK cells from peripheral blood.

Assessment of PKCθ expression levels. For stimulation of with K562 leukemic cells

NK-92 cells starved from IL-2 for 2 hours on ice and then mixed with paraformaldehyde- fixed K562 cells at a 5:1 ratio and stimulated for the indicated times and western blotted as previously described (2.4). anti-P-PKCθ was purchased from Cell Signaling

Technology (Boston, MA).

Calcium Flux Aassay: Intracellular Ca2+ flux was investigated by flow cytometry using

NK-92 cells loaded with indo-1-AM (Invitrogen), a Ca2+-sensitive fluorescent dye. Cells were then were preincubated with biotinylated anti-2B4 (ebiosciences), -NKp44 and

NKG2D (Biolegend) mAb for 2 min at 4 °C, followed by addition of streptavidin and cells were immediately run on a LSR II flow cytometer while in a 37 degree waterbath.

Samples were run until calcium returned to at or near baseline levels. Data was analyzed using FlowJo v7.6.1 (TreeStar).

3.5 Figures

Figure 9. PTEN mRNA among NK cell developmental subsets in human tonsil, peripheral blood and miR-26b-3P expression in CD56bright and CD56dim NK cells.

(A) Real-Time RT-PCR of PTEN for stages 1, 2, and 3 and 4 sorted from human tonsil by gating on CD3−CD19-CD14- and then gated on CD34+CD117-CD94- (stage 1),

CD34+CD117+CD94-(stage 2), CD34-CD117+CD94-(stage 3), and CD34-CD117-

CD94+(stage 4), showing average of two donors as a ratio over stage 1 (B) RT-PCR of

PTEN for three subsets of NK cells isolated from peripheral blood CD56brightCD94highNK cells, CD56dimCD94highNK cells, and CD56dimCD94low NK cells, showing average of 4 healthy donors, as a ratio of the CD56brightCD94highNK. (C) Nanostring results for miR-

26B-3P expression in CD56bright and CD56dim NK cells from peripheral blood, showing average of 4 healthy donors.

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Figure 9 PTEN mRNA among NK cell developmental subsets in human tonsil, peripheral blood and miR-26b-3P expression in CD56bright and CD56dim NK cells.

Figure 10. PKCθ expression and Ca 2+ flux in stimulated shPTEN-NK-92 cells and

Over-PTEN-NK-92 cells. (A) Western blot of NK lysates blotted P-PKC in empty vector (left) and shPTEN (right) NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes. (B) Western blot of NK lysates blotted for P-PKC in empty vector (left) and Over-PTEN (right) NK-92 cells after stimulation with fixed K562 target cells for 0, 1, 3, and 10 minutes. (C, D) Calcium flux after stimulation with biotinylated NK activating receptor cocktail (2B4, NKp44, NKG2D) with (C) empty vector and shPTEN-NK-92 and (D) empty vector and Over-PTEN-NK-92.

Figure 10 PKCθ expression and Ca 2+ flux in stimulated shPTEN-NK-92 cells and Over-PTEN-NK-92 cells.

3.6 Concluding Remarks

In this thesis we have provided evidence for the first time that the tumor suppressor

PTEN has important functions in NK cell activity. Further we believe that our discovery that PTEN is not simply an activator or inhibitor of NK cell signaling, but rather a dynamic regulator whose expression must be finely tuned for optimum cytolytic activity may hold implications beyond only PTEN in NK cells. The consequences of such a delicate balance make PTEN the ideal candidate for a yet to be described tumor evasion mechanism. Further if such a mechanism exists for PTEN we hope this offers a new avenue for targeted immunotherapy. Additionally, these findings and possible consequences should not be limited to only PTEN, but opens the possibility that other lipid phosphatases such as SHIP-1 may be a pathway for active tumor immune evasion.

Bibliography

1. Kiessling, R., E. Klein, and H. Wigzell, "Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol, 1975. 5(2): p. 112-7. 2. Herberman, R.B., et al., Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer, 1975. 16(2): p. 230-9. 3. Lee, S.H., T. Miyagi, and C.A. Biron, Keeping NK cells in highly regulated antiviral warfare. Trends Immunol, 2007. 28(6): p. 252-9. 4. Smyth, M.J., et al., New aspects of natural-killer-cell surveillance and therapy of cancer. Nat Rev Cancer, 2002. 2(11): p. 850-61. 5. Cella, M., et al., A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature, 2009. 457(7230): p. 722-5. 6. Hughes, T., et al., Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH 17 cytokine interleukin-22. Blood, 2009. 113(17): p. 4008-10. 7. Leibson, P.J., Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity, 1997. 6(6): p. 655-61. 8. Sun, J.C., et al., NK cells and immune "memory". J Immunol, 2011. 186(4): p. 1891-7. 9. Vivier, E., et al., Innate or adaptive immunity? The example of natural killer cells. Science, 2011. 331(6013): p. 44-9. 10. Sutlu, T. and E. Alici, Natural killer cell-based immunotherapy in cancer: current insights and future prospects. J Intern Med, 2009. 266(2): p. 154-81. 11. Blom, B. and H. Spits, Development of human lymphoid cells. Annu Rev Immunol, 2006. 24: p. 287-320. 12. Kondo, M., et al., Lymphocyte development from hematopoietic stem cells. Curr Opin Genet Dev, 2001. 11(5): p. 520-6. 13. Boos, M.D., K. Ramirez, and B.L. Kee, Extrinsic and intrinsic regulation of early natural killer cell development. Immunol Res, 2008. 40(3): p. 193-207. 14. Fehniger, T.A., et al., Fatal leukemia in interleukin-15 transgenic mice. Blood Cells Mol Dis, 2001. 27(1): p. 223-30.

15. Kennedy, M.K., et al., Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med, 2000. 191(5): p. 771-80. 16. Yokoyama, W.M., S. Kim, and A.R. French, The dynamic life of natural killer cells. Annu Rev Immunol, 2004. 22: p. 405-29. 17. Yu, J., et al., NKp46 identifies an NKT cell subset susceptible to leukemic transformation in mouse and human. J Clin Invest, 2011. 121(4): p. 1456-70. 18. Hughes, T., et al., Interleukin-1beta selectively expands and sustains interleukin- 22+ immature human natural killer cells in secondary lymphoid tissue. Immunity, 2010. 32(6): p. 803-14. 19. Cupedo, T., et al., Human fetal lymphoid tissue-inducer cells are interleukin 17- producing precursors to RORC+ CD127+ natural killer-like cells. Nat Immunol, 2009. 10(1): p. 66-74. 20. Satoh-Takayama, N., et al., Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity, 2008. 29(6): p. 958-70. 21. Crellin, N.K., et al., Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells. J Exp Med, 2010. 207(2): p. 281-90. 22. Hayakawa, Y. and M.J. Smyth, CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J Immunol, 2006. 176(3): p. 1517-24. 23. Vossen, M.T., et al., CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol, 2008. 180(6): p. 3739-45. 24. Cooper, M.A., et al., Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset. Blood, 2001. 97(10): p. 3146-51. 25. Chan, A., et al., CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J Immunol, 2007. 179(1): p. 89-94. 26. Huntington, N.D., et al., IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. J Exp Med, 2009. 206(1): p. 25-34. 27. Romagnani, C., et al., CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol, 2007. 178(8): p. 4947-55. 28. Yu, J., et al., CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood, 2010. 115(2): p. 274-81. 29. Cooper, M.A., T.A. Fehniger, and M.A. Caligiuri, The biology of human natural killer-cell subsets. Trends Immunol, 2001. 22(11): p. 633-40. 30. Ljunggren, H.G. and K.J. Malmberg, Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol, 2007. 7(5): p. 329-39. 31. Watzl, C. and E.O. Long, Signal transduction during activation and inhibition of natural killer cells. Curr Protoc Immunol, 2010. Chapter 11: p. Unit 11 9B. 32. Claus, M., et al., Regulation of NK cell activity by 2B4, NTB-A and CRACC. Front Biosci, 2008. 13: p. 956-65. 72

33. Caraux, A., et al., Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood, 2006. 107(3): p. 994-1002. 34. Tassi, I., et al., Phospholipase C-gamma 2 is a critical signaling mediator for murine NK cell activating receptors. J Immunol, 2005. 175(2): p. 749-54. 35. Cella, M., et al., Differential requirements for Vav proteins in DAP10- and ITAM- mediated NK cell cytotoxicity. J Exp Med, 2004. 200(6): p. 817-23. 36. Karre, K., et al., Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature, 1986. 319(6055): p. 675-8. 37. Caligiuri, M.A., Human natural killer cells. Blood, 2008. 112(3): p. 461-9. 38. Stebbins, C.C., et al., Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity. Mol Cell Biol, 2003. 23(17): p. 6291-9. 39. Vance, R.E., et al., Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J Exp Med, 1998. 188(10): p. 1841-8. 40. Borrego, F., et al., Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med, 1998. 187(5): p. 813-8. 41. Peterson, M.E. and E.O. Long, Inhibitory receptor signaling via tyrosine phosphorylation of the adaptor Crk. Immunity, 2008. 29(4): p. 578-88. 42. Jiang, K., et al., Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat Immunol, 2000. 1(5): p. 419-25. 43. Chen, X., et al., Many NK cell receptors activate ERK2 and JNK1 to trigger microtubule organizing center and granule polarization and cytotoxicity. Proc Natl Acad Sci U S A, 2007. 104(15): p. 6329-34. 44. Kim, N., et al., The p110delta catalytic isoform of PI3K is a key player in NK-cell development and cytokine secretion. Blood, 2007. 110(9): p. 3202-8. 45. Galandrini, R., et al., SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells. Blood, 2002. 100(13): p. 4581-9. 46. Parihar, R., et al., Src homology 2-containing inositol 5'-phosphatase 1 negatively regulates IFN-gamma production by natural killer cells stimulated with antibody- coated tumor cells and interleukin-12. Cancer Res, 2005. 65(19): p. 9099-107. 47. Dong, Z., et al., The adaptor SAP controls NK cell activation by regulating the enzymes Vav-1 and SHIP-1 and by enhancing conjugates with target cells. Immunity, 2012. 36(6): p. 974-85. 48. Trotta, R., et al., miR-155 regulates IFN-gamma production in natural killer cells. Blood, 2012. 119(15): p. 3478-85. 49. Trotta, R., et al., Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells. Blood, 2013. 121(16): p. 3126-34.

50. Wang, J.W., et al., Influence of SHIP on the NK repertoire and allogeneic bone marrow transplantation. Science, 2002. 295(5562): p. 2094-7. 51. Wahle, J.A., et al., Cutting edge: dominance by an MHC-independent inhibitory receptor compromises NK killing of complex targets. J Immunol, 2006. 176(12): p. 7165-9. 52. Wahle, J.A., et al., Inappropriate recruitment and activity by the Src homology region 2 domain-containing phosphatase 1 (SHP1) is responsible for receptor dominance in the SHIP-deficient NK cell. J Immunol, 2007. 179(12): p. 8009-15. 53. Fortenbery, N.R., et al., SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation. J Immunol, 2010. 184(9): p. 5065-74. 54. Orange, J.S., Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol, 2008. 8(9): p. 713-25. 55. Chen, S., et al., Suppression of tumor formation in lymph nodes by L-selectin- mediated natural killer cell recruitment. J Exp Med, 2005. 202(12): p. 1679-89. 56. Inoue, H., et al., Lipid rafts as the signaling scaffold for NK cell activation: tyrosine phosphorylation and association of LAT with phosphatidylinositol 3- kinase and phospholipase C-gamma following CD2 stimulation. Eur J Immunol, 2002. 32(8): p. 2188-98. 57. Davis, D.M., et al., The human natural killer cell immune synapse. Proc Natl Acad Sci U S A, 1999. 96(26): p. 15062-7. 58. Vyas, Y.M., et al., Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions. J Immunol, 2001. 167(8): p. 4358-67. 59. Riteau, B., D.F. Barber, and E.O. Long, Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J Exp Med, 2003. 198(3): p. 469-74. 60. Barber, D.F., M. Faure, and E.O. Long, LFA-1 contributes an early signal for NK cell cytotoxicity. J Immunol, 2004. 173(6): p. 3653-9. 61. Masilamani, M., et al., CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse. J Immunol, 2006. 177(6): p. 3590-6. 62. Orange, J.S., et al., The mature activating natural killer cell immunologic synapse is formed in distinct stages. Proc Natl Acad Sci U S A, 2003. 100(24): p. 14151-6. 63. Graham, D.B., et al., Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics. J Immunol, 2006. 177(4): p. 2349-55. 64. Chen, X., et al., CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells. Proc Natl Acad Sci U S A, 2006. 103(27): p. 10346-51. 65. Sancho, D., et al., The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing. J Cell Biol, 2000. 149(6): p. 1249-62. 66. Andzelm, M.M., et al., Myosin IIA is required for cytolytic granule exocytosis in human NK cells. J Exp Med, 2007. 204(10): p. 2285-91.

67. Roda-Navarro, P., et al., Dynamic redistribution of the activating 2B4/SAP complex at the cytotoxic NK cell immune synapse. J Immunol, 2004. 173(6): p. 3640-6. 68. Chiang, S.C., et al., Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production. Blood, 2013. 121(8): p. 1345- 56. 69. Marcet-Palacios, M., et al., Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line. Biochem Biophys Res Commun, 2008. 366(3): p. 617-23. 70. Arneson, L.N., et al., Cutting edge: syntaxin 11 regulates lymphocyte-mediated secretion and cytotoxicity. J Immunol, 2007. 179(6): p. 3397-401. 71. Bryceson, Y.T., et al., Defective cytotoxic lymphocyte degranulation in syntaxin- 11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. Blood, 2007. 110(6): p. 1906-15. 72. McCann, F.E., et al., The activating NKG2D ligand MHC class I-related chain A transfers from target cells to NK cells in a manner that allows functional consequences. J Immunol, 2007. 178(6): p. 3418-26. 73. Sivori, S., et al., 2B4 functions as a co-receptor in human NK cell activation. Eur J Immunol, 2000. 30(3): p. 787-93. 74. Sandusky, M.M., B. Messmer, and C. Watzl, Regulation of 2B4 (CD244)- mediated NK cell activation by ligand-induced receptor modulation. Eur J Immunol, 2006. 36(12): p. 3268-76. 75. Steck, P.A., et al., Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet, 1997. 15(4): p. 356-62. 76. Li, J., et al., PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science, 1997. 275(5308): p. 1943-7. 77. Maehama, T. and J.E. Dixon, The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5- trisphosphate. J Biol Chem, 1998. 273(22): p. 13375-8. 78. Chalhoub, N. and S.J. Baker, PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol, 2009. 4: p. 127-50. 79. Eng, C., PTEN: one gene, many syndromes. Hum Mutat, 2003. 22(3): p. 183-98. 80. Trotman, L.C., et al., Pten dose dictates cancer progression in the prostate. PLoS Biol, 2003. 1(3): p. E59. 81. Chen, Z., et al., Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature, 2005. 436(7051): p. 725-30. 82. Funamoto, S., et al., Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell, 2002. 109(5): p. 611-23. 83. Liliental, J., et al., Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Curr Biol, 2000. 10(7): p. 401-4.

84. Tamura, M., et al., Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science, 1998. 280(5369): p. 1614-7. 85. Martin-Belmonte, F., et al., PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell, 2007. 128(2): p. 383-97. 86. Raftopoulou, M., et al., Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science, 2004. 303(5661): p. 1179-81. 87. Trimboli, A.J., et al., Pten in stromal fibroblasts suppresses mammary epithelial tumours. Nature, 2009. 461(7267): p. 1084-91. 88. Bronisz, A., et al., Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320. Nat Cell Biol, 2012. 14(2): p. 159-67. 89. Shen, W.H., et al., Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell, 2007. 128(1): p. 157-70. 90. Puc, J., et al., Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell, 2005. 7(2): p. 193-204. 91. Mirmohammadsadegh, A., et al., Epigenetic silencing of the PTEN gene in melanoma. Cancer Res, 2006. 66(13): p. 6546-52. 92. Agrawal, S. and C. Eng, Differential expression of novel naturally occurring splice variants of PTEN and their functional consequences in Cowden syndrome and sporadic breast cancer. Hum Mol Genet, 2006. 15(5): p. 777-87. 93. Song, M.S., L. Salmena, and P.P. Pandolfi, The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol, 2012. 13(5): p. 283-96. 94. Poliseno, L., et al., A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature, 2010. 465(7301): p. 1033-8. 95. Vazquez, F., et al., Phosphorylation of the PTEN tail acts as an inhibitory switch by preventing its recruitment into a protein complex. J Biol Chem, 2001. 276(52): p. 48627-30. 96. Li, Z., et al., Regulation of PTEN by Rho small GTPases. Nat Cell Biol, 2005. 7(4): p. 399-404. 97. Okumura, K., et al., PCAF modulates PTEN activity. J Biol Chem, 2006. 281(36): p. 26562-8. 98. Chae, H.D. and H.E. Broxmeyer, SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells. Stem Cells Dev, 2011. 20(7): p. 1277-85. 99. Trotman, L.C., et al., Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell, 2007. 128(1): p. 141-56. 100. Yim, E.K., et al., Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell, 2009. 15(4): p. 304-14. 101. Wang, X., et al., NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell, 2007. 128(1): p. 129-39. 102. Di Cristofano, A., et al., Impaired Fas response and autoimmunity in Pten+/- mice. Science, 1999. 285(5436): p. 2122-5. 103. Suzuki, A., et al., T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity, 2001. 14(5): p. 523-34. 76

104. Soond, D.R., et al., Pten loss in CD4 T cells enhances their helper function but does not lead to autoimmunity or lymphoma. J Immunol, 2012. 188(12): p. 5935- 43. 105. Walsh, P.T., et al., PTEN inhibits IL-2 receptor-mediated expansion of CD4+ CD25+ Tregs. J Clin Invest, 2006. 116(9): p. 2521-31. 106. Haxhinasto, S., D. Mathis, and C. Benoist, The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J Exp Med, 2008. 205(3): p. 565-74. 107. Sauer, S., et al., T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc Natl Acad Sci U S A, 2008. 105(22): p. 7797-802. 108. Kishimoto, H., et al., The Pten/PI3K pathway governs the homeostasis of Valpha14iNKT cells. Blood, 2007. 109(8): p. 3316-24. 109. Canetti, C., et al., Activation of phosphatase and tensin homolog on chromosome 10 mediates the inhibition of FcgammaR phagocytosis by prostaglandin E2 in alveolar macrophages. J Immunol, 2007. 179(12): p. 8350-6. 110. Kuroda, S., et al., Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages. Eur J Immunol, 2008. 38(5): p. 1331-40. 111. Cao, X., et al., The inositol 3-phosphatase PTEN negatively regulates Fc gamma receptor signaling, but supports Toll-like receptor 4 signaling in murine peritoneal macrophages. J Immunol, 2004. 172(8): p. 4851-7. 112. Tassi, I., et al., p110gamma and p110delta phosphoinositide 3-kinase signaling pathways synergize to control development and functions of murine NK cells. Immunity, 2007. 27(2): p. 214-27. 113. Trotta, R., et al., Dependence of both spontaneous and antibody-dependent, granule exocytosis-mediated NK cell cytotoxicity on extracellular signal- regulated kinases. J Immunol, 1998. 161(12): p. 6648-56. 114. Trotta, R., et al., Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation. Blood, 2005. 105(8): p. 3011-8. 115. Scarpellino, L., et al., Interactions of Ly49 family receptors with MHC class I ligands in trans and cis. J Immunol, 2007. 178(3): p. 1277-84. 116. Banh, C., et al., Mouse natural killer cell development and maturation are differentially regulated by SHIP-1. Blood, 2012. 120(23): p. 4583-90. 117. Garcia-Cao, I., et al., Systemic elevation of PTEN induces a tumor-suppressive metabolic state. Cell, 2012. 149(1): p. 49-62. 118. Fehniger, T.A., et al., Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL- 12: implications for the innate immune response. J Immunol, 1999. 162(8): p. 4511-20. 119. Chiossone, L., et al., Maturation of mouse NK cells is a 4-stage developmental program. Blood, 2009. 113(22): p. 5488-96. 120. Stambolic, V., et al., Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell, 1998. 95(1): p. 29-39.

121. Kim, J.S., et al., Mechanistic analysis of a DNA damage-induced, PTEN- dependent size checkpoint in human cells. Mol Cell Biol, 2011. 31(13): p. 2756- 71. 122. Di Cristofano, A., et al., Pten is essential for embryonic development and tumour suppression. Nat Genet, 1998. 19(4): p. 348-55. 123. Gumbleton, M. and W.G. Kerr, Role of inositol phospholipid signaling in natural killer cell biology. Front Immunol, 2013. 4: p. 47. 124. Freud, A.G. and M.A. Caligiuri, Human natural killer cell development. Immunol Rev, 2006. 214: p. 56-72. 125. Leslie, N.R., et al., Understanding PTEN regulation: PIP2, polarity and protein stability. Oncogene, 2008. 27(41): p. 5464-76. 126. Heit, B., et al., PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils. Nat Immunol, 2008. 9(7): p. 743-52. 127. Mondal, S., et al., Phosphoinositide lipid phosphatase SHIP1 and PTEN coordinate to regulate cell migration and adhesion. Mol Biol Cell, 2012. 23(7): p. 1219-30. 128. Uner, A.H., et al., PTEN and p27 expression in mature T-cell and NK-cell neoplasms. Leuk Lymphoma, 2005. 46(10): p. 1463-70. 129. He, S., et al., MicroRNAs activate natural killer cells through toll-like receptor signaling. Blood, 2013. 130. Putz, U., et al., The tumor suppressor PTEN is exported in exosomes and has phosphatase activity in recipient cells. Sci Signal, 2012. 5(243): p. ra70. 131. P, H.J.H.N.L.K.G.N.S.A.T.J.R.C.a.K., AML Exosomes Regulate Stromal Bystander Cells and Provide a Novel RNA Biomarker Platform 54th ASH Annual Meeting and Exposition Atlanta, GA., 2012. 132. Wang, H., et al., Allele-specific tumor spectrum in pten knockin mice. Proc Natl Acad Sci U S A, 2010. 107(11): p. 5142-7. 133. Trotta, R., et al., The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells. Blood, 2011. 117(8): p. 2378-84. 134. !!! INVALID CITATION !!! 135. Trotta, R., et al., BCR/ABL activates mdm2 mRNA translation via the La antigen. Cancer Cell, 2003. 3(2): p. 145-60. 136. Freud, A.G., et al., A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity, 2005. 22(3): p. 295- 304. 137. Banerjee, P.P., et al., Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological synapse. J Exp Med, 2007. 204(10): p. 2305-20. 138. Awasthi, A., et al., Deletion of PI3K-p85alpha gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells. Genes Immun, 2008. 9(6): p. 522-35. 139. Saudemont, A., K. Okkenhaug, and F. Colucci, p110delta is required for innate immunity to transplantable lymphomas. Biochem Soc Trans, 2007. 35(Pt 2): p. 183-5. 78

140. Franke, T.F., et al., Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science, 1997. 275(5300): p. 665-8. 141. Scheid, M.P., et al., Phosphatidylinositol (3,4,5)P3 is essential but not sufficient for protein kinase B (PKB) activation; phosphatidylinositol (3,4)P2 is required for PKB phosphorylation at Ser-473: studies using cells from SH2-containing inositol-5-phosphatase knockout mice. J Biol Chem, 2002. 277(11): p. 9027-35. 142. Ma, K., et al., PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity. Cell Signal, 2008. 20(4): p. 684-94. 143. Di Paolo, G., et al., Recruitment and regulation of phosphatidylinositol phosphate kinase type 1 gamma by the FERM domain of talin. Nature, 2002. 420(6911): p. 85-9. 144. Ling, K., et al., Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal adhesions. Nature, 2002. 420(6911): p. 89-93. 145. Galandrini, R., et al., Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity. Blood, 2005. 106(2): p. 577-83. 146. Mace, E.M., et al., Elucidation of the integrin LFA-1-mediated signaling pathway of actin polarization in natural killer cells. Blood, 2010. 116(8): p. 1272-9. 147. Satoh-Takayama, N., et al., IL-7 and IL-15 independently program the differentiation of intestinal CD3-NKp46+ cell subsets from Id2-dependent precursors. J Exp Med, 2010. 207(2): p. 273-80. 148. Liu, X., et al., Identification of microRNA transcriptome involved in human natural killer cell activation. Immunol Lett, 2012. 143(2): p. 208-17. 149. Fehniger, T.A., et al., Next-generation sequencing identifies the natural killer cell microRNA transcriptome. Genome Res, 2010. 20(11): p. 1590-604. 150. Yamanaka, Y., et al., Aberrant overexpression of microRNAs activate AKT signaling via down-regulation of tumor suppressors in natural killer-cell lymphoma/leukemia. Blood, 2009. 114(15): p. 3265-75. 151. Page, K.M., et al., Natural killer cells from protein kinase C theta-/- mice stimulated with interleukin-12 are deficient in production of interferon-gamma. J Leukoc Biol, 2008. 83(5): p. 1267-76. 152. Tassi, I., et al., NK cell-activating receptors require PKC-theta for sustained signaling, transcriptional activation, and IFN-gamma secretion. Blood, 2008. 112(10): p. 4109-16. 153. Aguilo, J.I., et al., Protein kinase C-theta is required for NK cell activation and in vivo control of tumor progression. J Immunol, 2009. 182(4): p. 1972-81. 154. Krzewski, K., et al., Formation of a WIP-, WASp-, actin-, and myosin IIA- containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling. J Cell Biol, 2006. 173(1): p. 121-32. 155. Maul-Pavicic, A., et al., ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Proc Natl Acad Sci U S A, 2011. 108(8): p. 3324-9.