Regulation of IL-22 Production by Immature Natural Killer Cells and CD16 Expression During Their Maturation

Regulation of IL-22 Production by Immature Natural Killer Cells and CD16 Expression during their Maturation

DISSERTATION

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

By Aaron Robert Victor, B.S. Graduate Program in Biomedical Sciences Graduate Program

The Ohio State University 2016

Dissertation Committee: Professor Michael A. Caligiuri, MD, Advisor Professor Mark D. Wewers, MD Professor Natarajan Muthusamy, DVM, PhD Professor Jianhua Yu, PhD

Abstract

Natural killer (NK) cells are innate immune cells that play different roles depending on

their stage of development. NK cells develop through five stages that can be identified in

secondary lymphoid tissue. Stage 3 immature NK (iNK cells) cells play important roles in

homeostasis and defense against infection. They produce the cytokines interleukin (IL)-17

and IL-22 and express the transcription factor RORγt. Because IL-22 is critical to the antimicrobial immune response, we sought to identify factors that can promote proliferation and induce or maintain IL-22 production by iNK cells and determine a molecular

mechanism for this process. We identified IL-18 as a cytokine that cooperates with an iNK

cells cell survival factor, IL-15, to induce proliferation of iNK cells, as well as induce and

maintain IL-22 production. We found that this effect was mediated through the NFκB

pathway. Furthermore, we observed that IL-18-producing CD11c+ dendritic cells are

found proximal to iNK cells in human tonsil. Stage 4 and 5 mature NK cells are

distinguished from iNK cells in several ways. Mature NK cells play roles in antiviral and

antitumor responses. They express CD94, produce the cytokine IFN-γ, and display

cytotoxic activity. Stage 5 NK cells are distinguished from stage 4 NK cells by expression

of CD16. Because very little is known about how CD16 is regulated, we also sought to

identify pre-translational mechanisms governing this process. First, we used chromatin

immunoprecipitation (ChIP) to test for the association of STAT1, GATA3, SRF, and SP1 ii

with the CD16 promoter. While each of these transcription factors has a consensus binding sequence in the CD16 promoter, we did not find enrichment of any of these factors. We also investigated DNA methylation in the CD16 promoter and found that it was hypermethylated in stage 4 NK cells and hypomethylated in stage 5 NK cells. These data identify DNA methylation as a possible regulatory mechanism for CD16 expression in NK cells. Finally, we identified a microRNA, mir-218, that seems to negatively regulate CD16.

Mir-218 reduces translation of mRNA when it recognizes the CD16 3’ untranslated region in the human embryonic kidney 293t cell line. Furthermore, we found evidence that mir-

218 overexpression can reduce CD16 surface expression in mature NK cells; these data were confirmed in monocytes. These data are the first to show evidence of direct regulation of CD16 at a pre-translational level. Taken together, these discoveries from different stages of NK cell development contribute important elements that may be applied in the future to improve therapeutic targets in a variety of disease settings.

iii

To Sapna and my parents, Lois and Robert, for their enduring support

Acknowledgments

I would like to thank my advisor, Dr. Michael Caligiuri, for his insight, support, and

guidance. His unflinching confidence in my ability to find a way forward combined with

the wide variety of resources he opened up for me have been instrumental in my training.

Additionally, the lessons I have learned under his mentorship have proven invaluable and

I believe will continue to pay dividends in the years to come.

Thank you to my committee members, Dr. Mark Wewers, Dr. Natarajan Muthusamy, and

Dr. Jianhua Yu. They have challenged my thinking, pushed me to pursue new ideas, and

offered support and understanding. Thank you especially to Dr. Yu. His involvement

with my work from the beginning has driven me to develop many of the skills a scientific

investigator needs to drive his field forward.

I have received tremendous support and instruction from many excellent and talented

people. I would especially like to thank Dr. Shun He, Dr. Nicholas Zorko, and Min Wei

for teaching me so many of the skills and techniques I would require in the research

pursuits. I would also like to recognize Hsiaoyin Mao, Steven Scoville, Dr. Tiffany

Hughes, Dr. Aharon Freud, Dr. Bethany Mundy-Bosse, Dr. Wing Keung Chan, Dr.

Hongsheng Dai, Dr. Susheela Tridandapani, Benjamin Sunkel, and Dr. Qianben Wang.

Each of them has provided essential knowledge, counsel, and expertise to have made

critical advancements in my project possible. My thanks also go out to all the members of the Caligiuri lab.

I would like to thank my family members, who have supported my endeavors to improve myself and pursue my interests. They have always believed in me and encouraged me to go forward. Thank you especially to my parents for creating an environment where my curiosity was encouraged and always asking what they could do to help.

I owe a great debt of gratitude to my wife, Sapna, for her patience and support as I pursue my passion for biomedical research. Her support and love have propelled me to overcome challenge after challenge.

Vita

30 June 1986 ...... Born—Wheeling, West Virginia New Philadelphia High School B.S. Biochemistry, Case Western Reserve University 2009 to present ...... M.D. Student, The Ohio State University Medical Scientist Training Program, The Ohio State University

Publications Peer-reviewed articles Schrock JW, Glasenapp M, Victor A, Losey T, Cydulka, RK. Variables associated with discordance between emergency physician and neurologist diagnoses of transient ischemic attacks in the emergency department. Ann Emerg Med. 2012;59(1):19-26.

Schrock JW, Victor A, Losey T. Can the ABCD2 risk score predict positive diagnostic testing for emergency department patients admitted for transient ischemic attack? Stroke. 2009;40(10):3202-5.

Fields of Study

Major Field: Biomedical Sciences Graduate Program

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Table of Contents

Abstract ...... ii

Acknowledgments...... v

List of Figures ...... xiii

Chapter 1: Background ...... 1

1.1 Natural killer cell development and function...... 1

1.2 Fc receptors in immunity ...... 2

1.2.1 The activating Fc receptor FcγRIIIA ...... 3

1.2.2 Homology between FCGR3A and FCGR3B ...... 5

1.2.3 NK cell CD16 expression and cancer ...... 7

1.2.4 CD16 shedding following NK cell stimulation ...... 8

Chapter 2: Interleukin-18 drives ILC3 proliferation and promotes interleukin-22 production via NFκB...... 10

2.1 Introduction ...... 10

2.2 Materials and Methods ...... 12

2.2.1 Isolation of human NK precursors ...... 12

2.2.2 Flow cytometry ...... 12

2.2.3 Cell culture ...... 13

2.2.4 Cell lysis and immunoblotting ...... 13 viii

2.2.5 EMSA and antibody-supershift assays ...... 13

2.2.6 Transient transfection and luciferase assay...... 14

2.2.7 Immunohistochemistry ...... 14

2.3 Results ...... 16

2.3.1 IL-18 stimulation promotes iNK cell proliferation ...... 16

Expression of IL-18Rα and IL-18Rβ on the cell surface of iNK cells ...... 18

2.3.3 IL-18 sustains IL-22 protein expression in iNK cells ...... 20

2.3.4 IL-18 activates NFκB via phosphorylation of p65 and induces NFκB

binding to the IL22 promoter ...... 22

2.3.5 NFκB subunits p65 and p50 positively regulate transcription at the IL22

promoter ...... 24

2.3.6 CD117+ iNK cells reside near IL-18 producing dendritic cells (DCs) in

hSLT ...... 25

2.4 Discussion ...... 27

Chapter 3: Transcriptional regulation of FCGR3A ...... 30

3.1 Introduction ...... 30

3.2 Materials and Methods ...... 32

3.2.1 Isolation of primary NK cells from peripheral blood or tonsil ...... 32

3.2.2 Expansion of stage 4 NK cells ...... 32

3.2.3 Real-time RT-PCR ...... 33

3.2.4 Chromatin Immunoprecipitation Assays ...... 34

3.2.5 Cell culture and NK cell stimulations ...... 34

3.3 Results ...... 35

3.3.1 Predicted transcription factor gene expression in NK cells ...... 35

3.3.2 Chromatin Immunoprecipitation for predicted transcription factors ...... 37

3.4 Discussion ...... 46

Chapter 4: Epigenetic regulation of FCGR3A ...... 47

4.1 Introduction ...... 47

4.1.1 DNA methylation at the FCGR3A locus ...... 47

4.2 Materials and Methods ...... 48

4.2.1 Cell culture and treatment with 5- aza-2’-deoxycytidine ...... 48

4.2.1 DNA methylation analysis ...... 48

4.3 Results ...... 49

4.3.1 Demethylation of NK cells with 5-aza...... 49

4.4 Discussion ...... 53

Chapter 5: MicroRNA regulation of FCGR3A ...... 54

5.1 Introduction ...... 54

5.2 Material and Methods ...... 55

5.2.1 Isolation of primary NK cells from peripheral blood ...... 55

5.2.2 Isolation of primary monocytes from peripheral blood ...... 55 x

5.2.3 miRNA Expression Assay ...... 56

5.2.4 Expansion of stage 4 NK cells ...... 56

5.2.5 Real-time PCR ...... 57

5.2.6 Luciferase assay ...... 57

5.2.7 Lentiviral infection of primary human NK cells...... 58

5.2.8 Transient transfection of primary human NK cells with miRNA mimic or

inhibitor ...... 59

5.2.9 Electroporation of monocytes for transfection with mir-218 ...... 59

5.2.10 Antibodies and flow cytometric analysis ...... 60

5.3 Results ...... 61

5.3.1 Prediction of miRNAs that target FCGR3A ...... 61

5.3.2 Validation of putative miRNA regulators of FCGR3A ...... 65

5.3.3 Overexpression of mir-218 in human NK cells by lentiviral infection ...... 67

5.3.4 Modulation of mir-218 in human NK cells by transfection with miRNA

mimic or inhibitor ...... 69

5.3.5 Overexpression of mir-218 in human monocytes by transient transfection 71

Chapter 6: Extended discussion and future directions ...... 73

6.1 Time course of CD16 acquisition by NK cells is unclear ...... 73

6.2 Genomic landscape and the potential role of epigenetics in FCGR3A regulation 76

6.3 MicroRNA regulation of CD16 ...... 79

6.4 Concluding Remarks ...... 81

References ...... 82

xii

List of Figures

Figure 1. Promoter region determines tissue specificity of CD16 expression ...... 7

Figure 2. iNK cell proliferation in response to treatment with cytokines in the presence of

IL-15 ...... 17

Figure 3. Expression of IL-18Rα and IL-18Rβ on NK cell developmental intermediates freshly isolated from human tonsil ...... 19

Figure 4. iNK cells treated with IL-18 maintain expression of IL-22 ...... 21

Figure 5. NFκB is activated by IL-18 and binds to the IL-22 promoter ...... 23

Figure 6. NFκB subunits p65 and p50 positively regulate transcription through the IL22

...... 24

Figure 7. iNK cells reside in proximity to IL-18+CD11c+ DCs within the human tonsil 26

Figure 8. Gene expression in CD16neg and CD16pos NK cells ...... 36

Figure 9. Comparison of NK cell line FCGR3A expression to primary CD16neg NK cell

FCGR3A expression ...... 38

Figure 10. ChIP for STAT1 and SP1 at the FCGR3A promoter ...... 40

Figure 11. ChIP for SRF at the FCGR3A promoter ...... 41

Figure 12. Enrichment of PolII and GATA3 at the FCGR3A promoter, IL2RB promoter,

and IL2RA promoter in YT and huNK cells ...... 43

Figure 13. Enrichment at the FCGR3A promoter and IL2RB promoter with stimulated

and unstimulated huNK cells ...... 45

Figure 14. Analysis of FCGR3A promoter methylation ...... 51

Figure 15. Strategy to predict miRNAs targeting FCGR3A ...... 62 xiii

Figure 16. Candidate miRNA expression in NK cells ...... 64

Figure 17. MicroRNA regulation of the 3’ UTR of FCGR3A ...... 66

Figure 18. Infection of primary NK cells with mir-218 virus...... 68

Figure 19. Transfection of primary NK cells with mir-218 miRNA inhibitor or mimic .. 70

Figure 20. Mir-218 overexpression in primary monocytes ...... 72

xiv

Chapter 1: Background

1.1 Natural killer cell development and function

Natural killer (NK) cells play several important and varied roles as they develop from very early progenitor cells to terminally differentiated effector cells (1-4). Human NK cells develop in five stages (5). Stages 1 and 2 are known for their multi-lineage potential (6-8).

Stage 3 NK cells, also known as immature NK (iNK) cells, have effector functions that are

distinct from stage 4 and 5 NK cells, which are considered “mature” (2,9,10). iNK cells can be minimally defined as lin-CD34-CD117+ lymphocytes. These cells are phenotypically indistinguishable from ILC3s, which are the innate cognates of Th17 cells

(2,9-12). This suggests that human ILC3s are a developmental intermediate of NK cells rather than a distinct lineage, whereas in mice ILC3s and NK cells are separate lineages.

As innate cognates of Th17 cells, iNK cells are dependent on the transcription factors

RORγt and AHR and produce the cytokines IL-17 and IL-22 (4,11). IL-22 production is characteristic of iNK cells and is important for mediating iNK cell function. IL-22 mediates homeostasis, particularly in the gut by providing proliferative and pro-survival signaling to epithelial cells (13). IL-22 production is found at sites of inflammation and may mediate local wound healing and epithelial barrier repair (14). Moreover, while IL-22 is not critical for the formation of secondary lymphoid tissue (SLT) in the gut, in the absence of IL-22 during infection, SLT structures such isolated lymphoid follicles and mucosa-associated lymphoid tissue are lost (15). 1

Mature NK cells are found primarily in the peripheral blood (1,5,16). They are innate cognates of the cytotoxic T lymphocyte (11). Mature NK cells are minimally defined as lin-CD94+CD56+CD16- or lin-CD94+/-CD56+CD16+.Mature NK cells are dependent on the transcription factor TBET, produce the cytokines IFN-γ and TNF-α, and display potent cytotoxicity (11,17). Mature NK cells and iNK cells also play distinct roles in immunity.

Mature NK have antiviral and antitumor activity, while iNK cells are involved in antimicrobial immunity and the maintenance of homeostasis.

1.2 Fc receptors in immunity

In response to foreign antigen, the adaptive immune system undergoes a process that results in the production of antibodies that specifically recognize the foreign antigen (18).

Antibodies are able to target a diverse array of epitopes through their variable domains.

The tail of antibodies are composed of a constant region. Three fragments result from digesting an antibody with the cysteine protease papain. Two are identical and contain the variable domains that have antigen binding activity. These are called Fab fragments for

Fragment antigen binding (19). The third fragment contains the constant region, or Fc fragment for Fragment crystallizable, so named for its ability to readily crystallize. Many cell types express one or more receptors that recognize the Fc region of an antibody, and are called Fc receptors. Fc receptors can be activating or inhibitory (18,20). Some cells even express both activating and inhibitory receptors (18). The balance of ligation between antibody Fc and activating or inhibitory Fc receptors is an important determinant of the immune response to cancer (20). Clinically effective therapeutic antibodies such as

trastuzumab and rituximab engage both an activating Fc receptor and an inhibitory Fc receptor in vivo and modulate the cytotoxic response of effector cells (20). For example, the low affinity Fc gamma receptor type IIIA (FcγRIIIA or CD16) is an activating Fc receptor that is required for antibody-dependent cell-mediated cytotoxicity (ADCC) (20-

24). ADCC is ineffective in the treatment of human cancers in the absence of CD16 expression or when CD16 poorly binds the antibody Fc region (20,21).

In order to bias antibody engagement toward activating rather than inhibitory Fc receptors, and thereby improve clinical efficacy, several strategies are being employed in the design of therapeutic monoclonal antibodies (25). The attached sugar moieties are modified to reduce sialic acid and fucose. Furthermore, amino acids in the Fc region recognized by Fc receptors are changed to improve affinity for activating Fc receptors and reduce affinity for inhibitory Fc receptors. Fc receptors are broadly expressed on hematopoietic lineage cell types, such as B cells, mast cells, neutrophils, dendritic cells, monocytes, macrophages, and natural killer (NK) cells (18,25).

1.2.1 The activating Fc receptor FcγRIIIA

Among the cell types that express Fc receptors, NK cells are unique in that they are the only type to express only an activating Fc receptor (FcγRIIIA) and no inhibitory Fc receptor (25). Other cell types that express the activating Fc receptor FcγRIIIA are macrophages, and some monocytes. FcγRIIIA, also known as the surface antigen CD16, is coded by the gene FCGR3A (18,25).

NK cells are large granular lymphocytes that have two main effector functions, cytotoxicity

and cytokine production. One of the hallmark characteristics of NK cells is the potential

for spontaneous, or natural, killing target cells. Unlike other cell types such as cytotoxic T

lymphocytes, NK cells do not require priming or co-activation in order to kill target cells.

Thus, they are called “natural killer” cells. A second type of killing that NK cells engage

in is ADCC, which, as described above, requires engagement of antibody recognizing a

target cell. Not all NK cells mediate ADCC because not all NK cells express CD16. Five

stages of NK cell development have been identified (8). Stages 4 and 5 are considered mature and possess natural cytotoxicity and cytokine production capabilities. Stage 4 NK cells have lower cytotoxicity and higher capacity for interferon gamma production. Stage

5 NK cells are more cytotoxic. These mature NK cell stages are phenotypically distinguished by CD16, which is absent or poorly expressed by stage 4 NK cells and highly expressed by stage 5 NK cells. NK, NKT, and T cell lines broadly lack CD16 surface expression (26). CD16 is a marker of terminal differentiation, and seems to be lost as a cell takes on a less mature phenotype, such as that of a cancer cell line. For example, the NK- like cell line NKL is derived from an aggressive human natural killer cell leukemia (27).

The leukemia observed in the peripheral blood of the patient was composed of large granular lymphocytes with the surface phenotype CD3-CD16+CD56+. Isolated leukemia cells were able to mediate natural cytotoxicity and ADCC in vitro and exhibited proliferation similar to normal stage 5 NK cells. However, after prolonged culture, the

NKL cells lost surface expression of CD16 and CD57, both late markers of NK cell maturity.

CD16 has two extracellular immunoglobulin (Ig)-like domains. The distal domain is

recognized by the B73.1 antibody clone, while the proximal domain is recognized by the

3G8 clone. CD16 recognition with the 3G8 clone blocks ADCC while recognition with the

B73.1 clone blocks the association of CD16 with CD2, a process that seems to be important

for spontaneous killing of virus-infected cells (28). Each domain functions independently,

and therefore, the 3G8 clone does not impair NK cell natural cytotoxicity, while the B73.1

clone does not impair ADCC.

1.2.2 Homology between FCGR3A and FCGR3B

A highly homologous gene, FCGR3B, codes for the activating Fc receptor FcγRIIIB which

is expressed on neutrophils. These two genes, FCGR3A and FCGR3B, have greater than

95% sequence homology along the gene body but also in flanking regions (29). Because

of their high degree of sequence homology, flow cytometry antibodies that detect CD16 on

NK cells (coded by FCGR3A) also detect CD16 on neutrophils (coded by FCGR3B). Aside

from their tissue specificity, the two main differences between the two genes are their

membrane linkage and function. FcγRIIIB is glycophosphatidylinosital (GPI)-anchored to the cell surface while FcγRIIIA is a transmembrane receptor (30). The difference in cell membrane linkage arises from a single amino acid exchange, Phe-203 found in FcγRIIIA for Ser-203 found in neutrophils (31). Moreover, FcγRIIIA has a transmembrane domain and a short cytoplasmic tail which allow it to associate with the immunoreceptor tyrosine- based activation motif-containing adaptor proteins TCRζ and FcεRI (32). On NK cells and macrophages, CD16 mediates ADCC and phagocytosis. On neutrophils CD16 functions as

a sink for immune complexes (29). Neutrophils secrete toxic products in response to immune complexes bound by FcγRIIIB, but FcγRIIIB does not play a significant role in phagocytosis or killing of opsonized bacteria (33).

The proximal promoter regions of FCGR3A and FCGR3B differ by only 10 base positions

(29). Farther upstream, differences in promoter sequence become even rarer. Still, these small differences seem to account for the high degree of tissue specificity for the expression of each gene. Li et al created a series of transgenic mice to illustrate this point (29). Taking the human FCGR3A or FCGR3B genes and their promoter regions, they created transgenic mice that expressed the corresponding human Fc receptor. Interestingly, the human gene

FCGR3A was expressed only on natural killer cells as CD16. Likewise, the human gene

FCGR3B was expressed only on neutrophils. An additional pair of transgenic mice was created, this time with the promoters (along with exon 1 and intron 1) switched between the two transgenes (Figure 1). The tissue specificity of the transgene was determined by the promoter sequence plus exon 1 and intron 1. Transgenic mice with the FCGR3A promoter expressed CD16 on NK cells, despite having mostly the FCGR3B coding sequence. Likewise, transgenic mice with the FCGR3B promoter expressed CD16 on neutrophils, despite having mostly FCGR3A coding sequence. Importantly, this tissue specificity did not depend on the amino acid exchange at position 203, which is coded by exon 4. This study provides important evidence that the promoter sequence of FCGR3A, while being highly homologous to FCGR3B, is the key to regulation of CD16 expression in natural killer cells.

Figure 1. Promoter region determines tissue specificity of CD16 expression. Adapted Figure 1. Promoter region determines tissue specificity of CD16 expression from Li et al. Representation of the FCGR3 transgenes used to create the transgenic mice in Li et al. Each transgenic mouse displayed tissue specific CD16 expression, as

indicated. E = exon, I = intron.

1.2.3 NK cell CD16 expression and cancer

Understanding the regulation of CD16 in natural killer cells is crucial because NK cells

expressing CD16 can target and kill tumor cells via ADCC (34) to improve patient outcomes when monoclonal (mAb) immune therapy is used in conjunction with conventional chemotherapy (25,35-40). Some patients have a specific polymorphism at position 158 of FcγRIIIA (30,41). The dominant allele has a frequency of ~0.6 in the population and codes for a phenylalanine (F) at position 158 (41). The minor allele codes for valine (V) instead. Patients with two copies of the minor allele (V/V) are observed to have significantly longer progression-free survival when receiving antibody therapy compared with heterozygous patients (V/F) or patients homozygous for the dominant allele

(F/F) (21,39,42). Several factors seem to contribute to this effect. First, NK cells from V/V donors bind to antibody Fc much more strongly than F/F donors (41). As such, V/V NK 7

cells require less than a quarter as much antibody to kill target cells through ADCC as F/F

NK cells require (43). Furthermore, V/V NK cells express higher levels of FCGR3A

mRNA and CD16 surface expression compared to F/F NK cells, which also means that

V/V NK cells can bind more antibodies at once, leading to increased antitumor effector

function (44).

1.2.4 CD16 shedding following NK cell stimulation

While the downstream effects of CD16 ligation to antibody or Fc fragments have been well

documented (22,45-53), the mechanism by which CD16 expression is regulated in human

NK cells remains largely unknown. Because some cancers are able to decrease NK cell

expression of CD16 as a means of reducing NK-mediated ADCC (54-56), a better understanding of NK cell regulation of CD16 expression is critical to overcoming this method of tumor immunoevasion.

It has long been known that mitogen stimulation and co-culture with malignant targets can cause surface shedding of CD16 from NK cells (57-59). Recently, ADAM17 was identified as the metalloprotease responsible for proteolytic cleavage of the extracellular portion of

CD16 which is shed from NK cells in response to several types of stimulation, including phorbol 12-myristate 13-acetate (PMA), K562 cells, and combination IL-12+IL-18 (60).

Overnight stimulation of total peripheral blood-derived NK cells with combination IL-

12+IL-18 stimulation resulted in ~35% reduction in CD16 expression. Another group has

shown that NK cells cultured in IL-18 alone can reduce CD16 expression (61). Treatment

of NK cells with an ADAM17 inhibitor was shown to decrease CD16 shedding and increase NK cell activation (62).

Chapter 2: Interleukin-18 drives ILC3 proliferation and promotes

interleukin-22 production via NFκB

2.1 Introduction

Innate lymphoid cells (ILCs) play many roles in protective immunity and disease (63).

There are several types of ILCs, which produce characteristic cytokine profiles and transcription factors (64). Group 3 ILCs are composed of iNK cells and lymphoid tissue inducer cells (LTi) and are dependent on the transcription factors RAR-related orphan receptor gamma (RORγt) and aryl hydrocarbon receptor (AHR) and produce IL-17A and

IL-22 (65). iNK cells are enriched within human secondary lymphoid tissue (hSLT), such as the tonsil and lymph nodes (6,8). iNK cells are critical regulators of homeostasis and immunity (2,10,66,67). iNK cells are the primary steady state source of IL-22, a cytokine critical for tissue regeneration and for maintenance of barrier function in the gut, skin, oral mucosa, and lung (65,68). IL-22 signaling in epithelial cells drives genes involved in proliferation and wound healing

(13,69). In addition to bolstering the physical barrier of the epithelium, IL-22 stimulates epithelial cells to produce anti-microbial peptides necessary for barrier maintenance and prevention of infection by commensal bacteria (70). RORγt+ ILCs are also necessary for the formation of cryptopatches and isolated lymphoid follicles in the intestinal lamina

propria (71,72) as well as repair of lymph nodes following infection (73). IL-22 and IL-18

have recently been shown to cooperatively contribute to murine intestinal immunity to various

infections. IL-18 induces IL-22 during T. gondii infection in the murine ileum, while IL-22 induces

IL-18 during C. rodentium infection (74). A combination of IL-18 and IL-22 was shown to be critical for clearance infection by rotavirus-infected mice (75).

In this study, we identified IL-18 as a cytokine that can induce proliferation of and sustain

IL-22 production by iNK cells. IL-18 signals through the IL-18 receptor to activate NFκB

signaling. In the tonsil, iNK cells reside in close proximity to dendritic cells (DCs), a source

of IL-18. Together, these data support the hypothesis that DC-derived IL-18 stimulates iNK cell function by maintaining the population through proliferation and by sustaining production of IL-22 through an NFκB-dependent mechanism. Our study further clarifies the role of DCs in iNK cell function and identifies NFκB as a potential target for future therapies against IL-22-mediated diseases.

2.2 Materials and Methods

2.2.1 Isolation of human NK precursors

All procedures were performed with approval of the Ohio State University Institutional

Review Board. Normal pediatric tonsils were obtained following routine tonsillectomy

from Nationwide Children’s Hospital (Columbus, OH). Stage 1-3 NK developmental

intermediates were isolated as previously described (8). Briefly, total mononuclear cells

were depleted of CD19+ and/or CD3+ cells via magnetic negative selection (Miltenyi

Biotec). For some experiments, B and/or T cell depleted mononuclear cells were used

immediately for flow cytometric analysis. Alternatively, iNK cells were sorted directly

from the depleted fraction by gating on CD3-CD34-CD16-CD94-CD117+ events on a

FACSAria II cell sorter (BD Biosciences). Purity analysis routinely revealed that sorted

populations were ≥97% pure.

2.2.2 Flow cytometry

Antibodies for human CD34, CD3, and CD117 were purchased from BD Biosciences,

whereas those for CD94, IL-18Rα, IL-18Rβ, and IL-22 were purchased from R&D

Systems. Unless otherwise indicated, antibodies were used according to manufacturers’

instructions. Staining for intracellular IL-22 was performed following a 4-hour incubation in 2 μM GolgiPlug (BD Biosciences), and using the Cytofix/Cytoperm Plus

Fixation/Permeabilization Kit (BD Biosciences) and α-IL-22 PE (R&D Systems). Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences) and analysis was performed using FlowJo Software (Treestar, Inc.).

2.2.3 Cell culture

iNK cells purified by fluorescence activated cell sorting (FACS) were cultured in a round-

bottom 96-well plate (Costar) at a starting density of 2.5x104 cells/mL in α-MEM medium

containing 10% fetal bovine serum, penicillin G (100 μg/mL), and streptomycin (100

μg/mL) (Invitrogen). Cells were cultured with the indicated recombinant human cytokines,

including IL-15 (1 nM, Amgen), IL-18 (100 ng/mL, MBL), IL-12 (10 ng/mL, Genetics

Institute), IL-6 (20 ng/mL, R&D), IL-27 (10 ng/mL, R&D), IL-21 (100 ng/mL, R&D), IL-

23 (20 ng/mL, Miltenyi), IL-7 (10 ng/mL, Miltenyi), IL-10 (10 ng/mL, Shering), IL-25

(100 ng/mL, R&D), IFN-α (20 U/mL, Shering), IFN-γ (2 U/mL, Peprotech), TRAIL (10

ng/mL, R&D), and TGF-β (20 ng/mL, R&D). All cell culture was performed in the

presence of IL-15 because it serves as a survival factor for iNK cells (76).

2.2.4 Cell lysis and immunoblotting

Protein lysates and immunoblotting were performed as described previously (77).

Antibodies used were: rabbit polyclonal anti-human GAPDH antibody (Santa Cruz), rabbit

polyclonal anti-human p65 antibody (Rockland), and rabbit monoclonal anti-human

phospho-p65ser536 (Cell Signaling).

2.2.5 EMSA and antibody-supershift assays

Nuclear extracts were isolated using a Nuclear Extraction Kit (Active Motif).

Complementary oligonucleotides—probe 1 (nt 248 to 216) and probe 2 (nt 191 to 163)—

containing a putative NFκB binding site in the human IL22 promoter were synthesized. 13

Electrophoretic mobility shift assays (EMSA) were performed as previously described

(78). In brief, probe 1 and 2 were 32P-labeled and incubated with the nuclear extracts (2

μg) in the presence of poly[dI-dC] (1 μg). For the antibody gel supershift assays, nuclear

extracts were incubated with antibodies for p65 (Rockland) or p50 (Millipore) overnight at

4°C before the addition of the IL22 promotor probes.

2.2.6 Transient transfection and luciferase assay

293T cells were maintained in DMEM supplemented with 10% fetal bovine serum. The

cell line was obtained from ATCC in 2008; it has not been authenticated since receipt.

293T cells were seeded into 24-well plates at a density of 2.5x104 cells per well and grown

overnight. The IL22-Luc construct and p50, p65, STAT5, or empty expression vector or no

vector were co-transfected with Lipofectamine 2000 with Plus Reagent (Invitrogen). The

pGL3 basic reporter vector was used as a control for basal promoter activity. A renilla

luciferase vector, pRL-TK (Promega), was co-transfected to serve as an internal control for

transfection efficiency. Cells were harvested after 48 hours and assessed for luciferase

activity as previously described (79).

2.2.7 Immunohistochemistry

Paraffin-embedded tonsillar tissue sections (0.5 μm in thickness) were stained for immunohistochemistry as previously described (6,9,80) using the UltraView Universal system (Ventana Medical). Sections were stained with the indicated antibodies, including

α-CD11c (1:100, Abcam), α-IL-18 (1:500 Abcam), and α-CD117 (1:500, Dako). Images 14

were digitally captured using a DP12 camera, a BX50 microscope, and UPLANF1

objectives (Olympus). 3,3’-Diaminobenzidine and fast-red-stained sections were digitally converted to fluorescent green and red, respectively, using the Nuance FX system

(Cambridge Research & Instrumentation).

2.3 Results

2.3.1 IL-18 stimulation promotes iNK cell proliferation

To explore the role of IL-18 and other cytokines in the expansion of iNK cells, CD3-

CD34-CD94-CD117+ iNK cells were purified by FACS from fresh human tonsils. iNK

cells were then stimulated with cytokines as indicated, which could potentially act on

iNK cells (Figure 2A). All iNK cell cultures were performed in the presence of IL-15, which served as a survival factor, and for a duration 14 days. iNK cells, stimulated with

IL-6, TGF-β, IL-21, IL-27, IL-23, IFN-α, IL-10, IL-7, TRAIL, IL-25, IL-12, or TGF-β

plus IL-6, demonstrated little to no increased expansion—and in several cases

demonstrated decreased expansion—compared to controls with IL-15 alone. However,

when iNK cells were cultured with IL-18 they demonstrated ~12-fold increase in

expansion compared to that of control cultures (Figure 2A). We compared treatment of

iNK cells with IL-18, TGF-β, and IL-18 plus IL-12 or TGF-β to determine if IL-12 or

TGF-β enhanced or impaired iNK cell proliferation, respectively. The addition or IL-12 or TGF-β did not significantly alter the proliferation of iNK cells compared to stimulation with IL-18 (data not shown). Additionally, treatment of iNK cells with IFN-γ led to a decrease in cell number compared to controls (data not shown). To determine if the increase in cell number in the presence of IL-18 was due to proliferation rather than survival, we performed a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. After 14 days, cultured cells were harvested, counted, and examined for the incorporation of EdU by flow cytometry. An increase in EdU incorporation was observed following culture with IL-18 compared to controls (Figure 2B), suggesting that the quantitative expansion

of iNK cells following IL-18 stimulation is due, at least in part, to an increase in proliferation.

Figure 22. .iNK iNK cell cell proliferation proliferation in response in response to treatment to treatment with cytokines with incytokines the presence in the of

ILpresence-15 of IL-15. (A) Relative fold change was calculated and averaged from 6 donors

as the absolute number of cells enumerated (by Trypan blue exclusion) after 14 days of

culture in IL-15 plus cytokine divided by the absolute number of cells enumerated after 14

days of culture in IL-15 alone (p < 0.01, IL-18 vs other treatments; n=6). (B) Proliferation

was assessed via 5-ethynyl-2’-deoxyuridine (EdU) incorporation in total iNK cells treated

with IL-15 plus IL-18 or IL-15 alone. One of three experiments with similar results is

presented. 17

Expression of IL-18Rα and IL-18Rβ on the cell surface of iNK cells

Human IL-18 signals through a heterodimeric receptor composed of the IL-18Rα and IL-

18Rβ subunits (81). To validate that IL-18 can signal through its receptor on iNK cells, we examined the expression of IL-18Rα and IL-18Rβ on the surface of NK cell developmental intermediate by flow cytometry. Whereas surface IL-18Rβ was expressed by the majority of stage 1 pro-NK cells, stage 2 pre-NK cells and stage 3 iNK cells, robust surface expression of IL-18Rα was restricted to stage 3 iNK cells (Figure 3). This constitutive expression of the heterodimeric receptor on the stage 3 iNK cells is consistent with the above proliferation data and suggests that IL-18 signaling may have a unique role in regulating differentiation or homeostasis of iNK cells.

Figure 3. Expression of IL-18Rα and IL-18Rβ on NK cell developmental Figure 3. Expression of IL-18Rα and IL-18Rβ on NK cell developmental intermediates freshly isolated from human tonsil

intermediates freshly isolated from human tonsil. Histograms show a representative donor stained ex vivo with isotype (unfilled) or antibody specific for the indicated antigen

(filled). Stage 1 NK cells were defined as CD3-CD19-CD34+CD117-CD94-. Stage 2 NK cells were defined as CD3-CD19-CD34+CD117+CD94-. Stage 3 iNK cells were defined as CD3-CD19-CD34-CD117+CD94-.

2.3.3 IL-18 sustains IL-22 protein expression in iNK cells

Previous studies have demonstrated that freshly isolated iNK cells from tonsil

constitutively produce high levels of IL-22 (9,10,12). In order to determine whether IL-

18 plays a role in regulating IL-22 production, iNK cells were isolated from human tonsil

by FACS and cultured with or without IL-18 for 14 days. A minor fraction of cultured

cells expressed CD94 (which identifies mature NK cells) under both culture conditions.

We then sought to characterize the in vitro-derived cells generated in response to each treatment. Following culture, CD94 expression and IL-22 production were mutually exclusive, demonstrating that only iNK cells, and not in vitro-derived mature NK cells,

were capable of producing IL-22 in response to cultures containing IL-18 (Figure 4A).

While iNK cells cultured with IL-18 acquired CD94 surface expression at a rate similar to that of controls, the presence of IL-18 led to a larger fraction of cells expressing IL-22

(Figure 4B). Thus, IL-18 expands iNK cells and maintains their IL-22 production.

Figure 4. iNK cells treated with IL-18 maintain expression of IL-22. (A) Figure 4. iNK cells treated with IL-18 maintain expression of IL-22 Representative staining for surface CD94 and intracellular IL-22 protein at day 14 of culture. (B) Percentage of iNK cells staining positively for IL-22 was assessed via flow cytometry after a 4 hour culture with either IL-15 alone or IL-15+IL-18 (p < 0.05; n=4).

2.3.4 IL-18 activates NFκB via phosphorylation of p65 and induces NFκB binding to

the IL22 promoter

To determine a potential mechanism by which IL-18 may be regulating IL-22 production

in iNK cells, we considered the downstream intermediates through which IL-18 signals

(82,83), i.e. NFκB (84) and MAP kinases (85,86). We identified three NFκB binding sites in the promoter of the human IL22 gene, two of which are immediately adjacent to one another (Figure 5A). The addition of IL-18 to sorted iNK cells cultured in IL-15 revealed increased phosphorylation of NFκB p65 at serine 536 in iNK cells (Figure 5B). To test whether nuclear transcription factors in iNK cells bind to these sites, we synthesized two

DNA probes containing the NFκB binding sites in the IL-22 promoter (probe 1 containing the two adjacent NFκB binding sites and probe 2 containing the other). In electrophoretic mobility shift assays, more DNA-protein complexes were observed in nuclear lysates prepared from iNK cells treated with IL-18 in the presence of the survival cytokine IL-15

than from iNK cells treated with IL-15 alone (Figure 5C). Furthermore, an antibody

supershift experiment showed the presence of NFκB subunits p65 and p50 in the DNA-

protein complexes (Figure 5D). Taken together, these data demonstrate that

phosphorylated NFκB is capable of specific yet faint binding to the IL-22 promoter in iNK

cells stimulated with IL-15 alone, or specific and avid binding to the IL-22 promoter in

iNK cells stimulated with IL-15 plus IL-18. Taken together, these data suggest that NFκB probably regulates the expression of IL-22 through enhancer binding and that IL-18 activates NFκB by protein phosphorylation.

Figure 5. NFκB is activated by IL-18 and binds to the IL-22 promoter. (A) The IL22 promoter sequence. Boxes indicate predicted NFκB binding sites. Oligonucleotide probes were synthesized Figure 5. NFκB is activated by IL-18 and binds to the IL-22 promoter using the indicated ranges of the IL22 promoter sequence. (B) iNK cells were treated with either

IL-15 alone or IL-15+IL-18 and subjected to immunoblotting with the indicated antibodies. (C)

EMSA was performed with 32P-labeled predicted NFκB binding sites (probe 1 or probe 2) and nuclear extract (NE) prepared from iNK cells treated with IL-15 alone or IL-15 plus IL-18 or no

NE (-). The DNA-protein complexes are indicated with arrows. (D) Antibody gel supershift assay.

Probe 1 was incubated with NE from iNK cells treated with IL-15+IL-18 and pre-incubated with no antibody, p65 antibody, or p50 antibody, or without NE. The DNA-protein complexes and antibody gel supershifted complexes are indicated with arrows. 23

2.3.5 NFκB subunits p65 and p50 positively regulate transcription at the IL22 promoter

Since we established that NFκB can bind avidly to the IL22 promoter in an IL-18- dependent manner, we next set out to determine whether NFκB is also capable of regulating

IL-22 transcription. To this end, we performed a luciferase reporter gene assay in which the IL22 promoter was cloned immediately upstream of the luc gene in the pGL3 firefly luciferase construct and co-transfected with an expression vector containing a gene of interest, p65, p-50, or STAT5. We compared controls (empty expression vector or no expression vector) to co-transfection with p65 and p50. STAT5 was used as a presumed irrelevant control. Vectors overexpressing p65 and p50 resulted in a significant increase in luciferase activity compared to the empty expression vector and to no expression vector, as well as a vector containing STAT5 (Figure 6). These data further support the hypothesis that IL-18 positively regulates IL-22 transcription via NFκB.

Figure 6. NFκB subunits p65 and p50 positively regulate transcription through the IL22

Figurepromoter. 6. NFκB 293T subunits cells were p65 transfected and p50 positively with the regulate IL22-Luc transcription reporter construct through togetherthe IL22 with no expression vector, empty vector, p50, p65, or STAT5. ** denotes p < 0.01 (n=4). 24

2.3.6 CD117+ iNK cells reside near IL-18 producing dendritic cells (DCs) in hSLT

iNK cells were previously defined by immunohistochemistry (IHC) procedures as

lymphoid-shaped CD117+ cells residing within the parafollicular and lamina propria regions of the human tonsil (9,10,87). We hypothesized that CD11c+ DCs within the human

tonsil might be a physiological source of IL-18 for iNK cells. We performed IHC to

examine the expression of CD11c and IL-18 proteins within the parafollicular region of the

human tonsil. Indeed, CD11c and IL-18 appeared to be largely co-expressed by the same

cells (Figure 7A), suggesting that CD11c+ DCs are a primary source of IL-18 protein in hSLT. Furthermore, CD117+ iNK cells within the region of the tonsil studied did not

express IL-18 protein themselves, but were found in close proximity to IL-18 producing

DCs (Figure 7B). Together, these findings suggest that CD11c+ DCs are a robust

physiological source of IL-18, which we have shown is capable of stimulating CD117+

iNK cells resulting in quantitative expansion and production of IL-22.

Figure 7. iNK cells reside in proximity to IL-18+CD11c+ DCs within the human tonsil Figure 7. iNK cells reside in proximity to IL-18+CD11c+ DCs within the human tonsil.

Immunohistochemical staining depicted in sections of human tonsil. Original magnification is

4 00x. Blue is nuclear stain. (A) Brown indicates CD11c staining, red indicates IL-18 staining, and

brown indicates co-expression. (B) Brown indicates CD117 and red indicates IL-18.

2.4 Discussion

Human iNK cells play critical roles in antimicrobial immunity and homeostasis. As such, it is important to define the mechanisms by which these cells proliferate and regulate cytokine production. Of the thirteen cytokine conditions tested, only one (IL-18) resulted in significant expansion of iNK cells when cultured in combination with IL-15, a survival factor for iNK cells. Several cytokines we tested decreased iNK cell quantity, including

TGF-β, IFN-α, IFN-γ, IL-12, and IL-27. Interestingly, IL-18 was able to override the anti- proliferative effects of TGF-β.

IL-18 is an important regulator of ILC function. Group 1 ILCs—ILC1s and natural killer

(NK) cells—produce interferon-gamma (IFN-γ) in response to IL-18 stimulation (88-90).

Furthermore, IL-12 stimulation induces the IL-18 receptor on NK cells while IL-18 reciprocally induces the IL-12 receptor (80,84,88,91,92). This is hypothesized to be the mechanism by which the combination of IL-12 and IL-18 stimulation synergizes to activate

NK cells. Recent work in a murine rotavirus model showed that IL-18, IL-22, and ILCs are involved in clearing infection following flagellin challenge (75).

In this study we show that human iNK cells are responsive to IL-18 stimulation. In addition to driving iNK cell proliferation, we found that IL-18 can stimulate and sustain production of IL-22. IL-23 has been reported to stimulate IL-22 production in human (2) and murine

(93) iNK cells in the short term (<5 days), however, this effect does not appear to be sustained in human iNK cells (after 14 days), nor does IL-23 drive iNK cell proliferation

(9). Our earlier work showed that IL-1β induced iNK cell homeostasis (9), which is consistent with the data presented here, as both cytokines belong to the same cytokine

superfamily and are mediated by similar signaling pathways. However, here we also

provide a molecular mechanism (the NFκB signaling pathway) by which IL-18 induces the production of IL-22, and speculate that a comparable pathway exists for iNK cell activation by IL-1β.

NFκB mediates downstream processes induced by IL-18 in other immune cell types, such as B cells (94). Furthermore, these cells reside in the tonsil, and therefore may be able to receive IL-18 from the same source as iNK cells. In our study, we found that IL-18+ DCs

reside in close proximity to iNK cells in the tonsil. This suggests that DCs acting on iNK

cells may regulate the iNK cell response to various pathogens. High DC production of IL-

18 in hSLT may expand the pool of iNK cells ready to help combat microbial infection.

Together, our findings support a wealth of literature that suggests that dendritic cells have

important and complex interactions with ILCs (9,75,84,95,96).

Excess IL-22 production, which can be exacerbated by the presence of IL-18, can

contribute to immunological disease and cancer, likely through its tissue regenerative

effects (68,97). IL-22 is associated with a variety of cancers, including malignancies of the

skin, thyroid, lung, breast, stomach, pancreas, liver, cervix, and colon (68). iNK cells have

been shown to accumulate in gastrointestinal cancers and promote tumor progression

through IL-22 production (97,98). IL-18 is an important inflammatory mediator and has

also been associated with gastric cancer (99-101). Particularly in late stages of cancer, IL-

18 can drive angiogenesis (99,100), proliferation (101), and migration (100,101). IL-18’s

role in inflammation is a double-edged sword. On one hand it can play important roles in

activating immune cells to detect and kill pathogens (102,103) and cancer cells (61). On

the other hand, IL-18 can lead to immune dysfunction and contribute to a variety of diseases

(104) including autoimmune disease (105-108), inflammatory bowel disease (109-113),

cardiovascular disease (114-117), and cancer (118-120). It would be interesting to know whether these IL-18-associated diseases are mediated through IL-18’s action on iNK cells and IL-22.

By identifying NFκB as a positive transcriptional mediator of IL-22 production in iNK cells, we can now begin to explore the possibility of targeted therapy. Our findings suggest several mechanisms by which iNK cells may be targeted in anti-tumor therapy. IL-18 may be neutralized to prevent expansion of the iNK cell population and remove one of the drivers of IL-22 production. Furthermore, IL-22 production may be targeted more directly with the use of an inhibitor of NFκB.

In summary, IL-18 cooperates with IL-15 to promote iNK cell proliferation and IL-22 production. We describe an IL-18-induced, NFκB-mediated mechanism which regulates

IL-22 in iNK cells. At steady state, IL-18 produced by DCs mediates IL-22 production by iNK cells to help maintain normal tissue integrity. In some disease states such as autoimmune diseases, IL-18 production may be induced in order to commensurately increase the iNK cell population able to produce IL-22, thereby accommodating the increased demands for tissue repair and host defense.

Chapter 3: Transcriptional regulation of FCGR3A

3.1 Introduction

No transcription factor has been shown to regulate FCGR3A in human NK cells. To

address this gap in knowledge we sought to identify potential transcriptional regulators of

CD16 and validate their roles. In silico search tools predict few transcription factor (TF)

binding sites in the 5 kilobases (kb) upstream of the FCGR3A start codon. Searches through

the TRANSFAC database and the SABiosciences ChIP PCR primer design tool identified

only five factors. Most distal from the promoter, a POU2F1 (OCT1) binding site was

predicted ~3300 base pairs (bp) upstream of the FCGR3A start codon. A serum response

factor (SRF) consensus binding sequence ~2400 bp upstream of the start codon was also

identified. Both OCT1 and SRF binding sites are conserved at the FCGR3B locus.

Additionally, a consensus binding sequence for GATA-1, -2, and -3 was found ~1300 bp

upstream of the FCGR3A start codon, which is not found in the FCGR3B promoter. SRF

and GATA proteins have been implicated in cell cycle regulation and differentiation and

are known to interact with each other to regulate smooth and cardiac muscle-specific genes

(121). SRF is essential for mouse Treg and NKT-cell development (122). Three SP1 sites

were identified between positions -942 and -850. Most proximally, positions -121/-136

contain an interferon sensitive response element (ISRE). The ISRE site is conserved in the

FCGR3B promoter. In response to IFN-β, this element is bound by IRF7 or the complex interferon-stimulated gene factor 3 (ISGF3), which contains STAT1, STAT2, and IRF9. 30

We measured the expression levels of these predicted genes and attempted to identify one that specifically interacted with the FCGR3A promoter.

3.2 Materials and Methods

3.2.1 Isolation of primary NK cells from peripheral blood or tonsil

All human cell work was performed with approval of the Ohio State University

Institutional Review Board. Human NK cells were isolated from peripheral blood

leukopacks of healthy individuals (American Red Cross) by negative selection with

MACSxpress NK Cell Isolation Kit, human (Miltenyi). Briefly, blood was diluted with

1/5th volume of Hextend (BioTime, Inc) and gently rocked for 3 minutes. Samples were

centrifuged for 5 minutes at 90xg with low brake. Samples were allowed to settle for 20

minutes in a 37°C incubator cell culture incubator with 5% CO2. The top layer of cells were

collected into a new tube and resuspended in 1/10th the original blood volume. To each

sample 3.75mL of MACSxpress Cell Isolation Cocktail was added and gently mixed.

Samples were incubated for 20 minutes at room temperature. PBS + 2% FBS was added to

total volume of 40mL. Samples were placed on a magnet for 20 minutes. Cocktail-labeled

cells accumulated at the sides of the tube and the remaining cells were collected and labeled

for FACS sorting. Normal pediatric tonsils were obtained following routine tonsillectomy

from Nationwide Children’s Hospital (Columbus, OH). Stage 4 NK cells from tonsil were isolated as previously described (8).Cells were sorted to >97% purity.

3.2.2 Expansion of stage 4 NK cells

Peripheral blood mononuclear cells (PBMCs) from three different donors were isolated from peripheral blood leukopacks (American Red Cross) by Ficoll-Hypaque density gradient centrifugation. PBMCs were irradiated with 30 Gy of radiation and then mixed in

equal ratios. In a round bottom 96-well plate PBMCs were plated at a density of 1x104 mixed PBMCs per well in RPMI media supplemented with 10% human AB serum (Valley

Biomedical), 1x104 Dynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific), and 150 IU/mL rhIL-2 (Hoffman-LaRoche) at a total volume of 200μL per well. Stage 4

NK cells isolated from peripheral blood (or tonsil, as indicated) were sorted to >97% purity.

Then, these purified stage 4 NK cells were sorted into the previously prepared 96-well plate for co-culture with the mixed PBMCs. A total of 100 stage 4 cells were sorted into each well. Stage 4 cells were expanded ex vivo for three weeks. Every 7 days, half the media was removed and replaced with new RPMI media supplemented with 10% human AB serum, 150 IU/mL rhIL-2, 1x104 Dynabeads Human T-Activator CD3/CD28, and 1x104 irradiated, mixed PBMCs. Starting on day 10 and continuing every 3 days after that, media changes without Dynabeads and mixed PBMCs were performed.

3.2.3 Real-time RT-PCR

Total RNA was extracted using Total RNA Purification Plus Kit (Norgen Biotek Corp). cDNA was generated according to the manufacturer’s recommendations using

SuperScript® VILO Master Mix (ThermoFisher Scientific). cDNA was used with Power

SYBR Green Master Mix ((ThermoFisher Scientific). Data were analysis using the comparative δ-δ threshold cycle method and normalized to 18S RNA. Results are indicated as expression relative to internal control.

3.2.4 Chromatin Immunoprecipitation Assays

ChIP assays were performed as previously described (123) with the following modifications. NK cells were sonicated for 12 rounds of 10s sonication followed by 30s on ice. YT cells were sonicated for 6-8 rounds. The specific antibodies used in ChIP assays were the following: SP1 (sc-14027, Santa Cruz), SRF (sc25290, Santa Cruz), STAT1 (sc-

346, Santa Cruz), GATA3 (sc-9009, Santa Cruz), PolII (ab817, Abcam), and p-PolII

(ab5131, Abcam).

3.2.5 Cell culture and NK cell stimulations

YT cells were maintained in RPMI supplemented with 10% FBS. NK cells were cultured in RPMI supplemented with 10% human AB serum for 12 hours. Then media was changed to RPMI supplemented with 10% human AB serum, 10ng/mL rhIL-12 (Peprotech),

100ng/mL rhIL-15 (Amgen), and 100ng/mL rhIL-18 (MBL). NK cells were cultured an additional 12 hours and subjected to ChIP assay.

3.3 Results

3.3.1 Predicted transcription factor gene expression in NK cells

We sought to determine whether any of the TFs predicted by in silico tools do in fact bind

to the FCGR3A promoter and regulate its expression. We first measured FCGR3A mRNA expression levels in sorted primary NK cells to see if mRNA level correlated with CD16 surface expression. We found that freshly isolated CD16+ (CD16pos) primary NK cells express ~120-fold more FCGR3A mRNA than CD16- (CD16neg) NK cells (Figure 8A).

Additionally, tonsil derived ex vivo expanded NK cells sorted for CD16 show that

CD16pos gated cells express ~60-fold more FCGR3A mRNA than CD16neg gated NK cells (Figure 8B). Microarray expression data showed that GATA3 (but not other GATA proteins), SP1, STAT1, and SRF were moderately- to highly-expressed in both stage 4 and stage 5 NK cells (Figure 8C). These data were confirmed in human NK cells expanded and differentiated ex vivo (Figure 8D). OCT1 expression was below the level of sensitivity for the microarray and poorly expressed when measured by real-time PCR. We did not observe significant changes in any of these genes between stage 4 and stage 5 NK cells.

Figure 8. Gene expression in CD16neg and CD16pos NK cells Figure 8. Gene expression in CD16neg and CD16pos NK cells. (A) Real time PCR was

performed to measure expression of FCGR3A in freshly isolated NK cells sorted for CD16.

n= 6, p<0.05 (B) Real time PCR was performed to measure expression of FCGR3A in sorted CD16neg and CD16pos NK cells expanded ex vivo for three weeks. n=2, p<0.05 (C)

Microarray data was generated from pooled samples (n=1). Gene expression of TFs

predicted to bind the FCGR3A promoter is shown. The microarray did not include

FCGR3A, IRF7, or OCT1. (D) Real-time PCR to measure expression of TFs predicted to

bind the FCGR3A promoter. CD16neg and CD16pos NK cells were sorted from ex vivo

expanded NK cells after 3 weeks. SP1, n=3; IRF7, n=4; GATA3, n=6; SRF, n=4; OCT1,

n=5. Data presented in A, B, and C as mean ± SD. * indicates p<0.05.

3.3.2 Chromatin Immunoprecipitation for predicted transcription factors

Because transcriptional regulators of FCGR3A are unknown, we employed a number of different controls for our chromatin immunoprecipitation (ChIP) experiments. Because the fraction of CD16neg NK cells in the peripheral blood is small, we were not able to obtain enough cells from each donor for comparison with the CD16pos population. To overcome this limitation we employed either of the two following strategies. We obtained stage 4 NK cells from tonsil or peripheral blood and expanded them ex vivo for three weeks. This results in approximately one third of the total population acquiring high CD16 surface expression by flow cytometry. Then CD16pos and CD16neg NK cells were sorted to greater than 90% purity and used for chromatin immunoprecipitation experiments.

Alternatively, we used the NK-like cell line YT as a CD16neg NK cell population and used freshly isolated mature human NK cells from peripheral blood (enriched to >80% purity) as the CD16pos population. YT cells express no surface CD16 and very low amounts of

FCGR3A mRNA (even compared to CD16neg NK cells from peripheral blood, Figure 9).

More than 90% of peripheral blood NK cells express CD16 (89) so these cells did not undergo further purification to obtain a CD16pos population.

Figure 9. Comparison of NK cell line FCGR3A expression to primary CD16neg NK cell FCGR3A expression

Figure 9. Comparison of NK cell line FCGR3A expression to primary CD16neg NK cell FCGR3A expression. Real time PCR was performed to compare FCGR3A mRNA expression in each of the designated cell types. The p value comparing NK-92 and

CD16neg NK cells was not significant. For all other comparisons, p<0.05. Data presented as mean ± SD. * indicates p<0.05.

Mature human NK cells express IL2RB, the gene that codes for the beta subunit of the IL2

receptor, but not IL2RA, the alpha subunit (7). IL2RB is vital for NK cell proliferation and survival because NK cells are dependent on IL2 receptor signaling for survival. During maturation, NK progenitors lose IL2RA expression and gain IL2RB as mature NK cells.

YT cells, however, express both receptors (26). RNA polymerase II (PolII) is the RNA polymerase complex responsible for transcribing mRNA, microRNA, and most snRNA in the cell (124,125). Therefore, we expect to find PolII (or its active form, phospho-PolII) at the promoter of any actively transcribed gene. Furthermore, in addition to IL2RB, which should be expressed by both human NK cells and YT cells, we sometimes also probed for

GAPDH as an additional positive control. For chromatin immunoprecipitation experiments

(ChIP) we designed primer sets to amplify the promoter of each of the control genes, as well as several primer sets spanning FCGR3A positions -2450/+250, and primer sets designed to flank the predicted binding site of each transcription factor used.

We performed ChIP experiments to determine if and where the predicted TFs could bind to the FCGR3A promoter region. Using ex vivo expanded NK cells, we probed with antibodies against SP1 and STAT1. Neither SP1 nor STAT1 were enriched over non- specific controls in either CD16pos or CD16neg NK cells (Figure 10A). We used the

CCL5 promoter as a positive control and observed enrichment for STAT1 and a trend toward enrichment for SP1 (Figure 10B).

Figure 10. ChIP for STAT1 and SP1 at the FCGR3A promoter Figure 10. ChIP for STAT1 and SP1 at the FCGR3A promoter. Ex vivo expanded NK

cells were sorted into CD16pos and CD16neg population and subjected to ChIP assay.

Samples were probed with non-specific IgG control, STAT1, or SP1 antibody. (A) ChIP enrichment at the proximal FCGR3A promoter region. (B) ChIP enrichment at the CCL5 promoter. Data presented as mean ± SD. * indicates p<0.05. 40

Next, we probed with SRF in freshly isolated enriched NK cells and observed no enrichment above background (non-specific IgG control) at the predicted SRF binding site

(Figure 11A), nor at the proximal promoter region (Figure 11B).

Figure 11. ChIP for SRF at the FCGR3A promoter Figure 11. ChIP for SRF at the FCGR3A promoter. Freshly isolated mature NK cells from peripheral blood were enriched and subjected to ChIP assay with SRF antibody.

Enrichment of SRF is shown at (A) the putative SRF binding site in the FCGR3A promoter and (B) the proximal promoter region of FCGR3A. Data presented as mean ± SD.

Subsequent ChIP experiments used YT cells and freshly isolated peripheral blood-derived

human NK (huNK) cells as CD16neg and CD16pos NK cell populations, respectively.

To continue our ChIP experiments, we sought a positive control for the FCGR3A locus.

When CD16pos NK cells are sorted and assayed by real time PCR, we consistently observe

high FCGR3A mRNA expression, which suggests active transcription of the FCGR3A

gene. Therefore, we hypothesized that PolII would enrich at the promoter of any actively

transcribed gene in NK cells, such as FCGR3A. Therefore, we conducted ChIP

experiments to determine the suitability of PolII as a positive control in parallel with the

TFs of interest.

Unexpectedly, PolII did not enrich at the proximal promoter region of FCGR3A or the

more distal GATA3 binding site in primary huNK cells (Figure 12A and 12B). PolII

variably enriched at both promoter regions in YT cells (p=0.24 and 0.43). Likewise,

GATA3 did not enrich in huNK cells at either region of the FCGR3A promoter; YT cells again displayed variable enrichment. The positive control locus at the IL2RB promoter did not enrich for PolII in NK cells or YT cells. Interestingly, GATA3 showed highly variable enrichment depending on donor. All individual samples demonstrated enrichment compared to its own IgG control, however, due to the significant variability, these enrichments are not statistically significant (p=0.08 in YT cells and p=0.42 in huNK cells).

The YT cell positive control locus IL2RA enriched for PolII and GATA3 (p<0.05 for both pulldowns) while the huNK cell samples, as expected, did not enrich at this locus.

Figure 12. Enrichment of PolII and GATA3 at the FCGR3A promoter, IL2RB promoter, and IL2RA promoter in YT and huNK cells

Figure 12. Enrichment of PolII and GATA3 at the FCGR3A promoter, IL2RB promoter, and IL2RA promoter in YT and huNK cells. YT cells and freshly isolated peripheral blood-derived huNK cells were subjected to ChIP assay with PolII and GATA3 antibodies. Enrichment at the FCGR3A promoter (A and B), IL2RB promoter (C), and

IL2RA promoter (D) is shown. Data presented as mean ± SD. * indicates p<0.05.

The unexpected result of PolII not enriching at the huNK cell FCGR3A locus prompted us to consider whether the enrichment was too weak to detect with the PolII antibody. To address this concern we elected to utilize a different antibody against PolII. Specifically, this antibody recognizes the phosphorylated serine at amino acid position 5. PolII is in its active form when Serine 5 is phosphorylated, and therefore, allows us to be confident that any p-PolII we pull down is actively engaged in transcription. This antibody, which we abbreviate as p-PolII, is more sensitive than PolII in ChIP assays.

We also considered that mature NK cells in the peripheral blood may not be actively transcribing FCGR3A and may instead carry a large reserve of FCGR3A mRNA. As discussed above, NK cells may shed CD16 in the presence of strong activation. NK cells, however, can recover CD16 expression after a period of rest (60). Therefore, we decided to stimulate the NK cells with the cytokines IL-12+IL-15+IL-18 for 12 hours. These cells were compared with unstimulated controls rather than a CD16neg cell population.

Stimulation did not induce binding of either p-PolII or GATA3 at the FCGR3A promoter

(Figure 13A and 13B). Both transcription factors tended toward enrichment in unstimulated NK cells at the IL2RB promoter (Figure 13C). Stimulated NK cells demonstrated significant TF enrichment at the IL2RB promoter, but were not statistically different from unstimulated NK cells.

Figure 13. Enrichment at the FCGR3A promoter and IL2RB promoter with stimulated and unstimulated huNK cells

Figure 13. Enrichment at the FCGR3A promoter and IL2RB promoter with stimulated and unstimulated huNK cells. Freshly isolated huNK cells were incubated for 12 hours with IL-12+IL-15+IL-18 stimulation (stim) or without (unstim) and then subjected to ChIP assay with p-PolII and GATA3 antibodies. Enrichment was measured at the FCGR3A promoter (A and B) and the IL2RB promoter (C). Data presented as mean ±

SD. * indicates p<0.05.

3.4 Discussion

We find that no TFs, including PolII, enrich at the FCGR3A locus. It remains unclear why and how CD16pos huNK cells maintain large pools of FCGR3A mRNA without evidence of active transcriptional activity. One possibility is that CD16+ NK cells have already sequestered a large amount of CD16 mRNA before entering the peripheral blood.

Therefore, they might develop into stage 5 NK cells in the tonsil and then traffic to the periphery with a full complement of CD16 surface protein and mRNA. This seems unlikely because the stage 5 population in the tonsil is so small that it may not be able to generate the number of mature NK cells observed in peripheral blood. Another possibility is that the chromatin of NK cells, particularly in the vicinity of FCGR3A, is very tightly packed and therefore difficult to shear by sonication. This might leave very little intact DNA-protein interaction by the time that sonication treatment has sheared the DNA to small enough size for ChIP. We may detect other genes in NK cells simply because the chromatin in these region is less densely packed and easier to shear without disruption DNA-protein interactions.

We do find evidence that GATA3 binds to the IL2RB promoter in huNK cells. This suggests that GATA3 plays a previously unknown role in regulating NK cell survival and proliferation. We speculate that longer stimulations and/or longer recovery times may be necessary to induce transcription of FCGR3A by NK cells.

Chapter 4: Epigenetic regulation of FCGR3A

4.1 Introduction

All cells in an organism have the same genomic DNA. However, not all cells in an organism express the same genes. In fact, it is necessary for proper functioning of different tissues that some genes be active and others silenced. Likewise, as a stem cell differentiates into a mature cell, it too must activate some genes while silencing others. One mechanism by which this regulation occurs is through DNA methylation. The internal cytosine of the

DNA oligonucleotide sequence 5’-CCGG-3’ can be modified with the addition of a methyl group. This modification changes the transcriptional activity of nearby genes and thereby influences the pattern of gene expression in the cell.

4.1.1 DNA methylation at the FCGR3A locus

In the search for regulators of the FCGR3A gene in NK cells we have thus far been unable to identify transcription factor mediated mechanisms that fully explain the differences in expression between CD16neg and CD16pos NK cells. An alternative mechanism to investigate is epigenetic regulation of CD16. Here we focus specifically on DNA methylation. We wanted to know if there was a difference between the DNA methylation pattern in CD16neg NK cells and CD16pos NK cells.

4.2 Materials and Methods

4.2.1 Cell culture and treatment with 5- aza-2’-deoxycytidine

NKL cells were cultured in RPMI supplemented with 10% FBS and 150 IU/mL rhIL-2.

The indicated cell types were treated with the indicated dose of 5- aza-2’-deoxycytidine

(5-aza) solubilized in DMSO or DMSO alone (0μM 5-aza) for the indicated periods of time. Cells were cultured in 96-well plates at a density of 1x105 cells per well in a total volume of 200μL.

4.2.1 DNA methylation analysis

DNA was isolated from NKL cells treated with 5-aza at the indicated doses and time periods with the DNeasy Blood & Tissue kit (Qiagen). DNA methylation analysis experiments were performed by following the manufacturer’s guidelines for EpiJET DNA

Methylation Analysis Kit (ThermoScientific). Briefly, three digestions per sample were set up, undigested, HpaII digestion, or MspI digestion with 1μg of DNA each. Digestion reactions were performed for 1 hour and terminated by heat denaturation at 90°C for 10 minutes. Samples were purified by QIAquick PCR Purification Kit and amplified by PCR for 30 cycles with primer sets amplifying overlapping promoter regions of ~450 bp in size.

Samples were analyzed by agarose gel electrophoresis and imaging.

4.3 Results

4.3.1 Demethylation of NK cells with 5-aza

To investigate this question, we asked if we could increase FCGR3A mRNA expression in

the NKL cell line. NKL cells are an NK cell-like cell line that originally expressed several

mature NK cell markers, including CD16. However, after some time in culture, the NKL

cell line lost surface expression of many of these NK cell markers. To the best of our

knowledge there is no way to induce NKL cells to express surface CD16. NKL cells,

however, express more FCGR3A than other NK-like cell lines such as NK-92 and YT

(Figure 9). Importantly, NKL cells express about three times more FCGR3A mRNA than

CD56bright/CD16- NK cells isolated from peripheral blood, suggesting they retain potential to acquire CD16 expression if the right regulatory mechanism is lifted.

We treated NKL cells with 5-aza, a demethylating agent, to determine if methylation marks

in CpG sites could be removed. We assayed the FCGR3A promoter for methylated CpG

sites by performing HpaII/MspI digestions followed by PCR for regions of the FCGR3A

promoter. HpaII and MspI are isoschizomers that recognize the tetranucleotide sequence

5’-CCGG-3’. HpaII digestion is blocked when the internal C is methylated while MspI is not. These digestions are compared to uncut controls. There are two potential target sequences that can be found between -650 and -800 bp upstream of the FCGR3A start

codon. When the MspI band matches the uncut band, the external C is methylated. When

the MspI band is lost, MspI has recognized its target sequence and digested the DNA. When

the HpaII band matches the uncut band, the internal C is methylated and HpaII digestion

was blocked. We observe that at a dose of 1.0μM 5-aza, there is some evidence for

hypomethylation at the internal C because the HpaII band is weaker compared to the 0μM

and 0.5μM 5-aza doses (Figure 14A). At higher doses of 5-aza we observed significant

cell death after 3 days. While DNA demethylation was incomplete, we did observe a

modest increase in FCGR3A mRNA expression after 3 and 4 days of 5-aza treatment

(Figure 14B and 14C), but no surface expression of CD16 (data not shown). Next, we wanted to study methylation of FCGR3A promoter in primary NK cells. This DNA methylation analysis technique required significant initial DNA input, so we were unfortunately unable to perform this experiment with freshly isolated primary NK cells because the population of CD16neg NK cells was too small. Instead, we isolated CD16neg

NK cells, expanded and differentiated them ex vivo, and after three weeks, sorted CD16neg and CD16pos fractions for DNA methylation analysis. We observed that HpaII digestion was blocked, indicating that the 5’-CCGG-3’ internal C was blocked (Figure 14D). No difference in DNA methylation was observed between the two exNK cell populations.

Correspondingly, we observed no difference in FCGR3A mRNA expression at any of the indicated doses of 5-aza (Figure 14E). Treatment of exNK cells with 5-aza was highly toxic, resulting in very high levels of cell death after 3 days.

Figure 14. Analysis of FCGR3A promoter methylation

Figure 14. Analysis of FCGR3A promoter methylation. (A) DNA methylation analysis

by HpaII and MspI digestion was performed in NKL cells treated with 0μM, 0.5μM, or

1.0μM 5-azadeoxycitidine (5’aza) for 3 days. The FCGR3A promoter was analyzed with primers spanning the indicated region. (B and C) NKL cells treated for 3 or 4 days with

5’aza were assayed for FCGR3A expression by real time PCR. (D) Expanded NK cells

(exNK) were sorted for CD16pos and CD16neg populations and assayed for DNA methylation analysis by HpaII and MspI digestion at the indicated region of the FCGR3A 51

promoter. (E) exNK were sorted for CD16neg cells and treated for 2 days with 5’aza and then assayed for FCGR3A expression. Data in B, C, and E presented as mean ± SD. * indicates p<0.05.

4.4 Discussion

Primary NK cells expanded ex vivo and NKL cells exhibit differing FCGR3A expression responses to 5-aza treatment. NKL cells treated with 5-aza can be demethylated in the promoter region, resulting in increased FCGR3A mRNA expression. This effect comes at the cost of high levels of cytotoxicity. However, in exNK cells sorted for CD16 expression, we observe that both CD16neg and CD16pos fractions have a methylated internal C in the promoter region, and that 5-aza treatment does not induce FCGR3A mRNA expression.

In our studies of epigenetic regulation of FCGR3A we looked specifically at CpG methylation in a limited area of the promoter. The requirement for relatively large amounts of DNA isolated from primary NK cells is an important limitation because the CD16neg fraction of peripheral blood NK cells is so small. Moreover, the populations of all NK cell developmental intermediates that can be isolated from tonsil are also small. Our use of expanded NK cells represents an attempt to overcome the limitation of small cell numbers.

However, optimally, freshly isolated NK cells would be used.

Chapter 5: MicroRNA regulation of FCGR3A

5.1 Introduction

MicroRNAs (miRNAs) are a type of small, non-coding RNA found in eukaryotic

organisms (126). They are typically 20-25 nucleotides in length and play important

regulatory roles (127). MiRNAs execute their function by targeting mRNAs for

degradation or translational repression. By recognizing sites of antisense complementarity

found in the 3’ untranslated region (UTR) of the target mRNA, miRNAs can significantly

reduce protein production without changing expression levels of the target mRNA (128).

MiRNAs display diverse expression patterns based on tissue type and developmental stage,

which allow them to serve a variety of regulatory roles (127,129-131).

MiRNAs can bind through imperfect base pairing to the 3’ untranslated region (UTR) of target mRNAs and modulate their expression. As described above, CD16pos NK cells express high levels of FCGR3A mRNA, but this pool of transcripts cannot be easily increased with cytokine stimulations. Regulation at the posttranscriptional level is another possibility that may help explain how FCGR3A first becomes expressed in developing NK cells or how the pool of transcribed FCGR3A mRNAs are regulated by the cell.

5.2 Material and Methods

5.2.1 Isolation of primary NK cells from peripheral blood

All human cell work was performed with approval of the Ohio State University

Institutional Review Board. Human NK cells were isolated from peripheral blood

leukopacks of healthy individuals (American Red Cross) by negative selection with

MACSxpress NK Cell Isolation Kit, human (Miltenyi). Briefly, blood was diluted with

1/5th volume of Hextend (BioTime, Inc) and gently rocked for 3 minutes. Samples were centrifuged for 5 minutes at 90xg with low brake. Samples were allowed to settle for 20 minutes in a 37°C incubator cell culture incubator with 5% CO2. The top layer of cells were

collected into a new tube and resuspended in 1/10th the original blood volume. To each

sample 3.75mL of MACSxpress Cell Isolation Cocktail was added and gently mixed.

Samples were incubated for 20 minutes at room temperature. PBS + 2% FBS was added to

total volume of 40mL. Samples were placed on a magnet for 20 minutes. Cocktail-labeled

cells accumulated at the sides of the tube and the remaining cells were collected and labeled

for FACS sorting. Cells were sorted to >95% purity.

5.2.2 Isolation of primary monocytes from peripheral blood

Human monocytes were isolated from peripheral blood leukopacks of healthy individuals

(American Red Cross) by positive selection with CD14 MicroBeads, human (Miltenyi) and magnetic columns by follow the manufacturer’s protocol. Flow cytometric analysis determined that the enriched monocytes were greater than 85% pure.

5.2.3 miRNA Expression Assay

NK cells were enriched from 4 normal peripheral blood leukopack donors. NK cells were

sorted for as CD56bright/CD94high, CD56dim/CD94high, or CD56dim/CD94low. The

CD56bright populations poorly express CD16 while the CD56dim populations highly

express CD16. MicroRNA was extracted using the Total RNA Purification Plus Kit from

Norgen Biotek Corp. These RNA samples were subjected to nCounter ® miRNA

Expression Assay for a miRNA panel of 800 miRNAs (nanoString Technologies).

5.2.4 Expansion of stage 4 NK cells

Peripheral blood mononuclear cells (PBMCs) from three different donors were isolated

from peripheral blood leukopacks (American Red Cross) by Ficoll-Hypaque density gradient centrifugation. PBMCs were irradiated with 30 Gy of radiation and then mixed in equal ratios. In a round bottom 96-well plate PBMCs were plated at a density of 1x104

mixed PBMCs per well in RPMI media supplemented with 10% human AB serum (Valley

Biomedical), 1x104 Dynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific),

and 150 IU/mL rhIL-2 (Hoffman-LaRoche) at a total volume of 200μL per well. Stage 4

NK cells isolated from peripheral blood (or tonsil, as indicated) were sorted to >97% purity.

Then, these purified stage 4 NK cells were sorted into the previously prepared 96-well plate

for co-culture with the mixed PBMCs. A total of 100 stage 4 cells were sorted into each

well. Stage 4 cells were expanded ex vivo for three weeks. Every 7 days, half the media

was removed and replaced with new RPMI media supplemented with 10% human AB

serum, 150 IU/mL rhIL-2, 1x104 Dynabeads Human T-Activator CD3/CD28, and 1x104

irradiated, mixed PBMCs. Starting on day 10 and continuing every 3 days after that, media

changes without Dynabeads and mixed PBMCs were performed.

5.2.5 Real-time PCR

Total RNA was extracted using Total RNA Purification Plus Kit (Norgen Biotek Corp).

cDNA was generated according to the manufacturer’s recommendations using

SuperScript® VILO Master Mix (ThermoFisher Scientific) or TaqMan MicroRNA reverse transcription kit and RT primers specific for RNU44, mir-92a, mir-133a, or mir-218.

TaqMan Universal PCR Master Mix was used with microRNA cDNA samples while

VILO-generated cDNA was used with Power SYBR Green Master Mix ((ThermoFisher

Scientific). Data were analyzed using the comparative δ-δ threshold cycle method and normalized to internal control (18S RNA or RNU44). Results are indicated as expression relative to internal control.

5.2.6 Luciferase assay

The ~1400bp 3’UTR of FCGR3A was cloned into the pmirGLO luciferase vector immediately 3’ of the luc gene. The luc gene in pmirGLO is driven by a constitutively active CMV promoter. Mir-92a, mir-133a, and mir-218 were each cloned into the expression vector pCDH. Alternatively, mir-218 miRNA mimic or mir-218 miRNA inhibitor or control was used instead of expression vector. 293tn cells were maintained in

DMEM supplemented with 10% fetal bovine serum. 293tn cells were seeded into 24-well

plates at a density of 5x104 cells per well and grown overnight. The pmirGLO-3’UTR vector with or without one of the previously mention miRNA-expressing pCDH vectors were co-transfected with Lipofectamine 2000 with Plus Reagent (Invitrogen).

Alternatively, the mir-218 mimic, inhibitor, or negative control was co-transfected with pmirGLO-3’UTR instead of pCDH expression vector containing a miRNA. The pmirGLO-

3’UTR vector was used as a control for basal promoter activity. A renilla luciferase vector, pRL-TK (Promega), was co-transfected to serve as an internal control for transfection efficiency. Cells were harvested after 48 hours and assessed for luciferase activity as previously described (79).

5.2.7 Lentiviral infection of primary human NK cells

Human NK cells were isolated as described above and cultured overnight in RPMI+10% human AB serum in a round bottom 96-well plate under stimulation with 450 IU/mL rhIL-

2. NK cells were treated with lentivirus (MOI=1) and centrifuged for 2 hours at 32°C. NK cells were gently resuspended and centrifuged for two more rounds for a total of 6 hours.

After the third spin, virus media was removed and replaced with RPMI+10% human AB serum and 300IU/mL rhIL-2. Infected cells were incubated at 37°C for two days and FACS sorted for GFP positive cells.

5.2.8 Transient transfection of primary human NK cells with miRNA mimic or

inhibitor

Human NK cells isolated as described above were plated for 36 hours in a 24-well plate at a density of 1x105 per well and were cultured and maintained in RPMI+10% human AB serum and 300 IU/mL rhIL-2 Transient transfection of primary NK cells was performed with mir-218 miRNA, mir-218 miRNA Inhibitor, or miRNA negative control RNA oligos using the Lipofectamine 2000 transfection reagent according to manufacturer guidelines

(ThermoFisher Scientific). Each transfection well was treated with 15pmol of RNA oligo.

NK cells were collected after 48 hours and assayed by real-time PCR and flow cytometry for FCGR3A and CD16 expression, respectively. Mir-218 overexpression and knockdown were also assessed by real-time PCR.

5.2.9 Electroporation of monocytes for transfection with mir-218

Enriched monocytes were transiently transfected with either pCDH empty vector or pCDH- mir-218 using manufacturer guideline for monocyte Nucleofection (Lonza). Transfected monocytes were cultured for 48 hours in M-CSF and sorted for CD14+/GFP+ cells. Sorted cells were analyzed by real-time PCR for FCGR3A expression and mir-218 overexpression.

5.2.10 Antibodies and flow cytometric analysis

The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD

Biosciences), CD14 (TÜK4, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD

Biosciences), and CD56 (N901, Beckman Coulter). Flow cytometry data were analyzed with FlowJo v7.6.1 (Tree Star).

5.3 Results

5.3.1 Prediction of miRNAs that target FCGR3A

To investigate the question of whether miRNAs play a role in the regulation of FCGR3A, we followed a miRNA regulator prediction strategy described by Witkos et al (132)

(Figure 15). Using a nanoString miRNA expression assay, we obtained miRNA expression data for 800 miRNAs in populations of mature NK cells derived from 4 normal donors. In peripheral blood, the CD56bright NK cell subset lacks CD16 expression while the

CD56dim NK cell subset displays high CD16 expression. Therefore, these populations correlate closely with the CD16neg and CD16pos populations described above. For simplicity, we will refer to these populations as stage 4 and stage 5 NK cells, respectively.

The miRNA expression assay was cross-referenced with a list of miRNAs that were predicted to target the FCGR3A 3’ UTR. Two types of in silico prediction tools were used.

First, we utilized programs that consider site conservation between species for prediction of miRNAs that target the mRNA of interest. Sites that are conserved between species are likely to play important roles. This type of prediction misses newly evolved genes or recent mutations. Prediction tools of this type that we used were TargetScan and DIANA. The other type of miRNA prediction uses other parameters such as free energy of binding and does not consider conservation between species. These tools can detect new mutations that site-conservation prediction tools miss. Prediction tools that we used of this type were

PITA and mirSVR.

Figure 15. Strategy to predict miRNAs targeting FCGR3A Figure 15. Strategy to predict miRNAs targeting FCGR3A. Adapted from Witkos et al.

Using the prediction tools we compiled a list of 53 different miRNAs that were predicted to target FCGR3A mRNA (25 from TargetScan, 25 from DIANA, 15 from PITA, and 4 from mirSVR; some were redundant). We then compared the expression pattern of in silico predicted miRNAs to determine which would be likely to function as negative regulators

(high in stage 4 NK cells and low in stage 5 NK cells). We identified 3 miRNAs that showed this pattern of differential expression between stage 4 and stage 5 NK cells: mir-

92a, mir-133a, and mir-218. A value less than 1 indicates higher expression in stage 5 NK cells than in stage 4 NK cells. A value greater than 1 indicates the opposite. These expression patterns were validated in freshly isolated peripheral blood NK cells and ex vivo expanded NK cells (Figure 16). We found that this pattern held only for mir-218.

Figure 16. Candidate miRNA expression in NK cells

Figure 16. Candidate miRNA expression in NK cells. (A) Expression levels of miRNAs

predicted to target the FCGR3A 3’UTR were ranked by expression ratio as fold expression

in stage 5 NK cells over stage 4 NK cells. (B) Depiction of predicted binding site for each

miRNA with high expression in stage 4 NK cells and low expression in stage 5 NK cells.

(C and D) Real-time PCR was used to measure the expression of three candidate miRNA

regulators of FCGR3A. (C) MiRNA expression was measured in freshly isolated primary

NK cells or (D) ex vivo expanded NK cells. Data presented as mean ± SD. * indicates

p<0.05.

5.3.2 Validation of putative miRNA regulators of FCGR3A

In order to determine if these putative microRNA regulators could recognize and modulate the 3’ UTR of FCGR3A mRNA, we performed luciferase assays. We cloned the 3’ UTR of FCGR3A containing the putative binding sites for each of the three miRNAs, mir-92a, mir-133a, and mir-218 into the luciferase vector pmirGLO downstream of the firefly luc gene, which is driven by a constitutively active promoter. This 3’ UTR vector was co- transfected with an expression vector for one of each of the candidate miRNAs and a control vector expressing renilla luciferase for normalization. We found that only mir-218 showed a trend toward negatively regulating the FCGR3A 3’ UTR (Figure 17A). To confirm this observation, we repeated the luciferase assay, but, instead of using an expression vector to overexpress mir-218, we used a mir-218 miRNA mimic (mir-218 mimic). To show the inverse effect, we also used a mir-218 miRNA inhibitor (mir-218 inhibitor). We found that luciferase activity trended toward an increase in the presence of mir-218 inhibitor and decreased in the presence of mir-218 mimic (Figure 17B). The mir-

218 mimic strongly overexpressed mir-218 and the mir-218 inhibitor knocked it down

(Figure 17C), establishing an inverse relationship between FCGR3A and mir-218 expression.

Figure 17. MicroRNA regulation of the 3’ UTR of FCGR3A

Figure 17. MicroRNA regulation of the 3’ UTR of FCGR3A. Luciferase assay were

performed 293tn cells cotransfected with pmirGLO firefly luciferase plasmid containing

the FCGR3A 3’UTR, renilla luciferase control plasmid and either an expression plasmid

or miRNA mimic or inhibitor, as indicated. (A) Luciferase assay was performed with

miRNAs cloned into the expression vector pCDH or empty pCDH vector. Luminosity was

measured and normalized as firefly/renilla. (B and C) Luciferase assay was performed with

miRNA mimic (mir-218 Mim), inhibitor (mir-218 Inh), or anti-miR miRNA inhibitor

negative control (neg ctrl). Mir-218 expression was measured by qPCR. Data presented as

mean ± SD. *indicates p<0.05.

5.3.3 Overexpression of mir-218 in human NK cells by lentiviral infection

To further confirm this relationship, we overexpressed mir-218 in primary NK cells. First,

we infected NK cells with either lentivirus containing mir-218 or empty vector. In four

donors, we observed decreased CD16 expression by flow cytometry (Figure 18A and B).

There was significant variability between donors, but each individually had increased mir-

218 expression and decreased surface CD16 compared to its own control. FCGR3A mRNA

levels were significantly decreased, and mir-218 was overexpressed compared to the control in each donor (Figure 18C and D).

Figure 18. Infection of primary NK cells with mir-218 virus Figure 18. Infection of primary NK cells with mir-218 virus. (A) Flow cytometry

histogram plot of representative CD16 expression in infected NK cells gated on

GFP+/CD56+/CD3- cells. (B) Change in CD16 expression in mir-218-infected NK cells

compared to pCDH empty-infected NK cells (%CD16pos=percentage of CD16pos NK

cells). (C and D) Real time PCR expression of FCGR3A and mir-218 in infected NK cells.

Data in B, C, and D presented as mean ± SD, n=4. Two sided paired t tests were performed.

* indicates p<0.05.

5.3.4 Modulation of mir-218 in human NK cells by transfection with miRNA mimic

or inhibitor

Next, we transiently transfected primary NK cells with the mir-218 mimic or inhibitor. We observed modest modulation of CD16 expression by flow cytometry (Figure 19A). In each donor, CD16 MFI, MedFI, and % CD16pos cells trend up with the mir-218 inhibitor and down with the mir-218 mimic (Figure 19B). FCGR3A mRNA was unchanged in the presence of mir-218 inhibitor or mir-218 mimic, despite robust overexpression of mir-218 by the mimic (Figure 19C and D). We observed strong overexpression and knockdown, respectively, compared to negative control oligo. CD16 surface expression trended up with inhibitor treatment and down with mimic treatment. FCGR3A mRNA expression levels were unchanged.

Figure 19. Transfection of primary NK cells with mir-218 miRNA inhibitor or mimic Figure 19. Transfection of primary NK cells with mir-218 miRNA inhibitor or mimic.

(A) NK cells were sorted to >97% purity and transiently transfected with mir-218 miRNA inhibitor (Inh), mimic (Mim), or negative control miRNA (neg ctrl). 48hrs post transfection, flow cytometry was used to measure CD56 and CD16 expression in transfected NK cells. A representative set of plots is shown. (B) Change in CD16 expression in mir-218 Inh- and mir-218 Mim-treated NK cells compared to neg ctrl-treated

NK cells (MFI=mean fluorescence intensity, MedFI=median fluorescence intensity,

%CD16pos=percentage of CD16pos NK cells). (C and D) Real time PCR expression of

FCGR3A and mir-218. Data in B, C, and D presented as mean ± SD. * indicates p<0.05.

5.3.5 Overexpression of mir-218 in human monocytes by transient transfection

Additionally, we wanted to see if mir-218 may also regulate CD16 in monocytes, another

cell type that expresses FCGR3A rather than FCGR3B. We isolated primary monocytes and used nucleofection to transfect them with either empty pCDH vector or pCDH containing mir-218. We found observed variable transfection efficiency among monocyte donors. However, the fraction of CD16pos monocytes overexpressing mir-218 was greater than that of controls (Figure 20A). We observed a trend of increased mir-218 expression, which correlated with a trend toward decreased FCGR3A mRNA (Figure 20B and C).

We have found significant evidence that mir-218 regulates FCGR3A in primary NK cells and monocytes. MiRNAs are often an instrument of fine tuning gene expression. As such, even the very strong overexpression of mir-218 that we observe when 293tn or primary

NK cells are transiently transfected with miRNA mimic does not correspond to profound silencing of FCGR3A.

Figure 20. Mir-218 overexpression in primary monocytes Figure 20. Mir-218 overexpression in primary monocytes. Monocytes were enriched

and transfected with either pCDH empty vector or pCDH-mir-218 vector by Nucleofection.

Monocytes were cultured in M-CSF for 48hrs and subjected to flow cytometric analysis and sorted for GFP+ cells. (A) Flow cytometry measurement of CD16 expression in GFP+ cells. (B and C) Real time PCR expression of FCGR3A (p=0.071) and mir-218 (p=0.15).

Chapter 6: Extended discussion and future directions

6.1 Time course of CD16 acquisition by NK cells is unclear

As described earlier, NK cells acquire CD16 as part of normal development. In the

secondary lymphoid tissue (SLT), all five stage of NK cell development can be observed

(7). Both SLT- and peripheral blood-derived CD16neg NK cells can be isolated and

cultured ex vivo to generate CD16pos NK cells after two weeks. Because significant

expansion in the culture population does not occur until around this time, we have not

investigated these cells in detail. Therefore, we currently do not have a clear understanding

of the rate of differentiation of stage 4 NK cells to stage 5. This includes the caveat that the speed of maturation ex vivo may not match that of NK cells in vivo.

As we have shown in the previous studies, CD16neg NK cells express very low levels of

FCGR3A mRNA. Expanded NK cells can be sorted for CD16neg and CD16pos fractions which show significantly different expression levels, with CD16neg NK cells maintaining a low level of FCGR3A mRNA. The CD16pos fraction expresses similar amounts of

FCGR3A mRNA as freshly isolated CD16pos peripheral blood NK cells. Therefore, we observe a very clear correlation between mRNA expression and surface protein expression.

As evidenced by the ChIP experiments we performed, FCGR3A mRNA does not appear

to be actively transcribed, at least not at a high level. One explanation of this observation

is that early in the developmental transition from stage 4 to stage 5, NK cells massively 73

upregulate CD16 expression and accumulate a large pool of untranslated mRNAs. Instead

of actively transcribing new mRNAs, this pool is somehow preserved or sequestered until

needed. Testing this hypothesis poses a considerable technical challenge. There are two

general methods for measuring mRNA half-life. The first is a general inhibition of

transcription through treatment with a drug followed by several measurements of mRNA

over a time course to calculate the rate of decay. This is relatively simple to conduct, but

can result in severe toxicity with accompanying large changes in cell physiology (133-

135). Furthermore, many of these drugs have been shown to directly alter the stability of many mRNAs (135,136). A more physiologically relevant strategy is called transcriptional pulsing, which involves using inducible promoters to specifically promote transient transcription. This type of assay grants tight transcriptional control in a more physiologically normal context, but requires stable transfection with a construct containing an inducible promoter (134,137). Each could be attempted, but each comes with significant caveats and technical challenges.

If FCGR3A is truly not actively transcribed in CD16pos stage 5 NK cells, it may indicate that we are studying the cells at the wrong point in maturation. While it would not likely be feasible to obtain enough freshly isolated tonsil NK cells for ChIP, we may take actively expanding stage 4 NK cells and sort for low-to-moderate expression of CD16 instead of pure highly positive or highly negative populations. This subset may be in transition between CD16neg and CD16pos steps and exhibit active transcription of FCGR3A mRNA.

Follow-up experiments may immunoprecipitate with p-PolII and then subject the

transcriptional complex to mass spectrometry in order to identify transcription co- activators.

An alternative explanation for why p-PolII does not significantly enrich at the FCGR3A promoter is that p-PolII does not accumulate strongly at the promoter, but may be found to enrich at higher levels along the gene body. Primer sets could be designed to search for enrichment downstream of the transcription start site. It is also possible that the

stimulations of CD16pos NK cells were not sufficiently sustained to stimulate new

FCGR3A mRNA transcription. Future experiments may investigate the effects of

stimulations for 24, 48, or even 72 hours.

6.2 Genomic landscape and the potential role of epigenetics in FCGR3A regulation

We have examined the methylation pattern of the FCGR3A promoter in an NK-like cell line NKL and in ex vivo expanded NK cells sorted for CD16 expression. While we found some evidence of demethylation of NKL cells treated with 5-aza, the HpaII/MspI digestions may need to be optimized for clear answers. Furthermore, it would be optimal to analyze DNA methylation along an extended stretch of DNA along the FCGR3A promoter region. We would expect that CD16+ NK cells would be hypomethylated along this region and that methylated DNA would appear in higher frequency as we analyzed

DNA segments farther away from the promoter region. This would be in the pattern of a

“valley” of methylation around the demethylated region corresponding to the actively transcribed gene. In order to address this question of regional methylation status, one option would be to use an alternative technology to HpaII/MspI digestion in the study of DNA methylation around the FCGR3A locus. Currently, there are quantitative high-resolution

DNA methylation analysis technologies such as MassARRAY that can resolve DNA methylation at single CpG units (138). Furthermore, by designing a series of overlapping primer sets, one could investigate the methylation status of the FCGR3A promoter over several kilobases around the transcription start site. Furthermore, parallel analysis of the

FCGR3B promoter could be performed. We would expect hypermethylation of both loci in CD16- NK cells. In neutrophils, we would expect hypermethylation of FCGR3A only, and hypomethylation of FCGR3B because it should be actively transcribed. In CD16+ NK cells, we would expect the opposite pattern.

In the wider sense of epigenetic regulation are histone modifications and chromatin

architecture. The histone marks H3K4me3 and H3K27ac are recognized as active promoter

marks (139,140). The histone mark H3K27me3 is more ambiguous; there is evidence that

it is involved in transcription repression when found along a gene body, but is associated

with active transcription either in the context of H3K4me3 or when found at the gene

promoter (141). Each of these marks can be studied by ChIP. Therefore, further

investigation into the transcriptional and epigenetic regulation of FCGR3A could be

explored by looking at histone modifications around the FCGR3A locus.

The FCGR3A locus can be additionally interrogated through examination of the chromatin

architecture. CCCTC-binding factor (CTCF) is involved in chromatin looping by binding

two different chromatin elements and bringing them into close proximity (142). CTCF

binding sites are evolutionarily conserved and regularly interspersed throughout the

genome(143). Elements between a pair of CTCF binding sites are called CTCF blocks. The

looping structures help organize the genome and define discrete regulatory regions (142).

Elements within a region may all be co-activated or co-repressed (144,145). They can additionally serve as boundaries between activated and repressed chromatin regions.

FCGR3A is located within a CTCF block by itself (with the exception of an asparagine tRNA gene). These categories of CTCF blocks can direct cell type-specific gene expression

(144). The intergenic regions surrounding FCGR3A and FCGR3B are highly homologous.

The ~20 kb upstream of both genes and ~3 kb downstream have very high sequence homology, including CTCF binding sites ~17 kb upstream and ~2 kb downstream. These boundaries may signify CTCF blocks with distinct, cell type-specific regulation. There is

a single CTCF block separating the blocks containing FCGR3A and FCGR3B. ChIP experiments could also investigate the CTCF occupancy of these sites, which are conserved at both loci. These elements may reveal differences in histone modifications between the two presumed chromatin loops which may help explain their tissue specificity.

6.3 MicroRNA regulation of CD16

We have observed significant evidence that mir-218 is a negative regulator of CD16.

Experiments with primary cells consistently support this conclusion. However, small

sample numbers and donor variability prevent us from making firm conclusions. Future

studies must replicate the experiments we have performed with a larger number of donors

to achieve sufficient statistical power make claims with a high degree of confidence.

Furthermore, it will be important to examine the effect of mir-218 on effector function. We

expect that decreased surface CD16 expression would make the NK cells less able to

mediate ADCC or produce cytokine in response to antibody ligation. Work by others has

shown that CD16 can be cleaved from the surface of NK cells by ADAM17 following

activation. NK cells treated with IL12+IL-18 overnight cleave ~40% of their surface CD16

(60). However, when these cells are used in ADCC killing assays, they show no impairment

in specific lysis compared to NK cells treated with ADAM17 inhibitor. ADAM17

inhibition is very strong, resulting in virtually no CD16 shedding (60). Therefore, it would

be interesting to determine if mir-218 downregulation of CD16 impairs ADCC killing since surface cleavage does not despite a significant reduction in surface expression. It may be that NK cells have excess CD16 and therefore, loss of surface CD16 must meet a threshold before ADCC is impaired. Alternatively, the overnight stimulation may activate the NK cells to a great enough degree that they can overcome the loss of a large fraction of surface

CD16.

An additional question surrounding miRNA regulation of CD16 is how early in NK cell development does mir-218 play a role. Stage 3 NK cells express low levels of mir-218,

similar to stage 5 NK cells. If mir-218 were to be overexpressed in stage 3 NK cells, it may drive the cells to differentiate into stage 4 NK cells. If this happened, might mir-218

overexpression also cause a developmental block which would impair differentiation to

stage 5? Such an experiment might be accomplished by either lentiviral transduction with

a mir-218 expression vector or by transient transfection with mir-218 mimic. Transient

transfection with the small molecule oligos may be a more powerful technique because the

mir-218 inhibitor may also be used for comparison. With high transfection efficiencies, a

lag in development of stage 3 NK cells may be observable at two weeks. However, due to

the significant time course required to differentiate stage 3 cells to stage 4, a second or third

treatment of the cells with miRNA mimic or inhibitor may be necessary. Lentiviral

transduction of mir-218 would be stable enough to investigate its role in early NK cell

development by infecting stage 1 pro-NK cells and culturing in conditions conducive for

NK cell differentiation.

6.4 Concluding Remarks

The question of how CD16 is regulated in NK cells has been a challenging question for more than 20 years. In our investigations we have attempted to find some mechanism for pathway that could explain how CD16 is expressed and maintained on NK cells. Our inquiries into this question demonstrate that there are reasons little progress has been made.

We are challenged by the lack of a relevant model cell line, the very high degree of homology between FCGR3A and FCGR3B, and seemingly contradictory information about transcriptional activity. While several paths of investigation that we have pursued have not yielded promising insights, we feel that we have made several important early steps in fully understanding the mechanisms regulation CD16 expression. Importantly, we have identified a microRNA that consistently downregulates CD16 mRNA. There is still a large amount of work to be done to conclusively and exhaustively determine how mir-218 regulates CD16 expression and NK cell effector function. There is still more work to do in understanding how transcription factors and epigenetic changes influence CD16 acquisition and expression. We hope our contribution to addressing this question can help shape the framework for future progress.

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