The Role of Ikzf Factors in Mediating Th1/Tfh Development And

THE ROLE OF IKZF FACTORS IN MEDIATING TH1/TFH DEVELOPMENT AND

FLEXIBILITY

Bharath Krishnan Nair Sreekumar

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy In Translational Biology, Medicine and Health

Kenneth J. Oestreich Irving C. Allen Xin Luo Zhi Sheng

December 16, 2019 Roanoke Virginia

Keywords: CD4+ T helper 1 cell, CD4+ T follicular helper cell, interleukin-2, STAT transcription factors, IkZF transcription factors

The role of IkZF factors in mediating TH1/TFH development and flexibility

Bharath K. Sreekumar

ABSTRACT

The ability of cells within the adaptive immune system to develop into specialized subsets allow for a robust and tailored immune response in the advent of an infection or injury. Here, CD4+ T- cells are a crucial component within this system, with subsets such as TH1, TH2, TH17, TFH and

TREG cells playing vital roles in propagating cell-mediated immunity. For example, TH1 cells are essential in combating intracellular pathogens such as viruses, while TFH cells communicate with

B-cells to optimize antibody responses against an invading pathogen. The development (and functionality) of these subsets is ultimately dictated by the appropriate integration of extracellular cues such as cytokines with cell intrinsic transcription factors, thereby promoting the necessary gene profile. Moreover, the observation that T-helper cells could exhibit a flexible nature (i.e having shared gene profiles and effector functions) not only demonstrate the efficiency of our immune system but also how such flexibility could have unintended consequences during adverse events such as autoimmunity. An important mediator of such flexibility is cytokines. However, the complete network of factors that come together to co-ordinate cytokine mediated plasticity remain unknown. Thus, the work in this dissertation hope to delineate the factors that collaborate to regulate cytokine induced T-helper cell flexibility. As such, we see that in the presence of IL-2, the Ikaros Zinc Finger (IkZF) transcription factor Eos is upregulated in TH1 cells, with this factor playing a significant role in promoting regulatory and effector functions of TH1 cells. Moreover, we show that Eos forms a novel protein complex with STAT5 and promotes STAT5 activity in

TH1 cells. However, depleting IL-2 from the micro-environment leads to the upregulation of two other members within the IkZF family, Ikaros and Aiolos. Aiolos in turn collaborate with STAT3,

induces Bcl-6 expression within these cells, thus promoting these cells to exhibit characteristic features of TFH cells. The work in this dissertation hopes to advance our understanding of the regulatory mechanisms involved in cytokine mediated T-cell flexibility thereby hoping to open new avenues for the development of novel therapeutic strategies in the event of autoimmunity.

The role of IkZF factors in mediating TH1/TFH development and flexibility

Bharath K. Sreekumar

GENERAL AUDIENCE ABSTRACT

T-helper (TH) cells are an important component of the immune system, as these cells aid in the fight against pathogens by secreting factors that either accentuate the inflammatory response during infection or attenuate immune responses post infection. Such effects are made possible because T-helper cells can differentiate into a variety of subsets, with each subset being an important mediator in maintaining immune homeostasis. For example, the T-helper cell subset called TH1 plays a vital role in the fight against intracellular pathogens such as viruses and certain parasites, while T-follicular helper (TFH) cells aid in the production of antibodies specific to the invading pathogen. The development of such subsets occur when cell extrinsic signals, called cytokines, lead to the activation or induction of cell intrinsic proteins called transcription factors.

Interestingly, research over the years have shown that T-helper cells are highly adaptable in nature, with one subset having the ability to attain certain characteristic features of other subsets. This malleable nature of T-helper cells relies on several factors, with cytokines within the microenvironment being an important one. Although this form of flexibility is efficient and beneficial at times, it can also be detrimental, as such flexibility is known to promote certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and type 1 diabetes. Such detrimental effects are thought to be due to cytokines within the environment. Therefore understanding how cytokines influence the flexible nature of T-helper cells is important; as controlling such flexibility

(either by regulating cytokines or the transcription factors activated as a consequence) could prevent the propagation of undesired T-helper cell functions. As such, the work in this dissertation

hopes to uncover how one such cytokine, termed Interleukin-2 (IL-2) mediates the flexibility between TH1 and TFH cells. The work highlighted in this dissertation broadens our understanding of how cytokines influence T-helper cell development and flexibility, and consequently allows the design of novel therapeutic strategies to combat autoimmune diseases.

Dedication

To my Family. This would have been impossible without your love and support.

Acknowledgements:

First, I would like to thank Dr. Kenneth Oestreich for being an amazing mentor. Given the hurdle that is graduate school, I am eternally grateful for your guidance and patience. Your scientific acumen, work ethic and diligence are aspects that I hope to ingrain and take with me, wherever I go next. As you taught us, I will try to ‘be comfortable with being uncomfortable’.

I would like to express my sincere gratitude to my committee members: Dr. Allen, Dr. Luo and

Dr. Sheng. Thank you so much for your critiques and advice during my time here. Your guidance has significantly improved my capabilities as a researcher.

Next, thank you Kaitlin (Read) and Mike (Powell). You two taught me to think critically and showed me the meaning of hard work, dedication and kindness. The lab family would not have been as fun without you two. Shout out to Chandra Baker as well, for helping me out during the early days- teaching me important techniques- and for the amazing Christmas cookies.

Finally, I would like to thank all the people outside the lab that made my time in lab a little easier and my days in grad school, unforgettable. To my two roommates, friends from my cohort, friends from other labs and the extended family from Anna University and UNH. Thank you for being there.

Table of contents

Chapter 1- Introduction ...... 1 Introduction ...... 1 The two arms of the immune system ...... 1 The development of the adaptive immune system ...... 3

The activation, differentiation and function of T-helper (TH) cells ...... 4 Mechanisms of T-helper cell flexibility ...... 7

The role of IL-2 in mediating flexibility between TH1 and TFH cells...... 10

Novel Transcriptional regulators of TH1 and TFH cell development...... 11 References ...... 12 Chapter 2- Ikaros zinc finger transcription factors: Regulators of cytokine signaling pathways and CD4+ T helper cell differentiation...... 19 Abstract ...... 20 Introduction ...... 21

T helper 1 (TH1) cells ...... 25

IkZF factors in TH1 cell differentiation and function ...... 25

T helper 2 (TH2) cells ...... 28

IkZF factors in TH2 cell differentiation and function ...... 28

T helper 17 (TH17) cells ...... 30

IkZF factors in TH17 cell differentiation and function ...... 31

T follicular helper (TFH) cells ...... 32

IkZF factors in TFH cell differentiation and function ...... 33

Regulatory T (TREG) cells ...... 34

IkZF factors in TREG differentiation and function ...... 34 Concluding remarks ...... 39 References ...... 39 Chapter 3- Integrated STAT3 and Ikaros Zinc Finger transcription factor activities regulate Bcl-6 expression in CD4+ T helper cells ...... 47 Abstract ...... 48 Introduction ...... 49 Materials and Methods ...... 52 Results ...... 61 Expression of Aiolos and Ikaros correlate with that of Bcl-6 ...... 61

The Zinc Finger (ZF) DNA-binding domains of Aiolos and Ikaros are required to induce Bcl6 promoter activity ...... 66 Aiolos and Ikaros associate with the Bcl6 promoter ...... 69 IkZF/STAT factor association correlates with increased histone modification of the Bcl6 promoter ...... 70

Aiolos interacts with STAT3 in TFH-like cells ...... 72 N- and C-terminal ZF domains of Aiolos are required for induction of Bcl-6 ...... 74 Aiolos and STAT3 cooperatively regulate cytokine receptor expression ...... 77 Aiolos expression is increased in Ag-specific TFH cells after influenza infection ...... 79 Discussion ...... 81 Acknowledgements ...... 84 Disclosures ...... 84 References ...... 85

Chapter 4- The Ikros zinc finger transctiption factor Eos promotes TH1 differentiation through modulation of the IL-2-STAT5 signaling pathway ...... 90 Abstract ...... 91 Introduction ...... 92 Methods...... 94 Results ...... 101

Eos expression positively correlates with that of the TH1 gene program ...... 101

The TH1 transcriptional program is disrupted in the absence of Eos ...... 103 + Eos-deficient CD4 T cells exhibit reduced expression of TH1 genes and IFN-γ production ...... 105 Loss of Eos leads to attenuated T-bet levels in CD4+ T-cells during T.gondii infection. .... 107 Eos-deficient CD4+ T cells produce normal levels of IL-2, but express reduced levels of IL-2 receptor subunits ...... 109 IL-2-STAT5 signaling positively regulates Eos expression ...... 112

Eos and STAT5 form a novel transcription factor complex in TH1 cells ...... 115

STAT5 activation and association with TH1 target genes is enhanced in the presence of Eos ...... 118 Discussion ...... 122 Acknowledgements ...... 126 Disclosures ...... 126 References ...... 126

Chapter 5- Conclusions and Future Directions...... 130 Summary of findings...... 130 Insights into the regulatory mechanisms by which IkZF and STAT factors modulate T-cell development and plasticity...... 132 Non-transcriptional mechanisms of IkZF factors mediating T-helper cell flexibility ...... 134 IkZF/STAT factors modulating flexibility of other T-helper cell subsets ...... 135 References ...... 137

List of Figures

Figure 1.1. Differentiation of T-helper cell subsets...... 6 Figure 1.2. The flexible nature of T-helper cells...... 8 Figure 2.1. Structure of IkZF family members...... 24 Figure 2.2. Transcriptional regulation of Interleukine-2 locus by IkZF members ...... 38

Figure 3.1. Schematic depicting in vitro differentiation to TH1 and TFH-like cells...... 55 Figure 3.2.A Positive correlation exists between the expression of Aiolos, Ikaros, and Bcl-6. ... 64 Figure 3.3. Ikzf1 and Ikzf3 levels positively correlate with Bcl6 expression...... 65 Figure 3.4. DNA-binding activity of Aiolos or Ikaros is required to induce Bcl6 promoter activity ...... 68

Figure 3.5. Aiolos, Ikaros, and STAT3 associate with the Bcl6 promoter in TFH-like cells...... 71 Figure 3.6. Aiolos and STAT3 physically interact and regulate Bcl6 expression...... 73 Figure 3.7. Functional ZF domains are required for Aiolos-dependent induction of Bcl6 expression ...... 76 Figure 3.8. STAT3/Aiolos differentially regulate Il6rαand Il2rα expression...... 78

Figure 3.9. Aiolos expression is increased in antigen-specific TFH cells post-influenza infection...... 80

Figure 4.1. Eos expression positively correlates with that of the TH1 gene program...... 102

Figure 4.2. The TH1 transcriptional program is disrupted in the absence of Eos...... 104 + Figure 4.3. Eos-deficient CD4 T cells exhibit reduced expression of TH1 genes and IFN-γ production ...... 106 Figure 4.4. Loss of Eos attenuates T-bet levels in CD4+ T-cells during acute infection with T.gondii...... 108 Figure 4.5. Ikzf4-/- CD4+ T cells produce normal levels of IL-2, but express reduced levels of IL- 2 receptor ...... 110

Figure 4.6. Exogenous addition of IL-2 is unable to rescue TH1 development in Eos deficient cells ...... 111 Figure 4.7. IL-2-STAT5 signaling positively regulates Eos expression...... 114

Figure 4.8. Eos and STAT5 interact and form a transcription factor complex in TH1 cells...... 117

Figure 4.9. STAT5 activation and association with TH1 target genes is enhanced in the presence of Eos ...... 120 Figure 4.10. Loss of Eos has modest effects on genome accessibility...... 121 Figure 5.1. STAT and IkZF regulatory networks underlying the expression of T helper cell gene programs...... 136

Attributions

Chapter 2: Published review was authored by Mike Powell. Kaitlin Read and I assisted in literature review and editing.

Chapter 3: Published manuscript was co-authored by Mike Powell and Kaitlin Read. Me, Chandra Baker, Veronica Ringel-Scaia, Holly Bachus, Emily Martin, and Ian Cooley assisted with experiments and analyzed data.

Chapter 4: Me, Mike Powell, and Kaitlin Read helped with the design of the study, performed the experiments, analyzed data, and wrote the manuscript. Devin Jones, Jawad Zafar, and Mustafa Rasheed assisted with experiments. Dr. Lauren M. Childs and Patrick Collins assisted with analysis of data and bioinformatics

Chapter 1

Introduction

Historically, immunity has been defined as the ability to protect the body from infectious agents.

However, over time, functions attributed to the immune system have been vastly expanded. These functions include not only protecting the body from pathogens, but also having the ability to discriminate ‘self’ from ‘non-self’, the ability to maintain homeostasis and the capability to clear existing debris post injury or infection. For these very purposes, a diverse array of immune cells, complex immune cell systems and cross talk between these systems exist within our bodies. Failure to accomplish these tasks, either due to inherent defects within an immune cell or extrinsic dysregulation of cell systems can lead to the emergence of opportunistic infections and the progression of debilitating diseases such as allergies, autoimmunity and cancer. Therefore, an in- depth understanding of the immune system is needed as it would not only allow for improved diagnosis of disease onset but could also contribute significantly to generation of novel therapies.

The two arms of the immune system

Broadly, the immune system can be divided into two main arms: The innate and the adaptive immune systems. The innate immune system provides the first line of defense against an invading pathogen. Components within this system can range from physical barriers such as epithelial layer and mucosal linings of organs (which can harbor antimicrobial agents) to cells such as macrophages, Dendritic Cells (DCs) and Natural Killer (NK) cells 1, 2. Within the innate immune system, a key feature of is the manner in which immune cells recognize pathogens, which occurs

1 by sensing pathogen specific molecular patterns via Pattern Recognition Receptors (PRRs) 3, 4. The recognition of an infectious agent by innate immune cells lead to the release of specific signaling proteins, called cytokines, thus inducing a pro-inflammatory environment5. Although this method provides an excellent first line of defense, the innate immune system is limited as it lacks specificity (i.e. self vs non-self) and inability to form immunologic memory (the ability to respond faster to a pathogen upon reinfection) 6. These shortcomings are complemented by the adaptive immune system, which has characteristics that form the basis of tumor immunotherapy and vaccine synthesis.

The activation of the adaptive immune system occurs once the innate system has acknowledged infection, which allows for the appropriate induction of cells within the adaptive arm. For this purpose, a type of Antigen Presenting Cell (APC) called Dendritic Cells (DCs) exist to relay the necessary information from one arm over to the other. Once activated through PRRs, DCs secrete pro-inflammatory cytokines and engulf the pathogen. Following intracellular breakdown, a tiny fragment of the pathogen (defined as an antigen) is mounted onto a cell surface protein called the

Major Histocompatibility Complex II (MHC II) 7. The expression of Peptide-MHC II complexes on the surface of an APC is essential to kick-start the adaptive immune response. Additionally,

APCs also begin to express certain cell surface molecules (called co-stimulatory molecules) important for cell-cell crosstalk and acquire the ability to travel towards primary and secondary lymphoid organs8. It is within these lymphoid organs that DCs initiate the activation and differentiation of the adaptive immune system5, 9, 10.

The development of the adaptive immune system

The adaptive immune system comprises of multiple cell types which can be broadly categorized into T-cells and B-cells. While B-cells are responsible for propagating humoral-mediated immunity, such as antibody assisted pathogen clearance, T-cells provide cell-mediated immunity

11. Cell mediated immunity is carried out by two distinct subsets of T-cells (categorized by the presence of specific cell surface markers), namely CD4+ and CD8+ T-cells. Briefly, CD4+ T-cells

(or helper T-cells) work towards promoting/suppressing an inflammatory environment through cytokine secretion, such as interleukins (ILs) whereas CD8+ T-cells (cytotoxic T-cells) release factors that induce apoptosis within an infected cell 12, 13. The first step of T-cell activation requires

T-helper cells to come in contact with a DC that has engulfed a pathogen and contains a peptide-

MHC complex on its surface. The recognition of unique peptide-MHC complexes by the T-cell receptor form the basis of specificity.

The T-cell Receptor (TCR) is synthesized and expressed during the development of a T-cell from a lymphoid progenitor into a mature naïve state (i.e. a state in which these cells are not yet activated and differentiated but have gone through multiple stages of selection) 14. The synthesis of TCRs occurs through a process known as VDJ recombination, in which mutations (ergo, diversity) within the receptor are introduced through breaking and consequently, repairing the DNA 15, 16. VDJ recombination allows for tremendous receptor diversity between T-cells, resulting in a single receptor that is able to recognize peptide-MHCII complexes harbored by DCs. This recognition allows for activation and rapid expansion of a single T-cell into effector cells (known as clonal expansion) that is specific towards a pathogen.

Upon pathogen clearance, several T-cell clones specific to the pathogen perish. However, a small population of T-cells survive and remain quiescent. These cells form the memory pool of T-cells.

Hence in the event of reinfection, these T-cells become active and assist in the rapid clearance of the pathogen via their designated effector functions 17. The decision to forego apoptosis and remain in a quiescent state is highly dependent on environmental stimuli, cell intrinsic metabolism and the presence of specific transcription factors within the cell 17.

The activation, differentiation and function of T-helper (TH) cells

Helper T-cells, as the name suggests, aid in immune responses by secreting factors that modulate the inflammatory responses at the site of infection. Upon activation and clonal expansion, effector

T-cells express chemokine receptors that allow migration to the site of infection and also attain the ability to secrete cytokines that modulate immune responses as necessary 18.

In order to ensure the appropriate effects are exerted at the site of infection, mature T-cells can differentiate into various subsets, with each subset critical in providing cell mediated immunity towards combating a pathogen. Since the discovery of prototypical helper T-cell types, Type 1 and

Type 2 helper T-cells (denoted as TH1 and TH2 respectively), other prominent T-helper cell subsets

19, 20, 21, 22, 23, 24 have been discovered, including TFH, TH17,TH9, TH22 and TREG cells .

Here, certain T-helper cells play an important role in accentuating a pro-inflammatory environment. For example, TH1 cells play a key role in the fight against intracellular pathogens such as viruses 25. These cells enhance cell-mediated immunity by secreting the pro-inflammatory cytokine Interferon gamma (IFN-γ), thus promoting macrophage, CD8+ T-cell and Natural Killer

26 (NK) cell activation . More recently discovered TFH cells aid immune responses by interacting with B-cells and improve antibody specificity, thus a key player in vaccine mediated immune

27 therapy . On the other hand, TREG cells attenuate immune responses post infection by secreting

4 the anti-inflammatory cytokines interleukin 10 (IL-10) and modulating IL-2 levels within the environment 28.

The activation of a naïve T-helper cell is contingent upon contact with a DC. In addition to peptide:

MHCII-cognate TCR complexes, which activate T-cells, co-stimulation (achieved via co- stimulatory molecules on the surfaces of DCs and T-cells) allow for survivability and cell surface receptor expression 8. However, paracrine cytokine signaling, induces differentiation of T-cells into the subsets mentioned above 29. Classically, these cytokines are secreted predominantly by

DCs, because pathogen recognition and processing by DCs induces the secretion of specific cytokine profiles that promote a certain helper T-cell phenotypes 5.

Signaling via cytokines induces the activation of specific transcription factors called STAT (Signal

Transducer and Activator of Transcription) factors 30. These proteins subsequently dimerize and translocate into the nucleus, where they bind to DNA and induce (or in some cases, repress) the expression of another set of transcription factors termed Lineage Defining Transcription factors

(LDTFs). As the name suggests, LDTFs are essential for the development and function of helper

T-cell subsets 31. These two factors subsequently co-ordinate and bind to multiple regions within the genome, such as promoter regions near the transcription start site and enhancer regions within the gene loci to regulate expression 32, 33, 34. As such, LDTFs and STAT transcription factor(s) are usually attributed to a certain T-helper cell subset, where they work in tandem to promote a specific

T-helper cell gene profile. It is important to note here that although STATs and LDTFs are necessary, the concerted effort of additional regulatory pathways, complex and interlinked, are equally important for T-cell differentiation and function 34 (Figure 1.1).

Figure 1.1. Differentiation of T-helper cell subsets. Upon initial activation of naïve CD4+ T-helper cell by a dendritic cell, the presence of environmental cytokines activates specific STAT factors, which consequently lead to the induction of LDTFs. The collaborative effort of STAT and LDTFs promote the development of specific and context dependent T-helper cell subset. Image adapted from O’Shea et al. 2010 31.

Mechanisms of T-helper cell flexibility

Although it was previously thought that the differentiation of T-helper cells was unidirectional and terminal in nature, over time this paradigm has been superseded by one that revolves around flexibility (or plasticity). Mounting evidence indicated that effector T-cells can acquire aspects of other T-helper subsets35, 36. An important mechanism by which such flexibility is achieved is through a genomic architecture that is highly dynamic and one that can be regulated, in part, through downstream regulators of cytokine signaling (Figure 1.2).

Inactive regions of the genome (heterochromatin) are traditionally coiled around protein structures known as histones (such complexes are known as nucleosomes) 37. Subunits of the histone complex

(an octamer that consists of dimers of subunits H2A H2B H3 and H4) can be modified post- translationally by the addition of acetyl or methyl moieties to lysine residues within the histone 37.

The addition of these ‘marks’ modulates the binding affinity of DNA to the histones, causing DNA to be either unspooled or promote compaction 37. As such, these marks on histones are reflective of a permissive or repressive gene state. Marks such as H3K9Ac and H3K4Me3 indicate a region of active gene transcription, while marks such as H3K27Me3 are indicative of transcriptional silencing

38, 39, 40. Additionally, the existence of a bivalent chromatin state (i.e. a gene loci with concurrent permissive and repressive histone marks) makes such regions ‘poised’ for expression41.These post translational modifications are performed by enzymes such as histone acetyltransferases (HATs),

Histone Deacetylases (HDACs) and Histone Methyl-transferases (HMTs) 42. Such modifications lead to nucleosome remodeling by large multi-protein complexes such as Mi-2/NuRD (Mi-

2/nucleosomal remodeling and deacetylase) or SWI/SNF (SWItch/Sucrose Non-Fermentable) complexes, thereby regulating gene expression 43, 44, 45 .

IL-2 TH2

IL-4

TH2 TREG /TFH /TH2 TREG

TH1 /TH2

T TGFβ REG /TH17 CD4+ T-cell

T 17 TH1 T 1 TH17 H H /TFH /TFH

IL-12 IFNγ TFH IL-6 IL-21

Figure 1.2. The flexible nature of T-helper cells. Cytokine induced activation of STAT and LDTFs lead to the differentiation of T-helper cells into a polarized subset. However, dynamic changes in cytokines within the microenvironment can lead to co-activation and co-expression of STAT and LDTFs, respectively. This allows for T- helper cells to acquire certain shared characteristics during infection, consequently allowing for a relatively efficient immune system in the event of infection. Image adapted from DuPage et al. 201646.

With the advent of next generation sequencing techniques (NGS), researchers found that although genes encoding effector cytokines had the necessary histone marks based on T-helper cell subset, a bivalent histone state existed within genes encoding LDTFs (thus making these factors poised for expression) 47. Additionally, STAT factors were shown to modulate histone compositions, with

STAT deficiency deregulating remodeling and transcriptional complex binding within target gene loci 48, 49, 50, 51. These findings, coupled with the observation that T-helper cells can co-express

LDTFs (and regulate each other’s activity) and LDTFs can also modulate histones to prime effector gene loci, show that the spatiotemporal induction and activation of LDTF and STAT factors dictates flexibility and effector functions in T-cells 40, 52, 53.

Plasticity provides the body with a relatively efficient immune system and studies have shown that a greater degree of flexibility assists in context dependent immune responses 54, 55. However, several studies have shown that deregulation of cellular pathways in T-helper cells cause unusual inflammatory characteristics during chronic infection and conversion of TREG cells to pathogenic

T-cells in autoimmune diseases such as arthritis 56, 57. Therefore, understanding the mechanistic players (and the regulatory networks) involved in T-cell plasticity would allow us to direct immune responses to desired results in the advent of autoimmunity and infection 58.

The role of IL-2 in mediating flexibility between TH1 and TFH cells.

The flexibility between TH1 and TFH cells has been well documented. Early studies showed that

TFH cells grown in vitro could secrete IFN-γ, with TH1 cells capable of producing IL-21 and TFH

59, 60 effector modules existing during the differentiation of naïve T-cells to effector TH1 cells .

While IL-12 was shown to play an important role in this process, another key mediator of this flexibility is IL-2, a pleotropic cytokine that is necessary for TH1 proliferation and survival but

60, 61, 62 known to impede TFH development . Through STAT5 activation, IL-2 drives TH1 development by inducing the expression of critical cytokine receptors such as Il2ra and Il12rb2, transcriptional factors such as Blimp-1 (gene name Prdm1) and the TH1 LDTF T-bet (gene name

63, 64, 65, 66 Tbx21, while attenuating Bcl6 levels in TH1 cells . While T-bet is important for the production of IFN-γ in TH1 cells, Blimp-1 maintains TH1 polarity by repressing alternative T- helper cell fates65, 67, 68. An absence (or decrease) of IL-2 within the environment diminishes

STAT5 activity in TH1 cells, therefore inducing Bcl6 expression and consequently, drives the

69, 70 transition of these cells to exhibit a TFH gene profile .

The role of IL-2 in TH1/ TFH flexibility is just one example that illuminates the substantial influence cytokines have on T-helper cell polarity. These studies are important, as decoupling, or deregulations within cell intrinsic transcriptional networks can lead to T-cell mediated pathogenicity 46, 71. As such, an active area of investigation is aimed at identifying the entire range of regulatory factors and the molecular mechanisms that come together during IL-2 mediated

TH1/TFH development and flexibility.

Novel transcriptional regulators of TH1 and TFH cell development.

The Ikaros Zinc finger (IkZF) family of transcription factors plays an important role in hematopoiesis. These proteins, like STAT factors, can modulate gene expression by binding to conserved regions within the DNA and associate with nucleosome remodeling complexes, therefore regulating genome architecture 72, 73. While the role of STAT factors in effector T-helper cells have been reviewed extensively, in Chapter 2 we review the current literature regarding the role of IkZF factors in regulating effector T-helper cell development 74, 75. Following this, in

Chapters 3 and 4 we observe how members of the IkZF family are influenced by IL-2 signaling and collaborate with STAT factors to promote the development of TFH and TH1 cells respectively.

A major goal of the work in this dissertation is to delineate mechanisms by which IL-2 influences

TH1 and TFH development. By discovering previously undocumented signaling pathways and transcription factors that come together to influence plasticity, it would allow for the creation of therapeutic targets that can skew these T-cell populations towards a desired subset. As such, findings presented here may open doors to novel therapies in the future.

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Chapter 2 Ikaros zinc finger transcription factors: Regulators of cytokine signaling pathways and CD4+ T helper cell differentiation

Michael D. Powell1,4,6, Kaitlin A. Read1,5,6, Bharath K. Sreekumar1,4, and Kenneth J. Oestreich1,2,3,*

1Fralin Biomedical Research Institute at Virgina Tech Carilion, Roanoke, VA, USA 2Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

3Virginia Tech Carilion School of Medicine, Roanoke, VA, USA

4Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, VA, USA

5Biomedical and Veterinary Sciences Graduate Program, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

6These authors made equal contributions to the manuscript.

Published as:

Powell, M. D., Read, K. A., Sreekumar, B. K. & Oestreich, K. J. Ikaros Zinc Finger Transcription Factors: Regulators of Cytokine Signaling Pathways and CD4 T Helper Cell Differentiation. Frontiers in Immunology 10, (2019).

Abstract

CD4+ T helper cells are capable of differentiating into a number of effector subsets that perform diverse functions during adaptive immune responses. The differentiation of each of these subsets is governed, in large part, by environmental cytokine signals and the subsequent activation of downstream, cell-intrinsic transcription factor networks. Ikaros zinc finger (IkZF) transcription factors are known regulators of immune cell development, including that of CD4+ T cell subsets.

Over the past decade, members of the IkZF family have also been implicated in the differentiation and function of individual T helper cell subsets, including T helper 1 (TH1), TH2, TH17, T follicular

(TFH), and T regulatory (TREG) cells. Now, an increasing body of literature suggests that the distinct cell-specific cytokine environments responsible for the development of each subset result in differential expression of IkZF factors across T helper populations. Intriguingly, recent studies suggest that IkZF members influence T helper subset differentiation in a feed-forward fashion through the regulation of these same cytokine-signaling pathways. Here, we review the increasingly prominent role for IkZF transcription factors in the differentiation of effector CD4+ T helper cell subsets.

Introduction

The seminal discovery by Mosmann and Coffman that naïve CD4+ T cells could differentiate into either T helper 1 (TH1) or TH2 subsets launched an area of immunological investigation aimed at understanding the mechanisms underlying the functional diversity of CD4+ T helper cell

1 populations . In the past three decades, the original TH1 and TH2 dichotomy has been expanded to include additional subsets such as TH17, T follicular helper (TFH), and regulatory T (TREG) cell populations 2, 3, 4. The diverse functions performed by these populations permit a highly tailored pathogen-specific immune response to bacterial, viral, and parasitic infections. Conversely, dysregulated T helper cell responses have been implicated in a number of autoimmune disorders, including type 1 diabetes, multiple sclerosis, Crohn’s disease, and others 3, 5, 6, 7. Thus, due to the importance of these cell populations to human health, extensive efforts have been undertaken to better understand how CD4+ T helper cell subset differentiation is regulated.

Generally, it is recognized that the differentiation of effector CD4+ T cell populations requires three signals. Two of these signals are derived from direct cell-to-cell contact with an antigen- presenting cell (APC), in the form of T cell receptor and co-stimulatory receptor activation 8, 9.

Importantly, the third signal, derived from the cytokine environment, drives CD4+ T helper cell subset specification through the activation of cytokine-specific transcription factor networks.

Association of cytokines with their specific receptors results in the activation of Janus kinase/Signal Transducer Activator of Transcription (JAK/STAT) pathways, in which JAKs phosphorylate members of the STAT factor family 10. This ultimately leads to dimerization and translocation of STAT factors into the nucleus, where they activate the expression of subset- specific genes including those encoding ‘lineage-defining’ transcription factors, which are required for the differentiation of each T helper cell subset 11, 12.

As with STAT transcription factors, members of the Ikaros Zinc Finger (IkZF) transcription factor family have well-documented roles in the development of immune cell populations 13, 14, 15. Ikaros, the founding member of the family, was initially shown to be required for lymphoid cell development, as mice expressing a dominant negative form of Ikaros failed to produce early T and

B lymphocyte progenitors, as well as Natural Killer cells 16, 17. In the following decades, four proteins with a high degree of homology to Ikaros were identified and now comprise the IkZF family of transcription factors: Ikaros (encoded by the gene Ikzf1), Helios (Ikzf2), Aiolos (Ikzf3),

Eos (Ikzf4), and Pegasus (Ikzf5) 18, 19, 20, 21, 22.

Structurally, IkZF family members contain both an N-terminal zinc finger (ZF) DNA-binding domain and a C-terminal ZF protein-protein interaction domain (Figure 2.1) 23. This distinct structure confers diverse functional capabilities, as IkZF family members can both positively and negatively regulate gene expression through direct interactions with DNA, as well as by forming transcriptional complexes with other proteins. Mechanistically, IkZF factors have been shown to regulate gene expression by (1) remodeling chromatin structure through association with chromatin remodeling complexes such as the nucleosome remodeling deacetylase (NuRD), (2) interacting with and promoting the activity of the RNA Pol II transcription initiation complex, and

(3) inducing chromosome conformational changes by mediating interactions between distal cis- regulatory regions 14, 15, 24, 25, 26.

Recent research efforts have examined potential roles for IkZF family members in regulating the development of effector CD4+ T cell populations. Intriguingly, many of these studies point to mechanisms whereby IkZF factors propagate T helper cell subset differentiation via the

22 modulation of cytokine signaling pathways. Here, we review the literature describing roles for

IkZF members in the regulation of CD4+ T cell differentiation.

DNA-binding domain Protein Interaction Domain ZF1 ZF2 ZF3 ZF4 ZF5 ZF6

Ikaros (Ikzf1) NH2 COOH

Helios (Ikzf2) NH2 COOH

Aiolos (Ikzf3) NH2 COOH

Eos (Ikzf4) NH2 COOH

Pegasus (Ikzf5) NH COOH 2

ZF1: Binding site/target gene specificity ZF2, ZF3: Interaction with core IkZF DNA-binding motif (GGGAA) ZF4: Binding site/target gene specificity ZF5, ZF6: Homo and hetero-dimerization of IkZF family members

Figure 2.1. Structure of IkZF family members. Members of the Ikaros Zinc Finger (IkZF) family of transcription factors are known regulators of hematopoietic cell development, including that of CD4+ T cells. The IkZF family consists of five members: Ikaros (encoded by the gene Ikzf1), Helios (Ikzf2), Aiolos (Ikzf3), Eos (Ikzf4), and Pegasus (Ikzf5). These factors contain N-terminal zinc finger (ZF) domains, which are responsible for mediating direct interactions with DNA, and C-terminal ZFs, which facilitate homo- and heterodimerization between IkZF family members. Of the N-terminal zinc fingers, ZF2 and ZF3 mediate direct interaction with the core IkZF DNA binding motif (GGGAA), while ZF1 and ZF4 regulate factor binding site/target gene specificity.

T helper 1 (TH1) cells

The differentiation and function of TH1 cells is critical for effective adaptive immune responses against intracellular pathogens, including viruses, bacteria, and parasites. TH1 differentiation is dependent upon extracellular signals from the cytokine IL-12, which lead to activation of the transcription factor STAT4 27, 28, 29. Upon activation, STAT4 dimerizes and translocates to the nucleus where it directly activates expression of Tbx21, the gene encoding the TH1 lineage- defining transcription factor T-bet. T-bet directly induces the expression of the key TH1 effector cytokine Interferon-gamma (IFN-γ), which functions in a feed-forward fashion, as autocrine IFN-

γ signals lead to STAT1 activation and further T-bet expression 30. The production of IFN-γ allows effector TH1 cells to initiate anti-intracellular pathogen responses including increased activation and proliferation of macrophages, as well as the activation of CD8+ T cell populations, which are responsible for the elimination of infected cells. In addition to IL-12 signaling, TH1 cell differentiation is dependent upon autocrine signals from IL-2, the expression of which is induced upon T cell receptor/co-receptor activation. IL-2 signaling results in the activation of STAT5, which, like STAT4, induces expression of genes encoding both transcription factors (including

Blimp-1) and cytokine receptors (including components of the IL-12 and IL-2 receptor complexes)

31, 32 that are required for continued commitment to the TH1 lineage .

IkZF factors in TH1 cell differentiation and function

Of the IkZF family members, Ikaros has been most extensively linked to aspects of TH1 cell development. It has been shown in a number of experimental settings that loss of Ikaros expression and/or function results in increased T-bet expression, suggesting that Ikaros plays a role in

33, 34, 35 negatively regulating TH1 differentiation . Specifically, overexpression of a dominant

null negative form of Ikaros in TH2 cells resulted in increased T-bet expression, while Ikaros in vitro- differentiated TH2 cells expressed increased T-bet as compared to wildtype controls. Supporting a role for Ikaros in the direct regulation of T-bet expression, Ikaros has been shown to directly

33, 34 bind to the Tbx21 promoter in in vitro-polarized TH2 cells . However, Ikaros was noticeably absent from the Tbx21 promoter in in vitro differentiated TH1 cells, for which T-bet expression is required 34. Mechanistically, the association of Ikaros with the Tbx21 promoter may be related to alterations in chromatin structure, as another study found increased enrichment of the repressive chromatin mark H3K27me3 at this locus upon Ikaros binding in thymocyte populations 36. However, whether this mechanism is conserved in CD4+ T cell populations is unclear. Regardless, the collective data support a role for Ikaros in the negative regulation of TH1 cell differentiation through direct repression of T-bet expression.

In addition to regulating TH1 differentiation pathways, Ikaros has been shown to negatively regulate expression of the TH1 effector cytokine, IFN-γ. Ikaros enrichment was observed at predicted Ifng regulatory regions in TH2 cells, and the Ifng promoter displayed reduced methylation

33, 34 null in TH2 cells expressing a dominant negative form of Ikaros . Furthermore, Ikaros TH2 cells were shown to exhibit increased IFN-γ production, as well as an increase in both T-bet and STAT1 transcript expression as compared to WT controls 33, 34. In further support of a T-bet-independent role for Ikaros in regulating Ifng expression, it has been shown that overexpression of wildtype

null Ikaros in Ikaros TH2 cells results in reduced IFN-γ production in the absence of a significant impact on T-bet expression 37. Collectively, these data further support a repressive role for Ikaros in both TH1 cell differentiation and function.

It is important to note, however, that all of the above studies utilized germline mutant models to assess the role of Ikaros in regulating T helper cell differentiation programs. Providing further clarity regarding the role of Ikaros in T helper cell differentiation decisions, a recent study assessed the effects of conditional Ikaros knockout exclusively in mature T cell populations on CD4+ T cell

38 differentiation and function . Curiously, Ikaros-deficient mature T helper cells exposed to TH1- polarizing conditions did not exhibit increased T-bet or IFN-γ expression as compared to WT.

However, Ikaros-deficient TH2 cells displayed increased IFN-γ expression, possibly supporting a role for Ikaros in negatively regulating TH1 gene expression in alternative T helper cell subsets, consistent with previous findings 38.

Illustrating an expanded role for Ikaros in regulating TH1 cytokine signaling pathways, Ikaros has also been shown to directly associate with the Il2 promoter and repress its expression (Figure 2.2)

39. Loss of Ikaros function was found to result in increased acetylation at the Il2 promoter, which correlated with increased IL-2 production in anergic T helper cells undergoing TCR stimulation.

Similarly, Aiolos has also been shown to directly repress IL-2 expression 40. Given the importance of the IL-2/STAT5 pathway to TH1 cell differentiation, these data suggest that Ikaros and Aiolos may also negatively regulate TH1 differentiation by repressing autocrine IL-2 signaling.

Unlike Ikaros and Aiolos, the available literature suggests that Eos may function as a positive regulator of the TH1 gene program. Specifically, mixed bone marrow chimera studies utilizing a murine experimental autoimmune encephalomyelitis (EAE) model demonstrated that there was a reduction in IFN-γ production by Eos-deficient versus WT CD4+ T cells. Curiously, CD4+ T cells in Eos-/- animals displayed no difference in IFN-γ production as compared to those in WT animals during EAE, suggesting that there may be a compensatory mechanism at play in Eos-deficient animals 41. Furthermore, Eos was shown to regulate the expression of both IL-2 and CD25

+ 41 expression in conventional CD4 T (TCONV) cell populations . Thus, Eos may positively influence

TH1 gene expression patterns, at least in part, by promoting the IL-2/STAT5 signaling axis.

Additional work will be required to determine the precise role of Eos in promoting TH1 cell differentiation. Collectively, these studies support opposing roles for Ikaros and Aiolos versus

Eos in regulating TH1 cell differentiation and function.

T helper 2 (TH2) cells

TH2 responses both provide protection against parasitic infection, such as with helminthic worms, and also assist with tissue repair mechanisms following parasite- and inflammation-induced

42 damage . TH2 differentiation is dependent upon both paracrine and autocrine IL-4 signaling, which results in the activation of STAT6. Once activated, STAT6 directly induces the expression

43 of the TH2 lineage-defining transcription factor GATA3 . GATA3 then directly activates expression of the TH2 effector cytokines IL-4, IL-5, and IL-13, which recruit and activate additional immune cell types, such as macrophages and eosinophils, and promote IgE production to facilitate parasite clearance. As with TH1 cells, TH2 cell differentiation is also positively regulated by autocrine IL-2/STAT5 signaling, which, in the TH2 context, functions to induce

GATA3-indepdendent expression of IL-4, as well as the feed-forward induction of IL-4Rα 44.

IkZF factors in TH2 cell differentiation and function

Ikaros has been implicated in positively regulating IL-4 production in TH2 cells. Specifically,

+ null naïve CD4 T helper cells isolated from Ikaros mice exposed to TH2-polarizing conditions were found to exhibit reduced production of IL-4, and direct association of Ikaros with the Il4 locus was

28 accompanied by increased H3 acetylation 33. Unsurprisingly, this correlated with decreased

33 expression of the TH2 transcription factors GATA3 and cMaf . As mentioned previously, Ikaros has also been shown to promote TH2 lineage specification through direct repression of the TH1 lineage-defining transcription factor, T-bet 34. In support of this, in an in vivo experimental setting, mice heterozygous for a dominant negative form of Ikaros displayed an inappropriate TH1 response to Shistosoma mansoni parasitic infection 34. It is important to note that given heterodimeric interactions between IkZF family members, the dominant negative Ikaros protein, which lacks a functional N-terminal DNA binding domain, may disrupt the function of other IkZF factors in this experimental setting. Thus, the precise role of Ikaros in regulating the phenotypes observed in this study is unclear. Additionally, contrary to the above findings, a recent study utilizing a CD4+ specific conditional knockout of Ikaros exclusively in mature T cell populations found that Ikaros- deficient TH2 cells do not display a defect in either Gata3 or IL-4 expression, although they do produce more IFN-γ, as described above 38. Thus, it is possible that Ikaros deficiency during T cell development, versus the naïve to effector transition, results in an altered phenotype that makes analysis of individual effector T cell populations difficult. Ultimately, these data suggest that

Ikaros may support non-TH1 subsets by repressing the Th1 gene program during differentiation.

Similar to Ikaros, Helios expression is upregulated in TH2 cells generated in response to ovalbumin immunization in vivo, coinciding with the expression of GATA3, cMaf, and IL-4 45. However, unlike Ikaros, Helios does not seem to be required for induction of the TH2 phenotype, as the same study showed that loss of Helios expression had no effect on the expression of TH2-associated cytokines and transcription factors at the transcript level 45. Additionally, no difference in IL-4 production was observed between TH2-polarized WT and Helios-deficient T cells, further

46 establishing Helios as non-essential for TH2 development . Thus, although Helios is highly

29 expressed in TH2 cells, there is a lack of evidence to support Helios as a regulator of TH2 development.

In addition to the production of IL-4, TH2 cells mediate their effector functions via secretion of the pro-inflammatory cytokines IL-5 and IL-13 42. As with the IL-4 studies described above, Ikaros

+ appears to positively mediate TH2-associated functions as exposure of naïve CD4 T helper cells

null isolated from Ikaros mice to TH2 polarizing conditions resulted in reduced production of IL-5 and IL-13 compared to cells from wild-type mice 33. This is perhaps unsurprising, given the requirement for Ikaros for expression of GATA3, which directly regulates the expression of IL-5 and IL-13. Collectively, the above studies suggest that Ikaros positively regulates TH2 differentiation and function, both via the activation of IL-4 expression and through repression of the opposing TH1 differentiation program.

Finally, as mentioned above, Ikaros has also been implicated in the direct repression of Il2, which

+ 39 is critical for TH2 differentiation, in activated CD4 T cell populations . This is somewhat at odds with the positive role for Ikaros in regulating TH2 development and suggests that Ikaros may serve differential roles across T helper cell subsets.

T helper 17 (TH17) cells

TH17 cells are essential mediators of immunity at mucosal surfaces, and function to eliminate pathogenic extracellular bacteria and fungi 7, 47. A number of different cytokines have been implicated in TH17 cell differentiation, including TGF-β, and IL-6, among others. Signals from these cytokines result in the upregulation of TH17 transcriptional network that includes activation of STAT3, which directly regulates a number of genes required for TH17 differentiation including

48, 49 the TH17 lineage defining transcription factor RORγt . Both STAT3 and RORγt are required

30 for the production of the pro-inflammatory TH17 effector cytokines IL-17 and IL-22, which recruit and activate immune cells including neutrophils during the course of infection 2.

IkZF factors in TH17 cell differentiation and function

35 Recent work suggests that Ikaros is an important regulator of the TH17 gene program .

Specifically, loss of Ikaros expression in in vitro-generated TH17 cells has been found to result in decreased expression of TH17 genes including those encoding RORγt and IL-17. Consistent with the known role for Ikaros in modulating the epigenetic landscape, expression of Ikaros in TH17 cells has been shown to correlate with increased enrichment of permissive covalent histone modifications at these loci 35. More recent reports have described a somewhat conflicting role for

38, 50 Ikaros in TH17 function . In one study, inhibiting the DNA-binding capability of Ikaros had negligible effects on IL-17 production, while the production of IL-22 was increased 50. One possible explanation for the discrepancy presented by this study is the use of mice expressing a mutant Ikaros protein lacking a functional N-terminal ZF4 domain (Ikzf1∆f4/∆f4), as opposed to cells from Ikzf1Null mice. Zinc fingers 1 and 4 of the N-terminal zinc finger domain of Ikaros are important for binding to specific target genes, while zinc fingers 2 and 3 bind the core consensus sequence GGGAA 14, 15, 51 (Figure 2.1). Thus, the mutant utilized in the study may have retained some functionality regarding its DNA binding capability, which could explain the lack of an effect on IL-17 production in this setting. Furthermore, when Ikaros was conditionally knocked out in mature T cell populations, the expression of neither Il17 nor Rorc was impacted when Ikaros-

+ deficient CD4 T cells were exposed to TH17 polarizing conditions. Curiously, these cells exhibited increased expression of a pathogenic TH17 phenotype, including higher levels of T-bet

31 expression and increased IFN-γ production. These data once again support a role for Ikaros in

38 repressing TH1 gene expression during T helper cell differentiation .

Similar to Ikaros, recent work has established that Aiolos is necessary for the expression of TH17- associated genes including those that encode both IL-17a and IL-17f 40. Mechanistically, this study also determined that Aiolos aided TH17 lineage commitment, at least in part, through the direct

40, 52, silencing of the Il2 locus, as IL-2/STAT5 signaling negatively regulates TH17 development

53, 54 . Thus, both Ikaros and Aiolos appear to regulate TH17 differentiation through modulation of

TH17 gene expression and via repression of alternative gene programs.

In contrast to Ikaros and Aiolos, there is evidence to suggest that Eos may negatively regulate

TH17 differentiation. Specifically, inhibition of Eos expression by the miRNA miR-17 was shown

55 to enhance TH17 cell development . Furthermore, Eos-deficient TREG populations were found to gain the ability to produce IL-17 56. Consistent with this finding, Eos-deficient mice were also found to develop more severe EAE, which correlated with an increased presence of IL-17- producing cells in the central nervous system (CNS) 41. Taken together, the above findings suggest that Ikaros and Aiolos are positive regulators of TH17 differentiation and function, while Eos appears to functionally antagonize the development of TH17 cell populations.

T follicular helper (TFH) cells

TFH cells play critical roles in the generation of humoral immunity through their specialized ability to provide help to antibody-producing B cells. TFH cells engage in cognate interactions with B cells and produce the cytokine IL-21 to support the formation of germinal centers in secondary lymphoid tissues and B cell production of high-affinity, pathogen-specific antibodies 5, 57. As with

TH17 development, TFH cell differentiation can be driven by a number of cytokine signals, with

32 two of the more prominent being IL-6 and IL-21 58, 59, 60. Signals received from these cytokines result in subsequent activation of STAT3, which activates the expression of TFH genes including

61, 62, 63, 64 Bcl6, which encodes the lineage-defining transcription factor for the TFH cell subset .

IkZF factors in TFH cell differentiation and function

32, 65, 66, 67 IL-2 signaling is a potent inhibitor of TFH cell differentiation . Thus, it is not surprising that as antagonists of IL-2 signaling, Ikaros and Aiolos have been implicated as positive regulators

39, 40 of TFH cell differentiation .

As observed in other T helper cell populations, Eos appears to oppose the functions of Ikaros and

Aiolos in TFH cells as well. Indeed, unlike Ikaros and Aiolos, Eos expression inversely correlates

68 with that of the TFH gene program . A recent study utilizing Nr4a-deficient T cells, which display reduced Eos expression, more readily upregulate both TFH gene expression patterns and obtain the ability to support germinal center reactions 68. It is important to note that these cells also exhibit reduced expression of a number of genes in addition to Eos upon Nr4a loss, and thus this phenotype cannot be directly attributed to loss of Eos expression. An additional study demonstrated that Eos- deficient CD4+ T cells are less effective producers of IL-2 as compared to their wild-type counterparts. As IL-2 negatively regulates TFH cell differentiation, these findings suggest that Eos

41 may inhibit TFH differentiation via an IL-2-mediated mechanism (Figure 2.2) . Thus, further work is necessary to determine whether Eos functions to directly repress the TFH gene program or, rather, promotes the expression of alternative effector cell phenotypes.

Regulatory T (TREG) cells

Unlike the pro-inflammatory effector functions of other T helper cell subsets, the primary role of regulatory T cells is to maintain immune tolerance through a number of suppressive mechanisms,

69, 70 including the secretion of anti-inflammatory cytokines such as IL-10 . TREG development is driven by signals propagated through TGF-β engagement with its receptor and the resulting expression of TREG specific transcription factors, including the TREG lineage defining transcription factor Forkhead box P3 (FOXP3), and the IL-2 receptor α chain (CD25) 71, 72, 73. This stable expression of CD25, in conjunction with their inability to produce IL-2, allows TREGS to act as 'IL-

2 sinks', to restrain pro-inflammatory immune responses. While a number of TREG cell subsets have been identified, much recent work has focused on two major subsets: those that arise from

74 the thymus (tTREGS) and those that are generated in the periphery (pTREGS) .

IkZF factors in TREG differentiation and function

Gene expression analysis via microarray studies revealed that Eos is highly expressed in TREG populations and that it functions as a key component of the FOXP3-mediated gene repression complex. Mechanistically, it was shown that Eos forms a protein complex with FOXP3 and C-

75 terminal binding protein (CtBP) to promote gene silencing in TREG cells . These findings are consistent with another study demonstrating that Eos functions as a co-repressor in cooperation with Foxp3 to maintain the TREG phenotype and suppressive capabilities. Specifically, it was established that Eos downregulation occurs in TREG cells in response to inflammation, permitting their transition to a Foxp3-expressing T helper-like cell phenotype 56. Furthermore, another study found that knockdown of Eos expression resulted in decreased TREG function and a subsequent accentuation of colitis in mice 75. Intriguingly, a conflicting study found that Eos-deficient mice

34 did not exhibit defective TREG development or function, suggesting that another IkZF factor (or factors) may provide a certain level of redundancy (41).

Interestingly, one of the genes targeted by the Eos/FOXP3-repressive complex is the Il2 locus, which appears to be directly regulated by a number of IkZF factors across T helper cell populations

(Figure 2.2) 39, 40, 75. Furthermore, Eos-mediated repression of IL-2 seems to be dependent on the expression and activity of FOXP3, as Eos has also been shown to positively regulate IL-2 production in FOXP3- conventional T helper cell populations, as discussed previously (Figure

41 2.2) . Similar to Eos, Helios has also been implicated in repressing Il2 expression in TREG populations, supporting the possibility of redundant functions between these factors (Figure 2.2)

76.

Beyond its role in regulating IL-2 production in TREG cells, several studies have revealed that

+ 76 Helios is also required for the stability of a suppressive phenotype in FOXP3 TREG populations .

Consistent with these findings, the expression of pro-inflammatory cytokines, including IFN-γ,

TNF-α, and IL-17 by TREG populations is significantly increased in the absence of Helios, and

Helios-deficient mice display increased numbers of activated T cells and germinal center B cells,

77 as well as increased production of autoantibodies . Curiously, Helios-deficient TREG cells also exhibit reduced STAT5 activation and Foxp3 expression, the latter of which can be rescued upon overexpression of a constitutively active form of STAT5 77. Furthermore, the production of IL-17

+ - + + is significantly higher in human FOXP3 Helios memory TREGS as compared to FOXP3 Helios populations, supporting a role for Helios in negatively regulating IL-17 production in memory

78 TREG populations . However, it is important to note that some studies suggest that Helios expression does not always negatively correlate with inflammatory cytokine expression in TREG

+ populations. Indeed, Helios expression is consistent between conventional FOXP3 TREG cells and

+ IL-17-producing TREG cells that co-express FOXP3 and the TH17 lineage-defining transcription

+ + factor RORγt (FOXP3 RORγt TREG), indicating that IL-17 production may be unrelated to Helios expression in this population 79. Collectively, while many findings support a role for Helios in promoting TREG suppressive function by repressing effector cytokine production, further work is necessary to establish how Helios functions across diverse TREG subtypes.

A number of studies have implicated Ikaros in the regulation of TREG cell differentiation. To this

+ end, it has been shown that under iTREG-polarizing conditions, Ikaros-deficient CD4 T cells are unable to upregulate Foxp3 expression 35, 38, 80. Importantly, these studies include cells in which

Ikaros had been deleted in the germline 35, 80 and also exclusively in mature T cell populations 38.

Additionally, one group observed increased enrichment of the repressive chromatin mark

H3K27me2 at the Foxp3 promoter in Ikaros-/- naïve CD4+ T cells as compared to WT 35. In further support of a positive role for Ikaros in TREG differentiation, it has been shown that Ikaros-deficient animals exhibit reduced numbers of peripheral and natural TREG populations as compared to WT controls 80. Another study found that CD4+ T cells expressing a mutant form of Ikaros lacking the

N-terminal DNA-binding ZF4 (IkΔZF4) are unable to normally differentiate into iTREG populations 50. Curiously, while mice expressing the IkΔZF4 mutant were found to exhibit

+ increased numbers of total Foxp3 TREGS in vivo under steady-state conditions, the number of

50 pTREGS was reduced . Thus, the authors suggest that Ikaros may differentially regulate different

+ TREG cell compartments. It is important to note that this study found that CD4 T cells expressing the IkΔZF4 mutant produce significantly higher amounts of IL-21 than their WT counterparts,

50 which negatively regulates iTREG differentiation . However, Ikaros-deficient cells were not found to upregulate IL-21 production, indicating that the mechanism underlying the iTREG-deficient phenotypes differs between these two studies 50, 80. Indeed, the authors of the first study found that

CD4+ T cells isolated from the spleens of Ikaros-deficient mice exhibit a reduction in the expression of Foxo1, a transcription factor required for the generation of regulatory T cells 80. The different mechanisms observed between these studies may be attributed to use of the IkΔZF4 mutant, which is known to function as a dominant negative isoform. As discussed in previous sections, this may alter the function of other IkZF family members with which Ikaros interacts and may confound interpretation of the data in this system. Collectively, however, the available data support a role for Ikaros in positively regulating iTREG differentiation.

A B

FOXP3 FOXP3

Ikaros Aiolos Helios Eos Eos CtBP1

Il2 Il2

Figure 2.2. Transcriptional regulation of Interleukine-2 locus by IkZF members Signals from the pro- inflammatory cytokine interleukin-2 (IL-2) differentially regulate the expression of T helper cell programs. IL-2 signaling supports the differentiation of TH1, TH2, and TREG cell subsets, but represses the differentiation of TH17 and TFH populations. The Ikaros zinc finger family members Ikaros, Helios, Aiolos, and Eos have all been + implicated in regulating IL-2 expression. (A) In anergic CD4 and TH17 cells, respectively, Ikaros and Aiolos have been shown to directly associate with the Il2 promoter to repress IL-2 expression. For Ikaros, this association correlates with reduced H3 and H4 acetylation. Aiolos association has been linked to a decrease in both acetylation and the positive histone mark H3K4me3 at the Il2 promoter and a concurrent increase in H3K9me3, which is indicative of a transcriptionally inactive locus. In regulatory T (TREG) cells, Eos and Helios have both been implicated in IL-2 repression. Mechanistically, Eos forms a protein complex with the TREG lineage-defining transcription factor FOXP3 and C-terminal binding protein (CtBP) to repress Il2. As with Aiolos, this repression is associated with reduced H3 and H4 acetylation, reduced H3K4me3, and increased H3K9me3 enrichment at the Il2 promoter. Similarly, Helios and FOXP3 are co-enriched at the Il2 locus in TREG cells and correlate with reduced H3 acetylation, consistent with gene repression. (B) In conventional T helper cell populations, in the absence of FOXP3, Eos has been positively linked to the expression of IL-2. However, the mechanism by which Eos influences IL-2 expression is currently unclear. .

Concluding remarks

In the past decade, IkZF family members have emerged as key regulators of CD4+ T helper subset development and function. Interestingly, many of these studies have identified IkZF factors as regulators of cytokines, cytokine receptors, and other components of cytokine signaling pathways.

Likewise, it is becoming increasingly clear that cytokine signals reciprocally regulate the expression and activities of IkZF transcription factors. These points, coupled with the discovery that IkZF factors can engage in cooperative mechanisms with STAT transcription factors, suggest that IkZF factors may continue to emerge as central players in the regulation of T helper cell differentiation. Further work will be required to determine the extent to which IkZF factors may engage in similar mechanisms to regulate the differentiation and function of cells across the immune system. Importantly, cytokine signaling pathways have been popular targets of immunotherapeutic strategies to treat human diseases ranging from cancer to autoimmunity.

Therefore, continued study into the role of IkZF factors in the regulation of immune cell differentiation and function will inform the feasibility of targeting these factors in efforts to promote human health.

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Chapter 4

The Ikaros zinc finger transcription factor Eos promotes TH1 differentiation through modulation of the IL-2-STAT5 signaling pathway.

Bharath K. Sreekumar1,2, Michael D. Powell3, Kaitlin A. Read3,4, Devin M. Jones3,4, S. Jawad Zafar5, Mustafa N. Rasheed5, Lauren M. Childs6, Patrick Collins3 & Kenneth J. Oestreich3

1 Fralin Biomedical Research Institute, Roanoke, VA 24016, USA

2 Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, VA 24061, USA

3 Department of Microbial Infection and Immunity, The Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH; USA

4 Biomedical Sciences Graduate Program, Columbus, OH; USA

5 Virginia Tech Carilion School of Medicine, Roanoke, VA 24016; USA

6 Department of Mathematics, Virginia Tech, Blacksburg, VA 24061; USA

Abstract

The Ikaros Zinc Finger transcription factor Eos is required for the suppressive function of CD4+ regulatory T cells. However, the extent to which Eos regulates other CD4+ T cell populations is unknown. In the present study, we find that TH1 differentiation is compromised in Eos-deficient

CD4+ T helper cells and that these cells are poor producers of IFN-γ. Mechanistically, our data demonstrate that IL-2 signaling drives Eos expression in a STAT5-dependent manner and that Eos and STAT5 interact in TH1 cells. Interestingly, we also find that STAT5 phosphorylation is reduced in the absence of Eos. Consequently, we observe reduced enrichment of STAT5 at TH1 target genes in Eos-deficient CD4+ T cells, including those that encode CD25, Blimp-1, and IFN-

γ. Thus, our data define a novel role for Eos in regulating the differentiation and function of TH1 cells via modulation of the IL-2-STAT5 cytokine signaling pathway.

Introduction

Members of the Ikaros Zinc Finger (IkZF) transcription factor family are known regulators of hematopoiesis, including the development of T and B cell populations1, 2, 3, 4. The Ikaros family is comprised of 5 members: Ikaros (encoded by the gene Ikzf1), Helios (Ikzf2), Aiolos (Ikzf3), Eos

(Ikzf4), and Pegasus (Ikzf5), which each contain both N-terminal zinc finger (ZF) DNA-binding and C-terminal ZF protein-protein interaction domains5. Initially, these factors were described as transcriptional repressors through their interaction with chromatin remodeling complexes, including the nucleosome remodeling deacetylase (NuRD) complex6. However, subsequent studies have demonstrated that IkZF factors can also positively regulate gene expression by both interacting with and promoting the activity of the RNA Pol II transcription initiation complex, and through inducing chromatin looping by mediating interactions between distal cis-regulatory gene elements7.

While early studies described essential roles for Ikaros and Aiolos in the development of T and B cell populations, increasing evidence supports diverse roles for IkZF factors in regulating CD4+ T helper cell subset differentiation and function3, 8, 9, 10, 11, 12. Specifically, recent studies have described roles for Ikaros and Aiolos in the positive regulation of T helper 2 (TH2), TH17 and T follicular helper (TFH) cell populations, predominantly through the transcriptional regulation of both subset-specific cytokine signaling pathways and expression of lineage-defining transcription factors13, 14, 15, 16, 17. Helios has also been implicated in the regulation of cytokine expression,

18, 19, 20, 21 specifically in regulatory T (TREG) cell populations . Similarly, roles for Eos have been described primarily in the context of TREG cell subsets, where it works in concert with Foxp3 to

22, 23, 24, 25 promote TREG-specific gene expression patterns and facilitate suppressive capabilities .

More recently, Eos has also been implicated in the regulation of IL-2 and IL-17 production in

+ 23, 26 conventional (non-TREG) CD4 T cell populations . However, the extent to which Eos may regulate the differentiation and function of conventional CD4+ T cell subsets is currently unknown.

Here, we define Eos as a novel, positive regulator of TH1 cell differentiation and function. We

+ find that Eos-deficient CD4 T cells exhibit decreased expression of many TH1-associated markers, including the transcriptional regulators T-bet and Blimp-1. Conversely, expression of transcription factors associated with alternative T helper cell fates, including TFH and TH17 cells, are induced in the absence of Eos. We also find that Eos-deficient CD4+ T cells are poor producers of IFN-γ and express reduced levels of CD25 (IL-2Rα and CD122 (IL-2Rβ). We further demonstrate that IL-2 signaling is responsible for driving Eos expression in a STAT5-dependent manner.

Mechanistically, we find that Eos and STAT5 physically interact in TH1 cells and that STAT5 phosphorylation is reduced in the absence of Eos. Consequently, in Eos-deficient cells, we observe decreased STAT5 association with TH1 target genes including Ifng, Il2ra, and Prdm1. Taken together, these findings identify Eos as a novel regulator of TH1 differentiation and suggest that

Eos serves as an important modulator of the IL-2-STAT5 signaling pathway.

Methods

Mouse Lines, Primary T Cell Isolation, and Cell Culture

C57BL/6J mice were purchased from the Jackson laboratory. Ikzf4-/- mice were generously provided by Drs. Ethan Shevach (NIH) and Bruce Morgan (Harvard Medical School)26. For in vitro polarization experiments, naïve CD4+ T cells were isolated from the spleens and lymph nodes of 5-8 week old mice using the BioLegend Mojosort negative selection kit according to the manufacturer’s instructions. Harvested T cells were plated at a density of 0.9-2.5x105 cells/mL in complete IMDM ((IMDM [Life Technologies], 10% FBS [26140079, Life Technologies], 1%

Penicillin-Streptomycin [Life Technologies], and 0.05% 2-ME [Sigma-Aldrich]) and stimulated on plate-bound anti-CD3 (5µg/ml, BD Biosciences) and anti-CD28 (2µg/ml, BD Biosciences) in the presence of TH1-polarizing cytokines (anti-IL-4 (5µg/ml, BioLegend), rmIL-12 (5ng/ml, R&D

Systems)) for 1-4 days. In some cases, IL-2 receptor blocking antibodies (α-CD25 (PC61), α-CD-

122 (TM-beta1), 10µg/mL, BD Biosciences) were added to the culture.

For IL-2 titration studies, harvested naïve T-cells were stimulated in the presence of TH1 polarizing conditions for 72 hrs. , afterwhich cells were removed from stimulation and expanded in the presence of anti-IL-4 (5µg/ml, BioLegend), rmIL-12 (5ng/ml, R&D Systems) and varying concentrations of IL-2 as indicated.

EL4 cells were acquired from the American Type Culture Collection (TIB-39) and cultured in complete RPMI (RPMI-1640, 10% FBS, 1% Pen/Strep).

Toxoplasma gondii infection and in vivo analysis.

Age and sex matched WT C57BL/6J and Ikzf4-/- mice were infected intraperitoneally with 20 cysts of T. gondii (ME-49 F1) as described previously27, 28. 8 days post infection, primary CD4+ T-cells were isolated from the spleens of T.gondii infected mice (uninfected mice served as control)using the Biolegend Mojosort Isolation Kit (cat no.480033). Isolated cells were pelleted, washed with

FACS buffer, and incubated with Fc block (anti CD16/CD132 (Invitrogen)) for 5-10 minutes on ice. Cells were then washed, and stained with cell surface markers anti-CD44 (V450, cat. no.

560452; BD Biosciences) and anti-CD4 (AF488, cat. no. 56-0031; eBioscience) for one hour at

RT. Extracellular staining was followed by cell fixation and permeabilization for 20 minutes, with subsequent intracellular staining of T-bet (APC, cat no. 644813; Biolegend) for 30 minutes. After incubation, cells were washed with perm buffer, resuspended in FACS buffer and analyzed on the sony SH800 flow cytometer. Data was evaluated using Flowjo software.

RNA isolation and qRT-PCR

Total RNA was isolated using the Nucleospin RNA Isolation kit (Macherey-Nagel) according to the manufacturer’s instructions. cDNA was generated using the Superscript IV First Strand

Synthesis System (Thermo Fisher). qRT-PCR reactions were performed using 4-20ng cDNA per reaction with the following primers: Ikzf4 forward: 5’-GAC GCA CTC ACT GGC CAC CTC C, and reverse:5’-GGC ACC TCT CCT TGT GCT CCT CC; Prdm1 forward: 5’ –CTT GTG TGG

TAT TGT CGG GAC, and reverse: 5’- CAC GCT GTA CTC TCT CTT GG; Ikzf3 forward: 5’-

GCT GCA AGT GTG GAG GCA AGA C, and reverse: 5’- GTT GGC ATC GAA GCA GTG

CCG; Bcl6 forward: 5’- CCA ACC TGA AGA CCC ACA CTC, and reverse: 5’- GCG CAG ATG

GCT CTT CAG AGT C; Ifng forward: 5’- CTA CCT TCT TCA GCA ACA GC, and reverse: 5’-

GCT CAT TGA ATG CTT GGC GC; IL2ra forward: 5’- CCA CAA CAG ACA TGC AGA AGC

C, and reverse: 5’- GCA GGA CCT CTC TGT AGA GCC TTG; IL2rb forward: 5’- GGC CAT

GGC TGA AGA CAG TTC TC, and reverse: 5’- CGG CCT TGG AAT CTC CGT CGA G; Tbx21 forward: 5’- GTC CAA GTT CAA CCA GCA CC, and reverse: 5’- GTT GGT GAG CTT TAG

CTT CC; Ikzf1 forward: 5’- ACG CAC TCC GTT GGT AAG CCT C, and reverse: 5’- TGC ACA

GGT CTT CTG CCA TCT CG; Havcr2 forward: 5’-CAC ATT GGA GTG GGA GTC TC, and reverse: 5’-GCG AAT CCT GAC TGC TCC TG; IL2 forward: 5’- TGG ACC TCT GCG GCA

TGT TCT G, and reverse: 5’- GCT GAC TCA TCA TCG AAT TGG CA C-3’.

Over expression and Small interfering RNA knockdown experiments.

Nucleofection assays were performed with the Lonza 4D Nucleofection system (buffer SF, program CM-120). Expression vectors were made by cloning the coding sequences into the pcDNA3.1/V5-His-TOPO vector (cat. no. K4800; Life Technologies). Constitutively active

STAT5B was generated as previously described29. Mutant proteins as indicated were generated using the Agilent QuikChange Site-Directed Mutagenesis Kit (part number 200519) according to the manufacturer’s instructions. Wildtype and mutant coding sequences were transferred to the pEF1/V5-His vector (cat. no. V920; Life Technologies) for overexpression. Overexpression of proteins was assessed via immunoblot using both V5-tag- and protein-specific antibodies, and alterations in gene expression were assessed via qRT-PCR analysis.

+ Primary murine CD4 T cells were cultured under TH1-polarizing conditions for 5 days. TH1 cells were nucleofected with either siGENOME SMARTpool small interfering RNA (siRNA) targeting

Ikzf4 (Dharmacon, D-051517 (01-04) or siGENOME Non targeting siRNA (Dharmacon, D-

001210-01-20). Nucleofected cells were recovered for 48h in media containing IL-2 (20 ng/ml)

96 and rmIL-12 (5ng/ml). RNA was isolated and changes in gene expression were analyzed via qRT-

PCR. Gene expression changes for Ikzf3 and Ikzf4 were assessed to confirm knockdown specificity and efficiency, respectively.

Immunoblot analysis

An equivalent number of cells were harvested for each analysis, lysed directly in 1X SDS loading dye (50 mM Tris [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 15 minutes. Lysates were separated via SDS-PAGE and transferred to 0.45µm nitrocellulose membrane. Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Eos (W15169A; BioLegend, 1:1000), Blimp-1 (GenScript,

1:500), pSTAT5(Y694) (BD Biosciences, 1:5000), STAT5 (Cell Signaling, 1:5000), β-Actin

(GenScript, 1:15,000), goat anti-mouse (Jackson Immunoresearch, 1:5000-1:10,000), mouse anti- rabbit (Santa Cruz, 1:5000-1:10,000).

Co-Immunoprecipitation (Co-IP)

Coimmunoprecipitation analyses were performed using primary murine TH1 cells or EL4 cells overexpressing indicated proteins as previously described15, 30, 31. Briefly, lysates were immunoprecipitated with anti-STAT5A/B (5µg/IP, Santa Cruz) or isotype control overnight at

4C. The following day, samples were incubated in the presence of Protein A Sepharose beads

(Millipore) for 1-2h, and immunoprecipitated proteins were analyzed via subsequent immunoblot analysis. Antibodies to detect immunoprecipitated proteins were as follows: Eos (1:1000, Santa

Cruz), STAT5A/B (1:5000 Santa Cruz) and V5 (1:20,000; R960-25, Invitrogen).

Chromatin Immunoprecipitation (ChIP)

Chromatin Immunoprecipitation (ChIP): ChIP assays were performed as previously described32.

Chromatin fragments were immuno-precipitated with anti-bodies to STAT5 (R&D AF2168;

5µg/IP), RNA-pol II (Abcam ab5095; 1µg/IP) ,or IgG control (Abcam ab6709; 5µg/IP).

Enrichment of STAT5 on precipitated DNA was analyzed using quantitative real time PCR with the following primers; Prdm1 Forward: 5’- GCC TCT GTA CTT GTG TTT CCT ACA CC-3’,

Reverse: 5’- GGC AGG GTT CCA AGT ATT CAT CTG-3’, Ifng Forward: 5’- GG CTT CCT

CAC CAC ATT GGC-3’, Reverse: 5’- CTC TTG GGC TTC TCA AAC CAT GC-3’, Il2ra

Forward: 5’-CTG GTT TTC CAC AGG ACC CTG-3’, Reverse: 5’-GTG TAA GAG AAG ACA

GCA GC-3’, Negative control (a region within the Prdm1 gene locus) Forward: 5’- GTC ACC

ACT CAA CTT CAG ACC AGA G-3’, Reverse: 5’- GCA GTA TCC AGG ACA ACT TTC TGC

TG-3’. Samples were normalized to total DNA control followed by subtraction of readouts obtained when chromatin was immuno-precipitated with IgG, controlling for non-specific background. Data is represented as fold change over WT.

RNA-seq Analysis

+ Naïve CD4 T cells were cultured under TH1-polarizing conditions for 3 days. Cells were harvested and total RNA was isolated using the Macherey-Nagel Nucleospin RNA Isolation kit. cDNA was generated using the Superscript IV First Strand Synthesis System from Thermo Fisher, and 3ug of cDNA was submitted to GENEWIZ for library preparation and sequencing.

ATAC-seq Analysis

+ Naïve CD4 T cells were cultured in the presence of TH1 polarizing conditions for 3 days. Cell pellets containing 1x105 cells were prepared as recommended by Active Motif. Briefly, cell pellets were re-suspended in fresh cIMDM plus DNAase buffer (250mM MgCl2, 50mM CaCl2) and

DNAase solution (20,000 units/ml of DNAase (Worthington LS002006) in HBSS (Gibco, 14025-

092)) at 1:100. Samples were incubated at 37C for 30 minutes and washed with PBS. Final pellets were resuspended in cIMDM with 5% DMSO and frozen at -80C. ATAC-seq analyses of prepared samples were performed by Active Motif.

Flow Cytometry

Cells were washed 1x with FACS buffer (PBS w/ 2% FBS, 1% BSA) and incubated in the presence of fluorochrome-conjugated antibodies against mouse IL-2Rα PE (eBioscience), IL-2Rβ PE

(Biolegend), or the appropriate isotype control. Cells were incubated for 1 hour at room temperature and subsequently washed 2x with FACS buffer prior to being analyzed. For intracellular staining, the eBioscience Foxp3/Transcription Factor Staining Buffer Set was used to fix and permeabilize cells, according to the manufacturer’s instructions. Permeabilized cells were incubated with anti-Eos efluor 660 (Invitrogen) for 1 hour at 4C, washed 2x with FACS buffer, and analyzed. For analysis of cytokine production, cells were treated with Golgi stop (BD

Bioscience) and stimulated with PMA and Ionomycin for 2h. Stimulated cells were stained with

α-CD4 AF488 (R&D Systems) prior to being fixed and permeabilized using the BD

Cytofix/Cytoperm kit as suggested by the manufacturer, and then incubated with anti-IFN-γ

AF700 (R&D Systems) or Rat IgG2a AF700 (R&D Systems) isotype control for 30 minutes at

4C. Samples were analyzed on the BD Accuri C6 or the Sony SH800 flow cytometers and data evaluated using Flowjo software.

ELISA for cytokine production

Wildtype (WT) and Ikzf4-/- naive CD4+ T cells were cultured under TH1-polarizing conditions for 3 days. Supernatants were harvested and IL-2 expression was assessed for each population using the Mouse IL-2 DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

Statistical analysis

All data represent at least three independent experiments. Error bars represent the standard error of the mean or standard deviation as indicated. For statistical analysis, unpaired t tests or one-way

ANOVA with Tukey multiple comparison tests were performed to assess statistical significance, as appropriate for a given experiment. P values <0.05 were considered statistically significant.

Data Availability

The datasets produced in this study will be made available upon reasonable request. Requests should be sent to the corresponding author.

100

Results

Eos expression positively correlates with that of the TH1 gene program

In a prior study, we identified the Ikaros family member Aiolos as a novel regulator of Bcl-6

+ 15 expression and the TFH gene program in CD4 T helper cells . Through the course of the same study, we also made the observation that expression of the related family member Eos was elevated in TH1 cells relative to that of other IkZF transcription factors. As such, we sought to determine whether Eos may play a role in the regulation of TH1 cell differentiation. We began by assessing

+ Eos expression in naïve CD4 T cells and in vitro differentiated TH1 and TFH-like cell subsets. As with our previous study, we observed elevated Eos transcript and protein expression in TH1 cells relative to that expressed in naïve and TFH-like cells (Figure 4.1A and B). Eos expression positively correlated with Blimp-1 (encoded by Prdm1), a transcriptional repressor expressed in

TH1 cells, and inversely correlated with that of Aiolos (Ikzf3) and Bcl6 in the TFH-like cell population (Figure 4.1A). Next, we performed siRNA knockdown experiments to assess the effect of reduced Eos levels on TH1 gene expression. Upon knockdown of Eos, we observed significant decreases in expression for several notable TH1 genes including Prdm1, Ifng, Il2ra, and

Tbx21 (Figure 4.1 C). Collectively, these data are supportive of a role for Eos in promoting TH1 differentiation. Importantly, this effect appeared to be specific to TH1 genes, as we observed no significant changes in expression of Bcl6 or Ikzf1 (Figure 4.1 C). Collectively, these data are supportive of a role for Eos in promoting TH1 gene expression patterns.

101

1.2 1.2 5 6

) *** ** Naive

)

v *** *** e

1.0 1.0 i 5

t a 4 TH1

(

( ( 0.8 0.8 ( 4 TFH-like

3 n

s 0.6 0.6 s 3

2 r p

x 0.4 0.4 p 2

expression expression (relative)

e expression (relative) expression

expression (relative) expression

1 expression expression (relative)

f 1

f 0.2 m 0.2 1

I Bcl6

Ikzf4 Ikzf3

0.0 Prdm1 0.0 0 0

B TH1 6000 *** TH1 T -like FH 5000 Isotype TFH-like

) 4000

(

3000

E (MFI) Eos 2000 1000

Maximum% 0 Eos

C 1.2 1.2 1.2 1.2

)

v * *** * *

v 1.0 1.0 t 1.0 1.0

(

r 0.8 0.8 0.8 0.8

(

n o

s 0.6 0.6 0.6 s 0.6

x expression expression (relative)

0.4 0.4 x 0.4 0.4 expression expression (relative)

e expression (relative)

expression (relative)

a g

0.2 n 0.2 0.2 x 0.2

Ifng Il2ra Il2ra

I Prdm1 Prdm1

T Tbx21 Tbx21

P 0.0 0.0 0.0 0.0

NS 1.2 1.2 NS 1.2

)

e e ***

i i 1.0 1.0 1.0

l l siCtrl

(

( 0.8 0.8 ( 0.8

n siIkzf4 n

s 0.6 0.6 s 0.6

p 0.4 0.4 p 0.4

expression (relative) expression expression (relative) expression

e expression expression (relative)

f 0.2 0.2 f c 0.2

B Ikzf1

I Bcl6

I Ikzf4 0.0 0.0 0.0

Figure 4.1. Eos expression positively correlates with that of the TH1 gene program. (A) RNA was isolated + from murine CD4 Naïve T, TH1, and TFH-like cells and qRT-PCR was used to assess expression of the indicated genes. The data were normalized to Rps18 and presented as fold change relative to the TH1 sample (mean of n = 3 ± s.e.m.). *P < 0.05, ***P < 0.001; unpaired Student’s t-test. (B) Flow cytometry analysis of Eos expression in TH1 and TFH-like cell populations. Mean fluorescence intensity (MFI) is also shown (mean of n = 3 ± s.e.m.). ***P < 0.001; unpaired Student’s t-test. (C) TH1 cells were nucleofected with siRNAs specific to Eos (siIkzf4) or a control siRNA (siCtrl). After 48h, RNA was harvested and expression of the indicated genes was assessed by qRT- PCR. The data were normalized to Rps18 and presented as the fold change in expression relative to the control (mean of n = 3 ± s.e.m.). *P < 0.05, ***P < 0.001; unpaired Student’s t-test.

102

The TH1 transcriptional program is disrupted in the absence of Eos

To determine whether Eos may regulate the transcriptional program underlying TH1 differentiation, we performed RNA-seq analysis on naïve WT and Ikzf4-/- CD4+ T cells cultured under TH1-polarizing conditions. Initial hierarchical clustering analyses revealed that the WT and

Eos-deficient transcriptional programs exhibited distinct gene signatures (Figure 4.2A).

Consistent with this analysis, many TH1 genes were downregulated in Eos-deficient cells compared to their WT counterparts (Figsure 4.2B and C). These included notable transcriptional regulators (Prdm1 and Mxd1), cytokines (Ifng and Il10), and cell surface receptors (Havcr2, Ifngr1, and Il2ra). Interestingly, in the absence of Eos, a number of genes were upregulated, including transcriptional regulators linked to the differentiation of other T helper subsets including TH17

33, 34, 35, 36, 37 (Casz1) and TFH (Bcl6 and Pou2af1) cells (Figure 4.2B) . Hallmark gene set enrichment analysis revealed that the IL-2/STAT5 signaling pathway, which plays a key role in

TH1 differentiation, was downregulated in the Eos-deficient cells (Figure 4.2B). Conversely, pathways associated with the differentiation and function of other T helper cell populations, including MYC and E2F target genes, were upregulated (Figure 4.2D)38. Taken together, these

+ data indicate that TH1 differentiation and function is compromised in Eos-deficient CD4 T cells.

103

A B WT vs Ikzf4-/- DEGs Top 200 Significant DEGs 10.0 408 469 407 188 (P < 0.05) 2124 2130

7.5 Havcr2

-/- Ifng Tox

WT Ikzf4 Pvalue 5.0 Mxd1

2 10 Pou2af1 log

1 - 0 2.5 score Prdm1 - Casz1 -1 Z -2 Il2rb Bcl6 0.0 Il2ra -5 0 5 log2 Fold Change C WT Ikzf4-/- 2 Ikzf4 1 Mxd1 0

Prdm1 score -

Klf2 -1 Z

Factors Ets1 -2

Hopx Transcription Transcription Ccl3 Ccl4

Ifng Factors Secreted Il10 Il2ra Il2rb

Ifngr1 Surface Receptors D Estrogen Response Early Cellular Response to DNA Damage MYC Targets Locomotion E2F Targets Cell Cycle

Peroxisome Biological Adhesion

Upregulated Upregulated

Mitotic Spindle Positive Regulation of Gene Expression

Hallmark Hallmark Gene Sets GO GO Biological Process 0 5 10 0 5 10 15 -log10(Pvalue) -log10(Pvalue) IL-2 STAT5 Signaling Cell Activation KRAS Signaling Up Regulation of Immune System Process Complement Secretion

Coagulation Positive Regulation of Signaling Downregulated

Allograft Rejection Downregulated Regulation of Cell Activation

Hallmark Hallmark Gene Sets GO GO Biological Process 0 5 10 15 20 25 0 10 20 30 40 50 -log10(Pvalue) -log10(Pvalue) Figure 4.2. The TH1 transcriptional program is disrupted in the absence of Eos. Naïve WT or Eos-deficient + CD4 T cells were cultured under TH1-polarizing conditions for 3 days. RNA was isolated and RNA-seq analysis was performed to assess changes in the transcriptomes of WT and Eos-deficient cells. (A) Hierarchical clustering of the 200 most differentially expressed genes (DEGs) when comparing WT and Eos-deficient cells. P < 0.05 (n = 4). (B) A representative volcano plot displaying the gene expression changes in WT versus Eos-deficient cells. Genes were color-coded as follows: no significant changes in expression (black), >2-fold change in expression with a p value greater than 0.05 (purple), a <2-fold change in expression with a p value less than 0.05 (blue), or a >2-fold change in expression with a p value less than 0.05 (red). (C) Heat map display of differentially expressed TH1 genes in WT and Eos-deficient cells. (D) Gene set enrichment analysis (GSEA) was used to identify upregulated and downregulated hallmark gene sets and GO biological processes in WT and Eos-deficient cells.

104

+ Eos-deficient CD4 T cells exhibit reduced expression of TH1 genes and IFN-γ production.

To examine potential temporal roles for Eos in the regulation of TH1 differentiation, we cultured

-/- + naïve WT and Ikzf4 CD4 T cells under TH1-polarizing conditions and assessed TH1 gene expression over a 4-day time course. We began by examining expression of T-bet (encoded by

Tbx21), the lineage-defining factor for TH1 cells (Figure 4.3A). As compared to WT, we observed

-/- an early reduction in Tbx21 expression in TH1-polarized Ikzf4 cells. Conversely, expression of the TFH lineage-defining factor Bcl-6 was significantly elevated in Eos-deficient cells at early time- points (Figure 4.3A). Collectively, these data are suggestive of a disruption to the early transcriptional regulation of TH1 differentiation.

We next examined the expression of the transcriptional regulator Blimp-1, which supports TH1

30, 39, 40 differentiation through the repression of the TFH gene program in TH1 cells . Interestingly, we observed a stark reduction in Blimp-1 expression not only at early time-points, but also throughout the 4-day time-course (Figure 4.3B). We also assessed Blimp-1 protein levels at the day 3 time-point and similarly observed reduced expression in the Eos-deficient cells (Figure

4.3C). The alteration in key transcriptional regulators in the absence of Eos suggested that there may be a disruption to TH1 cell function as well. To assess this possibility, we next evaluated the expression of the TH1 cytokine IFN-γ. Indeed, Eos-deficient cells were poor producers of IFN-γ, as about half the number of Eos-deficient cells were IFN-γ+ as compared to WT controls (Figure

4.3D and E). Collectively, these data demonstrate that both expression of key transcriptional regulators involved in TH1 differentiation and production of IFN- γ are reduced in the absence of

Eos.

105

A 1.2 NS 2.0 1.2 ) WT

)

) e

e NS e

i -/-

1.0 t 1.0 Ikzf4

1.6 a

(

** (

0.8 0.8

1.2 n

0.6 s 0.6

0.8 r

p x 0.4 0.4

e expression expression (relative)

expression (relative) expression

expression expression (relative)

6 0.4

f 0.2 * l 0.2

z c

Bcl6 Ikzf4 Tbx21 Tbx21 0.0 0.0 0.0 1d 2d 3d 4d 1d 2d 3d 4d 1d 2d 3d 4d

B 1.2 C

) WT

v ***

i t 1.0 -/- -/-

a Ikzf4

l WT Ikzf4

r **

(

0.8 n Blimp-1

i 80 kDa -

s 0.6

r Eos

x 0.4 60 kDa -

e expression (relative)

1 b-actin m 0.2 ***

d 40 kDa -

P ** Prdm1 Prdm1 0.0 1d 2d 3d 4d

D E

1.2 50 * WT -/- WT ) WT Ikzf4

e ** -/- v 1.0 i Ikzf4 -/-

) t 40 Ikzf4

r 0.8 i

(

s 30

o n

i 0.6 34.0 13.4

( (% positive)

e 20

- g

p 0.4 -

N x

expression expression (relative) g * IFN 10 n 0.2

f I NS Ifng 0.0 0 1d 2d 3d 4d IFN-g

Figure 4.3. Eos-deficient CD4+ T cells exhibit reduced expression of TH1 genes and IFN-γ production. Naïve + WT or Eos-deficient CD4 T cells were cultured under TH1 conditions for 4 days, and cells were harvested at 24h intervals for analysis. (A, B, D) qRT-PCR was used to assess expression of the indicated genes in WT or Eos- deficient cells. Data were normalized to Rps18 and presented as fold change relative to the day 4 TH1 sample (mean of n = 3-4 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test. (C) Immunoblot analysis of Blimp-1 and Eos protein expression in WT and Eos-deficient cells cultured under TH1 polarizing conditions for 3 days. β -actin serves as a loading control. Shown is a representative blot of four independent experiments. (E) Flow cytometry analysis of intracellular expression of IFN-γ in the indicated cell populations at the day 3 timepoint. Shown are representative data from four independent experiments. The percentage of IFN- γ+ cells is also shown (mean of n = 4 ± s.e.m.). *P < 0.05; unpaired Student’s t-test.

106

Loss of Eos leads to attenuated T-bet levels in CD4+ T-cells during T.gondii infection.

Our data so far indicate that Eos plays an important role in the development and functionality of

TH1 cells, with loss of Eos impacting transcriptional regulators such as Tbx21 and Prdm1, and cytokine production such as IFN-γ. In order to assess whether loss of Eos caused such defects in

27, vivo, we examined mice infected with T.gondii, a parasite known to elicit robust TH1 responses

28. Consistent with our in vitro data during earlier time points, analyzing activated helper T-cells in mice during the acute phase of infection revealed a significant reduction of T-bet in Eos deficient mice when comparted to control (Figure 4.4). Thus, coupling our in vitro data with our in vivo findings, our observations so far suggests that Eos plays an important role in the mediating transcriptional factors important for TH1 development.

107

WT WT Ikzf4-/- Uninfected

Maximum% T-bet Ikzf4-/- WT -/- 2000 ** Ikzf4

1500

1000

bet bet (MFI) geo

CD44 -

T 500 CD4

Figure 4.4. Loss of Eos attenuates T-bet levels in CD4+ T-cells during acute infection with T.gondii. WT or Ikzf4-/- mice were infected with 20 cysts of T.gondii (ME-49 F1). 8 days post infection, CD4+ T-cells were isolated from harvested splenocytes and stained for flow cytometry analysis. T-bet was gated on CD4+CD44+ cells. Mean fluorescence intensity (MFI) is shown (mean of n=4±s.e.m). **P<0.01 unpaired student’s t-test. Uninfected mice served as control.

108

Eos-deficient CD4+ T cells produce normal levels of IL-2, but express reduced levels of IL-2 receptor subunits

As mentioned above, IL-2 signaling is a well-established positive regulator of TH1 differentiation41, 42. Importantly, a prior study demonstrated that IL-2 signaling was disrupted in

+ + 26 Eos-deficient conventional CD4 T cell populations (CD4 TCONVS) . Consistent with this study, our hallmark gene set analysis revealed that components of the IL-2/STAT5 signaling pathway were downregulated in the absence of Eos (Figure 4.2D). To explore this relationship further, we examined both expression of the IL-2 receptor subunits CD25 (IL-2Rα) and CD122 (IL-2Rβ), as well as the ability of Eos-deficient cells to produce IL-2. Indeed, we observed significant decreases in both CD25 and CD122 expression at late stages of TH1 differentiation (Figure 4.5 A-D). As

IL-2 signaling positively regulates CD25 expression, we considered the possibility that the reduced expression we observed in Eos-deficient cells was due to a defect in IL-2 production. However, we did not detect a difference in IL-2 expression or production between WT and Eos-deficient cells (Figure 4.5 E and F). Curiously, these findings were not consistent with the prior study, in

+ which they observed a reduction in the ability of Eos-deficient CD4 TCONVS to produce IL-2. To determine whether increased IL-2 signaling could compensate for the lack of Eos, and to rule out

IL-2 levels as a causative factor in disrupted TH1 differentiation, we cultured WT and Eos-deficient cells under TH1-polarizing conditions in the presence of high concentrations of IL-2. However, addition of exogenous IL-2 was not able to rescue TH1 gene expression in the absence of Eos, and overall trends were consistent between the IL-2-treated and non-treated cells (Figure 4.6).

Collectively, these data demonstrate that Eos-deficient cells exhibit reduced IL-2 receptor expression that is independent of signals from IL-2.

109

A B 1.2 200000 WT

) WT WT

e ** -/- v ** i Ikzf4 -/- t 1.0 Ikzf4-/- Ikzf4

a l * Isotype 150000

)

(

0.8

n (MFI)

(

0.6 a 100000

r 2R

2 -

NS L

x 0.4 IL

e 50000

expression expression (relative) a r 0.2

I % Maximum% Il2ra Il2ra 0.0 0 1d 2d 3d 4d IL-2Ra WT Ikzf4-/- C D 1.2 3000 WT WT WT ) ** e -/-

i 1.0 -/- Ikzf4 -/- t Ikzf4 Ikzf4

a l Isotype

)

( 0.8 2000

M (MFI)

(

0.6 b b

r 2R

- -

0.4 L 1000

I IL

e expression (relative)

r 0.2

I Il2rb 0.0 Maximum% 0 1d 2d 3d 4d IL-2Rb

E F 6 700 WT NS WT

)

e 600 -/- v 5 -/- i Ikzf4 Ikzf4

a l 500

)

r 4

(

/ 400

i 3 (

2 300

s 2 2 (pg/ml)

- -

2 I IL p 200 x

e expression expression (relative)

2 1

l 100

I Il2 0 0 1d 2d 3d 4d 3d

Figure 4.5. Ikzf4-/- CD4+ T cells produce normal levels of IL-2, but express reduced levels of IL-2 receptor + subunits. Naïve WT or Eos-deficient CD4 T cells were cultured under TH1 conditions for 4d. Cells were isolated at 24 hr intervals for analysis. (A, C, E) qRT-PCR was used to assess expression of the indicated genes in WT or Eos-deficient cells. The data were normalized to Rps18 and presented as fold change relative to the 4d TH1 sample (mean of n = 4 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test. (B, D) Flow cytometry analysis of IL-2Rα or IL-2Rβ cell surface expression in WT or Eos-deficient cells. Mean fluorescence intensity (MFI) is also shown (mean of n = 4 ± s.e.m.). **P < 0.01; unpaired Student’s t-test. (F) ELISA analysis of IL-2 production by WT and Eos-deficient cells (mean of n = 3 ± s.e.m.).

110

1.4 1.2 1.4 1.8 1.6 * )

) ) WT

)

e e

)

v 1.6

i ***

t i 1.4

1.2 t 1.2

v -/- 1.0 a

t NS i

a Ikzf4

l l

1.4 a

e e r 1.2

r r

e 1.0 1.0 (

(

r 0.8 1.2 (

(

n n

1.0

o o

i i o 0.8 0.8 o

i 1.0

s s

s s 0.6 0.8

e e

s 0.8

r r 0.6 0.6 e

p p

p 0.6

x x

p x 0.4 0.6 x

e e

expression (relative)

e 0.4 expression (relative) 0.4

2 1

expression (relative) expression 0.4 expression (relative)

expression (relative)

0.4 4 g

c m

0.2 n

x d 0.2 f 0.2 0.2

I r

a 0.2

P Ifng

Ikzf4 Prdm1 Prdm1 Havcr2 Havcr2 0.0 0.0 Tbx21 0.0 0.0 0.0 Cont.+IL-2 Cont.+IL-2 Cont.+IL-2 Cont.+IL-2 Cont.+IL-2

Figure 4.6. Exogenous addition of IL-2 is unable to rescue TH1 development in Eos deficient cells. Naïve WT + or Eos-deficient CD4 T cells were cultured under TH1 conditions in the presence or absence of exogenous IL-2 (100 U/mL), as indicated. After 3 days, RNA was isolated from WT and Eos-deficient cells cultured with additional (+IL-2) or without additional (Cont.) IL-2 and qRT-PCR was used to assess expression of the indicated genes. The data were normalized to Rps18 and presented as fold change relative to the WT control sample (mean of n = 4 ± s.e.m.). *P < 0.05, ***P < 0.001; unpaired Student’s t-test.

111

IL-2-STAT5 signaling positively regulates Eos expression

We next sought to determine the mechanism by which Eos expression is regulated in TH1 cells.

Given the important role of IL-2 signaling in TH1 differentiation, we considered the possibility that

IL-2 may positively regulate Eos expression. To test this possibility, we first exposed TH1 cells to variable IL-2 concentrations to determine the impact on Eos expression. Importantly, these experiments were conducted on resting TH1 cells (in the absence of stimulation) to reduce variability arising from IL-2 produced by the TH1 cells themselves. Indeed, as TH1 cells were exposed to decreasing amounts of IL-2, we observed corresponding decreases in Eos expression at both the transcript and protein level (Figure 4.7A and B). To further manipulate IL-2 signaling in TH1 cells, we used neutralizing antibodies specific to CD25 (IL-2Rα) or CD122 (IL-2Rβ) to alter and/or disrupt signaling through the IL-2 receptor complex. Indeed, when both CD25 and

CD122 were blocked, we observed a significant decrease in the expression of Eos at both the transcript and protein level (Figure 4.7C and D). Interestingly, we found that treatment with α-

CD25 alone was sufficient to significantly reduce Eos expression in TH1 cells (Figure 4.7C).

Collectively, these data suggest that signals downstream of the IL-2 receptor complex drive Eos expression in TH1 cells.

As signals received through IL-2 are propagated through the phosphorylation-dependent activation and subsequent nuclear translocation of STAT5, we next sought to determine whether STAT5 may directly regulate Eos expression43. First, we utilized an Eos (Ikzf4) promoter-reporter to determine whether STAT5 would be sufficient to drive Ikzf4 promoter activity. Indeed, overexpression of constitutively active STAT5B (STAT5BCA) resulted in a significant increase in Ikzf4 promoter activity (Figure 4.7E). Importantly, as a control, we did not observe an increase in promoter activity in response to a constitutively active STAT3 (STAT3CA) protein. To determine whether

112

STAT5 binds to the endogenous Ikzf4 promoter, we next used chromatin immunoprecipitation

(ChIP) analysis to assess STAT5 enrichment at the Ikzf4 locus. Indeed, we observed increased enrichment of STAT5 and RNA pol II at the Ikzf4 promoter in TH1 cells, compared to that observed in TFH-like cells (where Eos is expressed at low levels) (Figutr 4.6F). Collectively, these findings implicate the IL-2/STAT5 signaling pathway in the positive regulation of Eos expression in TH1 cells.

113

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k STAT5 STAT5 enrichment (%)

I RNA RNA Pol II enrichment (%) Ikzf4 0 0.0 0.0 Ikzf4 Ikzf4 Ikzf4 Ikzf4 CA CA Ikzf4 Ikzf4 Ikzf4 Ikzf4 5 3 Cont. T T Prom. Cont. Prom. Cont. A A Prom. Cont. Prom. Cont. ST ST

Figure 4.7. IL-2-STAT5 signaling positively regulates Eos expression. (A) Naïve CD4+ T cells were cultured under TH1 conditions and a range of IL-2 concentrations for 3 days. RNA was isolated and qRT-PCR was used to assess Ikzf4 (Eos) expression. Data were normalized to Rps18 and presented as fold change relative to the 500 U/ml IL-2 sample (mean of n = 6 ± s.e.m.). ***P < 0.001; one-way ANOVA with Tukey multiple-comparison test. (B) Cells were cultured as in ‘A’ and Eos protein expression was assessed by flow cytometry analysis. Data shown are representative of 3 independent experiments. Mean fluorescence intensity (MFI) is also shown (mean of n = 3 ± s.e.m.). **P < 0.01; one-way ANOVA with Tukey multiple-comparison test. (C) Naïve CD4+ T cells were cultured under TH1 conditions for 3 days. Where indicated, neutralizing antibodies for IL-2Rα (α-CD25) or IL-2Rβ (α-CD122) were added. RNA was isolated and qRT-PCR was used to assess Ikzf4 (Eos) expression. Data were normalized to Rps18 and presented as fold change relative to the control sample (mean of n = 3 ± s.e.m.). **P < 0.01; one-way ANOVA with Tukey multiple-comparison test. (D) Cells were cultured as in ‘C’ and Eos protein expression was assessed by flow cytometry analysis. Data shown are representative of 3 independent experiments. Mean fluorescence intensity (MFI) is also shown (mean of n = 3 ± s.e.m.). *P < 0.05; unpaired Student’s t-test. (E) EL4 T cells were transfected with an Ikzf4 promoter-reporter construct in combination with a constitutively active STAT5 (STAT5BCA) or STAT3CA expression vector or an empty vector control. Luciferase promoter-reporter values were normalized to a Renilla control and presented relative to the empty vector control sample (mean of n = 3 ± s.e.m.). **P < 0.01; one-way ANOVA with Tukey multiple-comparison test. (F) Chromatin Immunoprecipitation (ChIP) assays to assess STAT5 and RNA Pol II association with the Ikzf4 locus in TH1 or TFH-like cells. The Ikzf4 promoter, as well as an upstream control region, were interrogated for STAT5 enrichment. Data are presented as percent enrichment relative to a “total” input sample. (mean of n = 3 ± s.e.m.). *P < 0.05; unpaired Student’s t-test.

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Eos and STAT5 form a novel transcription factor complex in TH1 cells

In a prior study, we demonstrated that the IkZF factor Aiolos interacts with STAT3 to form a novel transcriptional complex that regulates Bcl-6 expression in CD4+ T cells15. Interestingly, the interaction between STAT3 and Aiolos was dependent upon the C-terminal ZF domain of Aiolos, which is conserved across Ikaros family members5, 15. As such, we hypothesized the Eos may similarly interact with STAT factors in TH1 cells. Given that TH1 cells exhibit both increased Eos expression and elevated STAT5 phosphorylation downstream of IL-2 signaling, we next sought to determine whether Eos and STAT5 may interact and promote gene expression in TH1 cells.

Indeed, co-immunoprecipitation (co-IP) analyses revealed interactions between Eos and STAT5 in TH1 cells. Furthermore, we were similarly able to detect interactions between Eos and

STAT5BCA in an overexpression system (Figutr 4.8 A and B). A key aspect of our previous study was that Aiolos and STAT3 could cooperate to induce Bcl-6 expression. As Eos expression and

STAT5 activation positively correlate with that of Blimp-1 (Prdm1) in TH1 cells, we hypothesized that Eos and STAT5 may cooperate to induce Prdm1 expression. Indeed, co-expression of

STAT5BCA and Eos resulted in a significant increase in Prdm1 expression, as compared to the effect of either factor alone (Figure 4.8C). As a control, we also assessed changes in expression of Bcl-6, which is known to directly repress Prdm1 expression. Importantly, we did not detect any significant differences in Bcl6 expression, suggesting that STAT5 and Eos may cooperate to directly induce Prdm1.

Given homology between IkZF family members, we hypothesized that, analogous to Aiolos and

STAT3, Eos and STAT5 interactions may be dependent upon the C-terminal protein interaction domain of Eos. To test this hypothesis, we constructed an Eos mutant lacking the C-terminal ZF domain (EosΔC) and performed co-immunoprecipitation analyses. Indeed, as with Aiolos and

115

STAT3, the EosΔC mutant was incapable of interacting with STAT5BCA (Figure 4.8D).

Furthermore, in contrast to WT Eos, we found that Prdm1 expression was diminished when EosΔC was co-expressed with STAT5BCA (Figure 4.8E). As before, we did not observe differences in

Bcl6 transcript expression, suggesting that the effects on Prdm1 were gene-specific. Taken together, these data demonstrate that the IkZF C-terminal ZF domain is a conserved regulatory feature that allows for functional IkZF and STAT factor interactions.

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T -like FH T 1 IB: H a-Aiolos IB: -60 kDa a-Eos -60 kDa

a-STAT3 a-p-STSTAATT55 -80 kDa -80 kDa Input IP: V5 IP: STAT3 Input IP: V5 IP: STpSTATA5T 5 A B IB: TH1 EL4 OverexpressionEL4 IB: IB: IB: a-Eos a-V5 (Aiolos) -60 kDa a-V5 (Eos) a-V5 (Eos) -60 kDa 60 kDa- -60 kDa a-STAT5 -80 kDa aa-ST-pSTATA5T 5 a-STAT3 a-STAT580 kDa- -80 kDa -80 kDa Input IP: IgG IP: STpSTATA5T 5 Input IP: IgG IP: STAT3

C * 6 ** 6 )

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i _ _

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Prdm1 Prdm1 0 _ _ 0 Eos + + _ _ Eos _ _ EosEos _+ ++ _ _ + + _ _ + STAT5CA _ _+ + CA _ _+ + STAT5BCA + + STAT5BSTAT5CA + +

D E 4 4 - - ) ** ** + Eos

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t i - - + EosDC

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1 1 1

expression (relative) 6 80 kDa -

r B -actin P b

Bcl6 40 kDa -

Prdm1 Prdm1 0 _ _ 0 _ _ Eos _ + _ Eos _ + _ Eos _ + Eos _ + STAT5CA _ + + STAT5CA _ + + STAT5BCA _ +_ + STAT5BCA _ _+ + EosDC + EosDC + _ _ + _ _ + EosDC EosDC Figure 4.8. Eos and STAT5 interact and form a transcription factor complex in TH1 cells. (A) Co- Immunoprecipitation (Co-IP) of endogenously expressed proteins in TH1 cells with control Ab (α-V5) or α- STAT5, followed by immunoblot with anti-Eos. Data shown are representative of five independent experiments performed. (B) Co-IP analysis of over-expressed Eos and tagless STAT5CA in EL4 T cells. Lysates were immunoprecipitated with anti-STAT5, followed by immunoblot analysis with α-V5 (for detecting Eos). Shown is a representative blot of five independent immunoprecipitation experiments performed. (C) EL4 T cells were transfected with STAT5CA, Eos, or STAT5CA and Eos in combination. After 24 h, RNA was isolated, and Prdm1 or Bcl6 expression was measured by qRT-PCR. Data were normalized to Rps18 as a control, and the results are presented as fold change in expression relative to the empty vector control sample. An immunoblot was also performed to assess the relative abundance of overexpressed proteins. (n = 3 ± s.e.m.). *P < 0.05, **P < 0.01; one- way ANOVA with Tukey multiple-comparison test. (D) Co-IP of overexpressed wild-type Eos or EosΔC and tagless STAT5CA in EL4 cells. Lysates were immune-precipitated with anti-STAT5, followed by immunoblot analysis with α-V5 (for detecting Eos proteins). Shown is a representative blot of three independent experiments performed. (E) EL4 T cells were transfected with Eos and STAT5CA, EosΔC and STAT5CA, or empty vector control. After 24 h, RNA was isolated, and Prdm1 or Bcl6 expression was measured by qRT-PCR. Data were normalized to Rps18 as a control, and the results are presented as fold change in expression relative to the empty vector control sample. Immunoblot with an anti-V5 Ab was performed to assess the relative abundance of overexpressed proteins. (n = 3 ± s.e.m.). **P < 0.01; one-way ANOVA with Tukey multiple-comparison test.

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STAT5 activation and association with TH1 target genes is enhanced in the presence of Eos.

Our findings thus far support a role for an IL-2/STAT5/Eos regulatory axis that promotes TH1 differentiation and function. Together, the hallmark gene set analysis and observed reduction in

IL-2 receptor expression suggested that STAT5 activation may be disrupted in the absence of Eos

(Figures 4.2C and 4.4A-D). Indeed, we observed a reduction in STAT5 activation (pSTAT5-

Y694) in Eos-deficient cells compared to WT cells cultured under TH1-polarizing conditions

(Figure 4.9A). To further determine whether STAT5 activation correlated with Eos expression, we overexpressed Eos, STAT5BCA, or Eos and STAT5BCA in combination and analyzed pSTAT5-

Y694 levels. Strikingly, pSTAT5B-Y694 was enhanced in the presence of Eos, but not the Eos mutant lacking its C-terminal ZF domain, suggesting that interaction between these factors may be required for the modulation of STAT activity (Figure 4.9B and C). Intriguingly, the increase in STAT5B activation was specific to co-expression with Eos, as co-expression with Aiolos or

Ikaros did not impact levels pSTAT5B (Figure 4.9D). Collectively, these data support a role for

Eos/STAT5 interactions in positively regulating STAT5 activation.

As STAT5 activation was disrupted in the absence of Eos, we hypothesized that association of

STAT5 with target genes may be compromised in Eos-deficient TH1 cells. Our data demonstrate that Eos/STAT5 complexes form in TH1 cells and that Eos/STAT5 cooperate to induce Blimp-1 expression (Figure 4.9A and 4.8C). Therefore, we next sought to determine whether STAT5 association with the Prdm1 locus was diminished in cells lacking Eos. We first used published

STAT5 ChIP-seq data (GSM2055718) to identify candidate binding sites for STAT5 in the Prdm1

44 locus and performed ChIP analysis of WT and Eos-deficient TH1 cells . In support of our hypothesis, we observed reduced STAT5 enrichment at a conserved Prdm1 intronic enhancer in

TH1 cells (Figure 4.9E). Similarly, we also observed reduced STAT5 association with both Ifng

118 and Il2ra regulatory elements in Eos-deficient cells compared to WT controls (Figure 4.9E).

Furthermore, ATAC-seq analysis of wildtype versus Eos-deficient cells revealed that while overall chromatin accessibility was only modestly affected by loss of Eos, areas exhibiting reduced chromatin accessibility in Eos-deficient cells corresponded with diminished STAT5 binding

(Figure 4.10A-C). Collectively, these data suggest that Eos is required for both optimal STAT5 activation and the association of STAT5 with its target genes in TH1 cells.

119

A B _ _ WT Ikzf4-/- + + Eos _ _ + + STAT5BCA Blimp-1 80 kDa - pSTAT5 80 kDa - pSTAT5 80 kDa - 80 kDa - STAT5 60 kDa - Eos STAT5 80 kDa - b-actin 40 kDa - 60 kDa - Eos

b-actin 40 kDa -

C D - + - Eos STAT5CA - - + EosDC _ _ - + + STAT5BCA pSTAT5 pSTAT5 80 kDa - 80 kDa - STAT5 STAT5 80 kDa - 80 kDa -

60 kDa - Eos 60 kDa - IkZFs (V5)

b-actin 40 kDa - b-actin 40 kDa -

E 1.2 1.2 1.2 WT

) * ** *** -/- % 1.0 1.0 1.0

( Ikzf4

t n 0.8 e 0.8 0.8

c i 0.6 0.6 0.6

5 0.4 0.4 0.4

T 0.2 0.2 0.2

STAT5 STAT5 enrichment (%) STAT5 STAT5 enrichment (%) STAT5 enrichment (%) 0.0 0.0 0.0 PPrdm1romoter Prdm1Control Ifng Il2ra Enh. Cont. Prom. Prom.

Figure 4.9. STAT5 activation and association with TH 1 target genes is enhanced in the presence of Eos. (A) Immunoblot analysis of Blimp-1, activated STAT5 (pSTAT5A/pY694), STAT5A/B, and Eos protein expression in WT and Eos-deficient ‘TH1’ cells. β-actin serves as a loading control. Shown is a representative blot of three independent experiments. (B) Immunoblot analysis of activated STAT5 (pSTAT5A/pY694), STAT5A/B, and Eos protein expression in EL4 T cells transfected with STAT5CA, Eos, or STAT5CA and Eos in combination. β-actin serves as a loading control. Shown is a representative blot of three independent experiments. (C) Immunoblot analysis of activated STAT5 (pSTAT5A/pY694), STAT5A/B, and Eos protein expression in EL4 T cells transfected with wild-type Eos or EosΔC and tagless STAT5CA in EL4 cells. β-actin serves as a loading control. Shown is a representative blot of three independent experiments. (D) Immunoblot analysis of activated STAT5 (pSTAT5A/pY694), STAT5A/B, and Eos protein expression in EL4 T-cells transfected with tagless STAT5CA alone or in combination with the indicated Ikaros transcription factor. β-actin serves as a loading control. Shown is a representative blot of three independent experiments. (E) ChIP assays to assess STAT5 enrichment at regulatory regions associated with the Prdm1, Ifng, and Il2ra loci in WT or Eos-deficient ‘TH1’ cells. Data are presented as percent enrichment relative to a “total” input sample and represented as fold change relative to the WT sample (n = 4 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student’s t-test.

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Merged Regions with Peaks from Samples

A B Color Key Peak Tag Numbers WT 2 Ikzf4-/- 1 (Pearson Coefficients) -1 0 1 Value -/- WT 1 1787 Ikzf4 2 WT 1 1224 2425 2087 4509 1911 5846 WT 2

-/- 48397 Ikzf4 1

2375 3404 -/- 1908 1575 Ikzf4 2

379

/ -

Merged Peaks 0 -

WT WT 1

WT WT 2 Ikzf4 Ikzf4

STAT5B (GSM2734693)

- 500 WT 1

- 500 WT 2

- 500 Ikzf4-/- 1

- 500 Ikzf4-/- 2

3’ 5’ Prdm1 IntronicP rEnhancerdm1

Figure 4.10. Loss of Eos has modest effects on genome accessibility. (A) ATAC-seq analysis of WT and Eos- deficient TH1 cells. ATAC-seq. was performed on in vitro derived WT and Ikzf4-/- TH1 cells (A) Venn diagram illustrating the overlap of accessible regions between WT and Ikzf4-/- samples. (B) Heatmap comparing ATACseq. peak sizes between indicated samples. Displayed is the Pearson Correlation coefficients of all pairwise comparisons colored from dark green (high correlation) to red (low or no correlation). (C) UCSC genome browser tracks at the Prdm1 locus displaying previously published STAT5B ChIP-seq dataset (GSM2734693) and ATAC- seq. tracks for WT and Ikzf4-/- TH1 cells. The intronic enhancer is highlighted in yellow.

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Discussion

For roughly a decade, Eos has been categorized as a transcription factor important for maintaining

+ 22, 23, 24, 25, 45, 46 both CD4 TREG cell identity and associated suppressive functions . However, recent work has suggested that Eos may also play additional roles in regulating the differentiation and function of effector T cell populations, though its precise role has been unclear23, 26. To this end, we have now identified Eos as a novel regulator of TH1 cell differentiation and function. We found

+ that Eos-deficient CD4 T cells display decreased expression of TH1 cell-associated transcription factors (e.g. T-bet and Blimp-1), cell surface receptors (IL-2Rα and IL-2Rβ), and diminished effector function as measured by production of IFN-γ. Furthermore, in the absence of Eos, we observed decreases in both the activation of STAT5 and its association with TH1 target genes.

Collectively, our findings demonstrate that Eos is an important regulator of TH1 differentiation and suggest that augmentation of STAT5 activity represents at least one mechanism by which Eos influences TH1 development.

Previous work from our laboratory demonstrated that another IkZF family member, Aiolos, physically interacts and cooperates with STAT3 to regulate Bcl-6 expression in CD4+ T cell

15 populations . Analogous to that finding, we now show that Eos interacts with STAT5 in TH1 cells and that these factors may cooperate to induce TH1 gene expression. Interestingly, we found that the C-terminal ZF domain of Eos, which is known to mediate homo- and hetero-dimerization amongst IkZF transcription factors, was required for its interaction with STAT5. This is consistent with our previous findings with regard to Aiolos/STAT3 regulatory modules, suggesting that that the C-terminal ZF domain of Ikaros family members may represent a conserved regulatory feature that enables IkZF and STAT factor interactions15. Furthermore, our data demonstrate that distinct

IL-2-dependent cytokine signatures regulate the formation and function of the Eos/STAT5 and

122

Aiolos/STAT3 complexes in TH1 and TFH cells, respectively. As such, it is intriguing to speculate that additional cytokine signals that drive the differentiation of TH1 and TFH populations (e.g. IL-

12-STAT4 or IL-6-STAT3), as well as those that direct the differentiation of other T helper subsets, may promote the formation and function of additional IkZF and STAT factor regulatory modules.

The mechanisms by which IkZF and STAT factors cooperate to regulate gene expression patterns remain somewhat enigmatic. Historically, IkZF factors have been associated with the recruitment of chromatin remodeling complexes and resultant changes to chromatin structure1, 4, 6. While we cannot rule this out as a potential mechanism, our ATAC-seq results suggest that chromatin accessibility is only modestly affected in the absence of Eos. Rather, our data in TH1 cells point to an additional mechanism whereby Eos regulates STAT5 phosphorylation, dimerization, and subsequent association with target genes. While the manner by which Eos modulates STAT5 activity is currently unclear, we hypothesize that there are three potential, non-mutually exclusive possibilities. First, and most obviously, our data indicate that Eos regulates expression of IL-2 receptor subunits, through which STAT5 is activated in the TH1 cell population. Indeed, a prior study described Eos-dependent regulation of CD25 (IL-2Rα) in undifferentiated, activated CD4+

26 TCONV cell populations . However, while much of the data we present in TH1 cells are consistent with this potential mechanism, it is important to note that the overexpression studies presented here were performed with a constitutively active STAT5 (STAT5CA) in the absence of signals from

IL-2. Thus, we speculate that Eos modulation of STAT5 activity may also be due to effects on the expression or activity of kinases or phosphatases that act upon STAT5. This includes the possibility that the physical interaction between Eos and STAT5 stabilizes STAT5 expression and/or protects it from phosphatase activity. Ultimately, further experimentation will be necessary

123 to understand the mechanism(s) by which Eos influences STAT5 phosphorylation. Given the importance of STAT signaling to a broad range of cell populations, including immune cells, it will also be of interest to determine whether other IkZF family members may similarly influence the activation and function of additional STAT family members (e.g. Aiolos and STAT3).

Another outstanding and related question is whether cooperative DNA-binding activities of Eos and STAT5 are required to activate TH1 gene expression. In a previous study, we demonstrated that Aiolos and STAT3 were co-enriched at the Bcl6 promoter and that the presence of both transcription factors was required to induce Bcl-6 expression15. Unfortunately, the reagents required to answer this question with regard to Eos are currently unavailable. We have attempted to perform ChIP experiments in efforts to determine whether Eos and STAT5 are co-enriched at

TH1 gene regulatory elements. While we did observe elevated levels of both Eos and STAT5 enrichment, additional analyses in Eos-deficient cells also detected a modest amount of background enrichment that could not be attributed to Eos. Subsequent experiments revealed that the polyclonal Eos antibody used exclusively for the ChIP experiment was also capable of weakly detecting Ikaros. Unfortunately, the Eos-specific antibodies utilized in immunoblotting and flow cytometry experiments herein are not sufficient for IP. Thus, future studies with immunoprecipitation antibodies that are specific to Eos will be necessary to address whether it directly associates with TH1 genes.

Finally, it is interesting to compare and contrast the known roles of Eos in TREG populations versus those described here in effector TH1 cells. Thus far in TREG cells, Eos has been described primarily as a transcriptional repressor that works in concert with Foxp3 to regulate TREG cell-specific

22, 24, 25 24 expression patterns . Interestingly, a notable Eos target in TREG cells is the Il2 locus . The findings from our study, as well as those from a prior report, suggest that Eos-mediated repression

124 of IL-2 is not conserved in CD4+ effector T cell populations26. In fact, while we certainly cannot rule out a repressive role for Eos in TH1 cells, our findings here suggest that Eos plays an important role in activating the TH1 gene program through modulation of STAT5 activity. However, it is not unreasonable to expect that Eos may play a similar role in regulating the elevated levels of STAT5 activation that are required for the differentiation and stability of TREG cell populations. Curiously,

Eos expression is much higher in TREG cells as compared to that observed in conventional T cell populations26. Thus, determining whether there are conserved and/or divergent roles for Eos in regulating TREG versus TH1 cell differentiation is an area which requires further study.

Finally, future studies are also required to assess whether Eos/STAT5 regulatory modules function downstream of additional cytokine signals. While our current study examined Eos and STAT5 downstream of IL-2 signaling, STAT5 is also activated downstream of signals from the cytokines

IL-7 and IL-1541, 47. Furthermore, as IL-2, IL-7, and IL-15 signaling exhibit diverse functions across immune cell populations, it will be of interest to assess potential cooperative roles for Eos and STAT5 in the regulation of CD8+ T, B, Natural Killer (NK), and Innate lymphoid cell (ILC) populations48. In doing so, we will gain increased insight into the mechanisms by which Ikaros family members regulate the differentiation and function of immune cell populations and how these activities may be leveraged in targeted immunotherapy approaches to treat human disease.

125

Acknowledgements

We thank Dr. Ethan Shevach (NIH) and Dr. Bruce Morgan (Harvard Medical School) for kindly providing Eos-deficient mice. This work was supported by grants from The National Institutes of

Health (AI134972 and AI127800) to K.J.O. This work was also supported by a grant from the

Jeffress Memorial Trust to K.J.O and L.M.C. K.A.R. is supported by Nell Mondy and Hartley

Corporation graduate fellowships from the Graduate Women in Science (GWIS) foundation.

Disclosures

The authors declare no competing interests.

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Chapter 3

Integrated STAT3 and Ikaros Zinc Finger transcription factor activities regulate Bcl-6 expression in CD4+ T helper cells Michael D. Powell1,4,6, Kaitlin A. Read*1,6, Chandra E. Baker1, Bharath K. Sreekumar1,4, Veronica M. Ringel-Scaia3,4, Holly Bachus5, R. Emily Martin1, Ian D. Cooley1,3, Irving C. Allen3, Andre Ballesteros-Tato5 & Kenneth J. Oestreich1,2,3

1 Virginia Tech Carilion Research Institute, Roanoke, VA 24016, USA

2 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA

3 Virginia Tech Carilion School of Medicine, Roanoke, VA 24016, USA

4 Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA 24061, USA

5 Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA

6 These authors contributed equally to this work.

Published as:

Integrated STAT3 and Ikaros Zinc Finger Transcription Factor Activities Regulate Bcl-6 Expression in CD4 + Th Cells. J Immunol. 2017;199(7):2377-2387.

Abstract

B cell lymphoma-6 (Bcl-6) is a transcriptional repressor that is required for the differentiation of

T follicular helper (TFH) cell populations. Currently, the molecular mechanisms underlying the transcriptional regulation of Bcl-6 expression are unclear. In this study, we have identified the

Ikaros zinc finger transcription factors Aiolos and Ikaros as novel regulators of Bcl-6. We found that increased expression of Bcl-6 in CD4+ Th cell populations correlated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Furthermore, overexpression of Aiolos or

Ikaros, but not the related family member Eos, was sufficient to induce Bcl6 promoter activity.

Intriguingly, STAT3, a known Bcl-6 transcriptional regulator, physically interacted with Aiolos to form a transcription factor complex capable of inducing the expression of Bcl6 and the TFH- associated cytokine receptor Il6ra. Importantly, in vivo studies revealed that the expression of

Aiolos was elevated in Ag-specific TFH cells compared with that observed in non-TFH effector Th cells generated in response to influenza infection. Collectively, these data describe a novel regulatory mechanism through which STAT3 and the Ikaros zinc finger transcription factors

Aiolos and Ikaros cooperate to regulate Bcl-6 expression.

Introduction

CD4+ Th cells are responsible for coordinating a wide array of immune responses. Upon activation, naive CD4+ T cells differentiate into specific Th cell subtypes that are critical for coordinating individual activities as part of a pathogen specific immune response. These include TH1, TH2,

1, 2, 3, 4 TH17, TH9, TH22, and T follicular helper (TFH) cell populations . The armamentarium provided by these subsets is diverse, ranging from the TH1-mediated secretion of pro-inflammatory cytokines, such as IFN-γ, to the critical role of TFH cells in promoting the generation of pathogen- neutralizing Abs by B cells. This level of CD4+ T cell subtype specialization depends upon unique lineage-defining transcription factors that direct Th cell development by activating cell-specific gene expression programs and repressing alternative Th cell fates5, 6, 7, 8.

One such example is the transcriptional repressor B cell lymphoma-6 (Bcl-6). Bcl-6 is a member of the broad-complex, tramtrack and bric- à -brac–zinc finger family of proteins and has been identified as a lineage-defining transcription factor required for TFH cell differentiation and the formation of germinal centers9, 10, 11, 12, 13. Additionally, Bcl-6 is important for numerous aspects of B cell development and function, as well as the differentiation of CD4+ and CD8+ memory T cell populations. A conserved role for Bcl-6 in the generation of these populations is to repress the expression of a second repressor, B lymphocyte–induced maturation protein-1, a direct antagonist

5, 14, 15, 16 of TFH cell– and memory cell–associated genes . Other Bcl-6 target genes include those that encode the TH1 and TH2 cell lineage-defining transcription factors T-bet and Gata3, as well as genes associated with cell cycle and metabolic regulation10, 11, 12, 14, 17. Thus, through its ability to modulate a litany of developmental and regulatory pathways, Bcl-6 has emerged as a key driver of immune cell differentiation and function.

As with other transcriptional regulators, the expression and activity of Bcl-6 is regulated by cell- intrinsic signaling cascades that are initiated by extracellular cytokine signals. For example, it is recognized that the cytokines IL-6, IL-12, and IL-21 promote Bcl-6 expression in CD4+ T cell18,

19, 20, 21, 22, 23, 24. In contrast, signaling cascades initiated downstream of IL-2 and IL-7 impede Bcl-

6 expression25, 26, 27,28,29,30. The differential effects of these cytokines are propagated through the activation of specific STAT factors known to associate with regulatory regions within the Bcl6 locus. Specifically, STAT1, STAT3, and STAT4 have been shown to positively regulate Bcl-6 expression, whereas STAT5 is a demonstrated repressor of Bcl-621, 31. Beyond STAT factors, additional transcriptional regulators, including Batf and Tcf-1, have been shown to induce Bcl-6 expression32, 33, 34,35. Despite these important insights, many questions remain regarding the identity of the transcriptional network that regulates Bcl-6 expression in CD4+ T cell populations.

Similar to STAT factors, the five members of the Ikaros zinc finger (IkZF) family of transcription factors have been implicated in the differentiation of numerous immune cell types, including Th cell subsets 36,37,38,39. In the current study, we found that the expression patterns of two IkZF factors, Aiolos and Ikaros, correlated with Bcl-6 levels in in vitro–generated TFH-like and in vivo–generated TFH cell populations. Mechanistically, we found that Aiolos and Ikaros were enriched at the Bcl6 promoter and that their association was coincident with chromatin remodeling events, consistent with gene activation, including increased histone acetylation and histone 3 lysine

4 trimethylation (H3K4Me3). Surprisingly, we found that Aiolos physically interacted with the known Bcl-6 activator STAT3 to form a novel transcription factor complex capable of inducing

Bcl-6 expression.

Importantly, we found that Aiolos expression was elevated in Ag specific TFH cells compared with non-TFH effector cells that are generated in response to influenza infection. Collectively, our findings identify Aiolos as a novel regulator of Bcl-6 expression and uncover an unexpected cooperative relationship between IkZF and STAT transcription factors that may be an important regulatory feature in the specification of Th cell–differentiation programs.

Materials and Methods

Primary cells, cell culture and nucleofection

Naive CD4+ T cells were isolated from the spleens and lymph nodes of 5–8-wk-old age- and sex- matched C57BL/6 mice using the MagCellect CD4+ T cell isolation kit (R&D Systems), per the manufacturer’s instructions. Cells were plated at a density of ∼5×105 cells per well in complete

IMDM (IMDM [Life Technologies], 10% FBS [catalog number (cat. no.) 26140079, Life

Technologies], 1% Penicillin-Streptomycin [Life Technologies], and 0.05% 2-ME [Sigma-

Aldrich]) and stimulated using plate-bound anti-CD3ε (5 µg/ml) and anti-CD28 (10 µg/ml; both from BD Biosciences) in the presence of TH1-polarizing conditions: 5 ng/ml IL-12 (R&D

Systems), 5 µg/ml anti–IL-4 (11B11; BioLegend), and 20 ng/ml IL-2 (PeproTech). After 72 h,

5 cells were removed from stimulation and expanded to plate at ∼5×10 cells per well in TH1- polarizing conditions containing high IL-2 (20 ng/ml) or low IL-2 (0.8 ng/ml) for an additional 2

28,30 d to generate TH1 or TFH-like cell populations, respectively (Figure 3.1) . To generate TFH-like cells as described by Awe et al., CD4+ T cells were cultured as previously described40. Briefly, isolated naive CD4+ T cells were stimulated on plate-bound anti-CD3ε (5 µg/ml) and anti-CD28

(10 µg/ml) for 72 h in complete IMDM in the presence of TFH-polarizing conditions (10 µg/ml anti–IFN-γ (XMG1.2; BioLegend), 10 µg/ml anti–TGF-b (1D11; Bio X Cell), 10 µg/ml anti–IL-

2 (JES-1A12; eBioscience), 10 µg/ml anti–IL-4, 100 ng/ml recombinant mouse IL-6 (R&D

Systems), and 50 ng/ml rmIL-21 (PeproTech). Subsequently, cells were expanded and placed in fresh media containing 0.8 ng/ml IL-2, 100 ng/ml rmIL-6, and 50 ng/ml rmIL-21 for an additional

48 h. TH2 cells were generated under the following polarizing conditions (10 ng/ml IL-4, 20 ng/ml

IL-2, and 10 µg/ml anti–IFN-g). All studies involving mice were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved

52 by the Institutional Animal Care and Use Committee of Virginia Tech and the University of

Alabama at Birmingham.

The murine EL4 T cell line (TIB-39; American Type Culture Collection) was cultured as previously described28, 41. EL4 cell nucleofection was performed using the Lonza 4D

Nucleofection system (Program CM-120; Buffer SF). Overexpression of proteins was confirmed via immunoblot, and endogenous gene expression changes in response to overexpressed proteins were assessed using quantitative RT-PCR (qRT-PCR) analysis. Expression vectors were generated by cloning the coding sequence of genes of interest into the pcDNA3.1/V5-His-TOPO vector (cat. no. K4800; Life Technologies). Sequences were confirmed by sequencing with T7 and BGH primers, followed by transfer of the coding sequence into the pEF1/V5-His vector (cat. no. V920;

Life Technologies). The constitutively active STAT3 (STAT3CA) expression vector was generated using the methods above in conjunction with the Agilent QuikChange Site-Directed Mutagenesis

Kit (part number 200519), as previously described42. Expression of each protein was confirmed via immunoblot using V5- and protein-specific Abs.

Figure 3.1. Schematic depicting in vitro differentiation to TH1 and TFH-like cells. Schematic depicting in + vitro differentiation of TH1 and TFH-like cells. Naïve CD4 T cells were isolated from the spleen and lymph nodes of C57BL/6 wild-type mice and cultured as indicated above to generate TH1 and TFH-like cells.

RNA purification and qRT-PCR

RNAwas isolated using the NucleoSpin RNA Kit (MACHEREY-NAGEL), and cDNA was generated using the SuperScript IV First-Strand Synthesis System (Life Technologies), according to the manufacturers’ instructions. cDNA was used at a concentration of 20 ng per qRT-PCR reaction with gene-specific primers (Rps18 forward: 5’-GGA GAA CTC ACG GAG GAT GAG-

3’, Rps18 reverse: 5’-CGC AGC TTG TTG TCTAGA CCG-3’; Bcl6 forward: 5’-CCA ACC TGA

AGA CCC ACA CTC-3’, Bcl6 reverse: 5’-GCG CAG ATG GCT CTT CAG AGT C-3’; Ikzf1 forward: 5’-ACG CAC TCC GTT GGT AAG CCT C-3’, Ikzf1 reverse: 5’-TGC ACA GGT CTT

CTG CCATCT CG-3’; Ikzf2 forward: 5’-ACG CTC TCA CAG GAC ACC TCA G-3’, Ikzf2 reverse: 5’-GGC AGC CTC CAT GCT GAC ATT C-3’; Ikzf3 forward: 5’-GCT GCA AGT GTG

GAG GCA AGA C-3’, Ikzf3 reverse: 5’-GTT GGC ATC GAA GCA GTG CCG-3’; Ikzf4 forward:

5’- GAC GCA CTC ACT GGC CAC CTC C-3’, Ikzf4 reverse: 5’-GGC ACC TCT CCT TGT GCT

CCT CC-3’; Ikzf5 forward: 5’-TCG GTA CTG CAA CTA TGC CAG C-3’, Ikzf5 reverse: 5’-AGG

TGG CGC TCG TAA GCA GAT G-3’; Il6ra forward: 5’-CCA CATAGT GTC ACT GTG CG-

3’, Il6ra reverse: 5’-GGTATC GAA GCT GGA ACT GC-3’; and Il2ra forward: 5’- CCA CAA

CAG ACATGC AGA AGC C -3’, Il2ra reverse: 5’-GCA GGA CCT CTC TGT AGA GCC TTG-

3’) and SYBR Select Master Mix for CFX (Life Technologies). All samples were normalized to

Rps18 as a control and are represented relative to Rps18 expression or relative to the indicated control sample.

Small interfering RNA nucleofection

Day-5 primary murine TFH-like cells were nucleofected with siGENOME SMARTpool small interfering RNA (siRNA) (D-051247, D-064214; Dharmacon) targeting Ikzf1, Ikzf3, or both, using

55 the Lonza 4DNucleofector system and buffer P3, per the manufacturer’s instructions. siGENOME nontargeting siRNA was used as a control (D-001210-01; Dharmacon). Following nucleofection, cells recovered in TH1-polarizing conditions containing low IL-2 (TFH-like–polarizing conditions) for 48 h. RNA was isolated, and changes in gene expression were analyzed via qRTPCR, including

Ikzf1 and Ikzf3 to establish knockdown efficiency.

Immunoblot analysis

Immunoblot analysis of endogenous and overexpressed proteins was performed using standard procedures. Briefly, cell pellets were lysed in 1X SDS-PAGE buffer (50 mM Tris [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and immediately boiled for 15 min.

Separation of lysates from an equivalent number of cells by SDS-PAGE was followed by immunoblot analysis on a 0.45-mm nitrocellulose membrane, which had been blocked using 2% instant nonfat dry milk in TBS-T (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20). Abs used to detect proteins of interest were as follows: Aiolos (1:500; cat. no. 39657; Active Motif; 1:5000; cat. no. sc-18683X; Santa Cruz Biotechnology), Ikaros (1:5,000; cat. no. sc-13039X; Santa Cruz

Biotechnology), Eos (1:1,000; cat. no. ABE1331; Millipore), Bcl-6 (1:500; cat. no. 561520; BD

Pharmingen), and V5 (1:20,000; code R960; Invitrogen). β-actin (1:15,000; cat. no. A00730;

GenScript) expression was used as a control to ensure equivalent protein loading.

Promoter-reported analysis

A Bcl6 promoter-reporter construct (pGL3-Bcl6) was generated by cloning the regulatory region of Bcl6 (positions 21573 to 0 bp) into the pGL3- Basic vector (Promega). EL4 T cells were nucleofected with expression vectors for Ikaros, Aiolos, Eos, or the indicated mutants, in

56 conjunction with pGL3-Bcl6 and a SV40-Renilla vector as a control for transfection efficiency.

Following 20–24 h of recovery, samples were harvested, and luciferase expression was analyzed using the Dual-Luciferase Reporter system, according to the manufacturer’s instructions

(Promega). Abundance of overexpressed proteins was assessed via immunoblot.

Chromatin Immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed as published28, 30. Briefly, chromatin was harvested from TH1 and TFH-like cells and immune-precipitated (i.p.) with antibodies to Aiolos (cat. no. sc-18683X; 5 µg/i.p.), Ikaros (cat. no. sc-9859X; 5 µg/i.p.), STAT3

(cat. no. sc-482X; 5 µg/i.p.; all from Santa Cruz Biotechnology), H3K4Me3 (cat. no. 39160, 1

µl/i.p.; Active Motif), histone 4 acetylation (H4Ac; cat. no. 39926, 1 µl/i.p.; Active Motif), histone

3 lysine 27 acetylation (H3K27Ac; cat. no. 07-360; 1 µl/i.p.; Millipore), histone 3 lysine 9 acetylation (H3K9Ac; cat. no. 06-942; 1 µl /i.p.; Millipore), or IgG control (cat. no. ab6709; 5

µg/i.p.; Abcam). Precipitated DNA was analyzed by quantitative PCR with gene-specific primers

(Bcl6 “A” forward: 5’-GTA CTC CAA CAA CAG CAC AGC-3’, and reverse, 5’-GTG GCT CGT

TAA ATC ACA GAG G-3’; Bcl6 “B” forward: 5’-CGA CCT TGA AAC GAA CCC AG-3’, and reverse: 5’-GTG TGG GTA CGT GTA ATG TTT GCC-3’; Bcl6 “C” forward: 5’-CGA GTT TAT

GGG TAG GAG AGG-3’, and reverse: 5’-GTC TTC GCT GTA GCA AAG CTC G-3’; Bcl6 “D” forward: 5’-GCG GAG CAA TGG TAA AGC CC-3’, and reverse: 5’-CTG GTG TCC GGC CTT

TCC TAG-3’; and Il2 forward: 5’-CTG CCA CAC AGG TAG ACT C-3’, and reverse: 5’-GGT

CAC TGT GAG GAG TGA TTA GC-3’). Samples were normalized to a standardized total input

DNA control, followed by subtraction of the IgG Ab as a control for the nonspecific binding. The

57 final value represents the percentage enrichment of Aiolos, Ikaros, STAT3, H3K4Me3, H4Ac,

H3K27Ac, and H3K9Ac.

Co-immunoprecipitation (Co-IP) assay

Co-Immunoprecipitation assays were performed as previously described17, 30. Briefly, lysates from primary murine TFH-like cells or EL4 cells overexpressing the indicated proteins were immune- precipitated with an experiment-specific antibody or antibody control. Lysates were then incubated at 4C in the presence of Protein A or Protein G sepharose beads (Millipore, 16157,

16201) for 1-2 hours. Immuno-precipitated proteins were detected by subsequent immunoblot analysis. The following antibodies were used for immunoprecipitation at 5 g/i.p., for both overexpression and primary T cell Co-IP analyses: STAT3 (Santa Cruz, sc-482X), Ikaros (Santa

Cruz, sc-9859X). Antibodies used to detect immuno-precipitated proteins were as follows: Aiolos

(Active Motif, 39293), Aiolos (Santa Cruz, sc-18683X), STAT3 (Santa Cruz, sc-8019), Ikaros (sc-

13039X), and V5 (Invitrogen, R960-25).

Influenza virus infections and in vivo analysis

Influenza virus infections were performed intranasally with 6500 VFU A/PR8/34 in 100 ml of

PBS. Cell suspensions from mediastinal lymph nodes (mLNs) were prepared by passing tissues through nylon mesh. Cells from mLNs were resuspended in 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA for 5 min to lyse red cells. Cell suspensions were then filtered through a 70- mm nylon cell strainer (BD Biosciences), washed, and resuspended in PBS with 5% donor calf serum and 10 mg/ml Fc Block (2.4G2; Bio X Cell) for 10 min on ice before staining with fluorochromeconjugated Abs or tetramer reagents. Fluorochrome-labeled anti–Bcl-6 (clone K112-

91; dilution 1:50), anti-Cxcr5 (clone 2G-8; dilution 1:50), anti-Aiolos (clone S48-791; dilution

1:50), and anti-CD4 (clone RM4-5; dilution 1:200) were from BD Biosciences. The IAbNP311–

325 MHC class II tetramer (dilution 1:100) was obtained from the National Institutes of Health

Tetramer Core Facility. Intracellular staining for Bcl-6 and Aiolos was performed using the Mouse

Regulatory T Cell Staining Kit (eBioscience), according to the manufacturer’s instructions. Flow cytometry was performed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo software.

Listeria monocytogenes infections and analysis

All studies used 6-10 week-old age and sex-matched C57B6/J mice that were maintained in specific pathogen–free facilities. Listeria monocytogenes was obtained from the American Type

Culture Collection (ATCC BAA-679TM) and cultured in brain heart infusion agar/broth, following product guidelines. Prior to in vivo inoculation, bacteria density was estimated in liquid broth culture by measuring the absorbance at 600 nm (0.4 OD600 = 7.9×108 bacteria/ml). Cultures of L. monocytogenes were pelleted, washed twice in PBS, and resuspended in PBS. Mice were inoculated via. Intravenous injection with 5000 CFUs of L.monocytogenes in 50 ml of PBS. L. monocytogenes inoculation dosages were confirmed for each experiment by plating an aliquot of homogenized liver from randomly selected mice 24 h after inoculation and calculating the resultant

CFUs/mg of tissue.

After 6 days of infection (d.p.i), primary CD4+ T cells were isolated from the spleens of

L.monocytogenes infected mice using the BioLegend MojoSort Isolation Kit (cat. no. 480033), per the manufacturer’s instructions. For subsequent flow cytometry analysis, isolated cells were

59 stained with the following fluorochrome-labeled antibodies: anti-CD44 (cat. no. 560452; BD

Biosciences), anti–PD-1 (cat. no. 11-985; eBioscience), and anti-Cxcr5 (cat. no. FAB6198P; R&D

Systems), anti-CD4 (cat. no. 56-0031; eBioscience), and the respective isotype controls (R&D).

Briefly, harvested cells were pelleted, washed, and stained with the indicated fluorochromeconjugated Abs. Following staining, cells were washed three times with FACS buffer and then resuspended for analysis and sorting on a Sony SH800 flow cytometer. All of the obtained data

+ + hi + were analyzed using FlowJo software. CD4 CD44 Cxcr5 PD1 TFH cell transcript analysis was conducted as described above in RNA purification and qRT-PCR.

Statistics

All data represent at least three independent experiments. Error bars represent the standard error of mean (SEM) or standard deviation (SD), as indicated. For statistical analyses, an unpaired t-test or one-way ANOVA with the Tukey multiple-comparison test were performed to assess statistical significance, as appropriate for a given experiment. The p values <0.05 were considered statistically significant.

Results

Expression of Aiolos and Ikaros correlate with that of Bcl-6

Although it was once widely believed that initial commitment to an individual effector T-cell subset was a terminal event, a large body of literature has emerged demonstrating that a substantial degree of flexibility exists between effector Th cell populations43, 44, 45, 46, 47. It has been postulated that this phenomenon, termed Th cell plasticity, allows Th cell subsets to respond to the cellular microenvironment in real-time to provide a more efficient and effective immune response. For example, many studies have demonstrated that TH1 cells maintain flexibility with the TFH cell

19, 30, 48 49, 50 type . More recently, there have also been reports of TH1-biased TFH cell populations .

Corroborating these studies, our laboratory has previously demonstrated that TH1 cells are capable

28, 30 of upregulating a Bcl-6 dependent TFH-like cell state in response to altered IL-2 signaling .

Mechanistically, in TH1 cells exposed to low IL-2 environments, increased Bcl-6 expression allows for the repression of the TFH antagonist B lymphocyte–induced maturation protein-1 and the

28, 30 subsequent expression of a TFH-like cell program (Figure 3.2A and B ). Because the induction of Bcl-6 expression is a critical step for the transition from the TH1 to the TFH-like state, our current study aimed to identify the IL-2–sensitive transcription factors that are responsible for regulating

Bcl-6 expression.

Members of the IkZF transcription factor family have previously been implicated in the differentiation of specific effector Th cell subsets, including TH1, TH17, and regulatory T cell populations37, 51, 52, 53, 54. As such, we began by assessing whether these factors were differentially expressed in in vitro–generated TH1 versus TFH-like cells (Figure 3.1). We found that two IkZF factors, Ikaros (Ikzf1) and Aiolos (Ikzf3), were upregulated in TFH-like cells, whereas a third IkZF family member, Eos (Ikzf4), was more highly expressed in TH1 cells (Figure 3.2B). Although two

61 additional IkZF family members, Helios (Ikzf2) and Pegasus (Ikzf5), also displayed altered expression, their overall transcript levels were much lower than those of the other IkZF factors

+ (Figure 3.2B). A time course encompassing the naïve CD4 T cell transition to differentiated TH1 or TFH-like cell populations further demonstrated that increased Aiolos expression correlated with the highest expression of Bcl-6, whereas Eos expression was elevated in day-3 and day-5 TH1 cells

(Figure 3.2C, Figure 3.1). Although there was a slight increase in Ikaros expression in TFH-like cells, the expression was relatively abundant across CD4+ T cell populations, perhaps implying a broader role for this factor in Th cell differentiation (Figure 3.2C). In addition to our in vitro– generated TFH-like cells, other laboratories have generated TFH-like cells using alternative in vitro culturing conditions (40, 48). Importantly, analysis of Ikaros and Aiolos expression revealed a similar trend among the TFH-like populations compared with that observed for in vitro generated

TH1 or TH2 cells (Figure 3.3A). Furthermore, similar expression patterns were observed in TFH cells generated in vivo in response to L. monocytogenes infection (Figure 3.3B). Collectively, these gene-expression analyses indicated that a positive correlation exists among Aiolos (Ikzf3),

Ikaros (Ikzf1), and Bcl6 expression.

To determine whether protein expression correlated with the observed changes in transcript, we performed immunoblot analyses of Aiolos, Ikaros, Eos, and Bcl-6 expression in TH1 and TFH-like cells. Indeed, we observed a sharp increase in Aiolos expression that was consistent with increased expression of Bcl6 in TFH-like cells (Figure 3.2D-F). As with the transcript data, Ikaros expression was moderately elevated in TFH-like cells, whereas Eos protein expression inversely correlated with that of Bcl-6 and the other two IkZF family members (Figure 3.2D-F). Given the positive correlation among Aiolos, Ikaros, and Bcl-6, we focused on elucidating possible mechanisms by which these IkZF factors may contribute to the induction of Bcl6 expression.

To further assess whether Aiolos or Ikaros may be involved in promoting Bcl-6 expression, we used siRNAs to knockdown Ikaros and/or Aiolos to determine whether reduced expression of either factor affected Bcl6 expression in TFH-like cells. Indeed, upon knockdown of Aiolos (Ikzf3) or Ikaros (Ikzf1) individually, we observed decreased expression of Bcl6 transcript, whereas combined knockdown of Ikzf1 and Ikzf3 resulted in a further reduction in Bcl6 expression (Figure

3.2G). Importantly, Eos (Ikzf4) levels remained unchanged, demonstrating the specificity of the

Aiolos- and Ikaros-dependent effects upon Bcl6 expression. These data also demonstrate the specificity of the siRNAs for the intended Ikzf1 and Ikzf3 targets, an important consideration given the high degree of conservation among IkZF family members (36). Collectively, these data support a role for Aiolos and Ikaros in the positive regulation of Bcl6 expression.

+ Figure 3.2.A Positive correlation exists between the expression of Aiolos, Ikaros, and Bcl-6. Primary CD4 T cells were cultured in TH1 polarizing conditions and exposed to either high (TH1 cells) or low (TFH-like cells) environmental IL-2 (20 ng/ml or 0.8 ng/ml, respectively). (A, B) RNA was isolated from TH1 and TFH-like cells and the expression of the indicated genes was determined by quantitative RT-PCR. Data in ‘A’ are normalized to the Rps18 control and represented as the fold increase relative to the TH1 sample. Data in ‘B’ are normalized and represented relative to the Rps18 control (A, B mean of n = 4 ± s.e.m.). (C) RNA was isolated from Naïve, Day

3 TH1, and Day 5 TH1 and TFH-like cells and the expression of the indicated genes was determined by quantitative RT-PCR. Data were normalized to Rps18 as a control and the results are represented as fold change in expression relative to the TH1 sample (mean of n = 3 ± s.e.m.). (D-F) An immunoblot analysis was performed to assess changes in protein expression in response to alterations of environmental IL-2. Expression for Bcl-6, Aiolos, Ikaros, and Eos was measured with β-actin serving as a control for equal protein loading. Shown is a representative blot of three independent experiments performed. (G) TFH-like cells were nucleofected with either siRNA specific to Ikaros (siIkzf1), Aiolos (siIkzf3), both (siIkzf1/siIkzf3), or a control siRNA. Following a 48-hour time period, RNA was harvested and expression of the indicated genes was assessed by qRT-PCR. The data were normalized to Rps18 and presented as fold change in expression relative to the control (mean of n = 4 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (A, B unpaired Student’s t-test; C, G one-way ANOVA with Tukey multiple comparison test).

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Figure 3.3. Ikzf1 and Ikzf3 levels positively correlate with Bcl6 expression. (A) Primary CD4+ T cells were cultured in TH1, TH2, TFH-like (Read et al.), or TFH-like (Awe et al.) conditions throughout a five day time-course. RNA was isolated and the expression of the indicated genes was determined by quantitative RT-PCR. Data are normalized and represented relative to Rps18 control (mean of n = 3 ± s.e.m.). **P < 0.01 (one-way ANOVA with Tukey multiple comparison test). (B) Mice were infected with Listeria monocytogenes. Six days post-infection, + + HI + mice were sacrificed and TFH cells were sorted (CD4 CD44 Cxcr5 PD-1 ). RNA was isolated and the expression of the indicated IkZF family members was assessed via quantitative RT-PCR. Data were normalized and represented relative to Rps18 as a control (mean of n = 3 ± s.e.m.).

The Zinc Finger (ZF) DNA-binding domains of Aiolos and Ikaros are required to induce

Bcl6 promoter activity

To determine whether Aiolos and Ikaros may interact with regulatory regions of the Bcl6 locus, we performed an in silico analysis of the Bcl6 promoter and located several predicted binding sites containing the core IkZF DNA-binding motif “GGGAA” (Figure 3.4A). To test the functionality of these sites, we created a Bcl6 promoter-reporter construct encompassing the predicted sites to examine the effect of Aiolos, Ikaros, or Eos overexpression on Bcl6 promoter activity. Importantly, upon Aiolos or Ikaros overexpression, we observed a significant increase in Bcl6 promoter activity

(Figure 3.4A). Conversely, as a control, there was no increase in Bcl6 promoter activity in the presence of Eos. These data suggest that Aiolos and Ikaros may induce Bcl6 expression via effects upon the Bcl6 promoter.

Aiolos and Ikaros contain N-terminal zinc finger (ZF) domains, which mediate their DNA-binding capabilities. Of the four ZFs that make up the N-terminal domain, ZF2 and ZF3 are required for

DNA binding, whereas ZF1 and ZF4 appear to modulate sequence specificity55, 56. To determine whether ZF-mediated DNA binding was required for Aiolos- and Ikaros-dependent Bcl6 promoter activation, we constructed expression vectors with point mutations in select N-terminal ZFs

(Aiolos: AiΔZF1, AiΔZF1, 2, and AiΔZF4; Ikaros: IkΔZF1, IkΔZF1, 2, and IkΔZF4) (Figure

3.4B, C). We then compared the ability of wild-type Aiolos or Ikaros and their corresponding ZF mutants to induce Bcl6 promoter activity. As with our previous data, Bcl6 promoter activity was readily induced by wild-type Aiolos or Ikaros (Figure 3.4B, C). However, we did not observe increases in Bcl6 promoter activity with the AiΔZF1,2 or IkΔZF1,2 mutants, suggesting that direct

DNA binding by Aiolos or Ikaros may be required for Bcl-6 induction (Figure 3.4B, C).

Interestingly, overexpression of a subset of the Aiolos and Ikaros mutants with a single ZF

66 mutation in either the first or fourth ZF (AiΔZF1, IkΔZF1, and IkΔZF4) resulted in only a modest increase in Bcl6 promoter activity, suggesting that there may be differential requirements for individual ZFs in mediating promoter activation. Taken together, these results suggest that the N- terminal ZF DNA-binding domain is of functional importance in the Aiolos- and Ikaros-dependent induction of Bcl6 promoter activity.

Figure 3.4. DNA-binding activity of Aiolos or Ikaros is required to induce Bcl6 promoter activity (A) EL4 T cells were transfected with the Bcl6 promoter-reporter construct in combination with wild-type Aiolos, Ikaros, or Eos expression vector, or an empty vector control. Luciferase promoter-reporter values were normalized to a renilla control and presented relative to the empty vector control sample for each experiment (mean of n = 3 ± s.e.m.). Aiolos, Ikaros, and Eos protein levels were assessed by immunoblot analysis with an antibody against the V5 epitope tag. Shown is a representative blot of three independent experiments performed. Also shown is a schematic of the Bcl6 promoter with predicted IkZF binding sites depicted as gray ovals. (B, C) EL4 T cells were transfected with the Bcl6 promoter-reporter construct in combination with wild-type (B) Aiolos, or (C) Ikaros expression vectors, the corresponding Aiolos or Ikaros ZF mutants (ΔZF), or an empty vector control (mean of n = 3 ± s.e.m.). Luciferase promoter-reporter values were normalized to a renilla control and expressed relative to the empty vector control sample for each experiment. Wild-type and mutant Aiolos or Ikaros protein levels were assessed by immunoblot analysis. Shown is a representative blot of three independent experiments performed. Also shown are schematics depicting wild-type Aiolos or Ikaros and the indicated Aiolos or Ikaros zinc finger (ZF) mutants used in the Bcl6 promoter-reporter experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey multiple comparison test).

Aiolos and Ikaros associate with the Bcl6 promoter

Our previous data suggested that Aiolos and Ikaros may induce the activation of Bcl6 expression and that direct DNA binding may be required, we performed ChIP assays to determine whether either factor directly associates with the endogenous Bcl6 promoter in TFH-like cells (Figure 3.5A).

Indeed, we observed association of Aiolos and Ikaros with a region of the Bcl6 promoter proximal to the transcriptional start site (TSS) (Figure 3.5B, C). Importantly, this association was significantly enriched in TFH-like cells compared with that observed for TH1 cells (Figure 3.5B,

C). Furthermore, the binding observed proximal to the TSS was specific, because enrichment of these factors was markedly reduced at a distal location from the Bcl6 locus. Interestingly, further

ChIP analysis for the known Bcl-6 regulator STAT3 revealed a similar enrichment pattern to that observed for Aiolos and Ikaros in TFH-like cells, suggesting the intriguing possibility that these factors may collaboratively regulate Bcl-6 expression (Figure 3.5D).

Quintana et al. recently demonstrated that Aiolos directly represses IL-2 production during TH17 cell differentiation37. Because IL-2 signaling negatively regulates Bcl6 expression, we considered the possibility that Aiolos could contribute to increased levels of Bcl-6 indirectly in a similar IL-2 dependent fashion. To address this, we examined Aiolos association within the IL-2 (Il2) locus, as described by Quintana et al.37. Interestingly, although there was a slight increase in Aiolos binding at the Il2 locus in TFH-like cells, overall enrichment was much less than that observed at the Bcl6 locus, suggesting that the effect of Aiolos on Bcl6 expression is mediated by a direct mechanism rather than indirectly via an IL-2 dependent effect (Figure 3.5B-D). Collectively, these data support a model in which Aiolos, Ikaros, and STAT3 associate with the Bcl6 promoter to induce

Bcl6 expression in TFH-like cells.

IkZF/STAT factor association correlates with increased histone modification of the Bcl6 promoter

IkZF and STAT factors are known to exert their effects, at least in part, through their association with chromatin-remodeling complexes that are capable of directing histone modifications indicative of gene activation and repression. To assess whether Aiolos and Ikaros binding was associated with changes to chromatin structure at the Bcl6 promoter, we performed a ChIP assay to examine alterations in histone acetylation and methylation. Consistent with a role in activating

Bcl6 expression, we observed increased H3K4Me3, H3K9Ac, H3K27Ac, and H4Ac at the Bcl6 promoter in TFH-like cells compared with TH1 cells (Figure 3.5E-H). Importantly, significant increases in H3K4Me3, H3K9Ac, and H3K27Ac were detected at regions where Aiolos, Ikaros, and

STAT3 were most highly enriched (Figure 3.5E-H). Collectively, these data indicate that association of Aiolos, Ikaros, and STAT3 with the Bcl6 promoter correlates with changes in chromatin structure consistent with gene activation.

Figure 3.5. Aiolos, Ikaros, and STAT3 associate with the Bcl6 promoter in TFH-like cells. (A) A schematic of the Bcl6 locus indicating the location of PCR amplicons used in the chromatin immunoprecipitation (ChIP) analyses, indicated as “A”, “B”, “C”, and “D”. (B-D) ChIP assays to assess Aiolos, Ikaros, and STAT3 association with the Bcl6 locus in TH1 or TFH-like cells. Data are represented as percent enrichment relative to a “total” input sample (mean of n = 3 ± s.e.m.). (E-H) ChIP assays to assess alterations to histone modifications within the Bcl6 promoter in TH1 or TFH-like cells. Data are represented as percent enrichment relative to a “total” input sample (mean of n = 4 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).

Aiolos interacts with STAT3 in TFH-like cells

Given the similar enrichment patterns observed for Aiolos, Ikaros, and STAT3 at the Bcl6 promoter, we considered the possibility that they may cooperate to induce Bcl6 expression. To this end, we performed Co-IP assays to determine whether these factors interact in TFH-like cells. It is well established that Aiolos and Ikaros form heterodimers via their C-terminal ZF domains36, 56.

Indeed, Co-IP analysis demonstrated that Aiolos and Ikaros form heterodimeric complexes in TFH- like cells (Figure 3.6A). Intriguingly, additional Co-IP assays revealed the presence of novel

Aiolos/STAT3 complexes in TFH-like cells (Figure 3.6B). Despite the large degree of amino acid conservation between Aiolos and Ikaros, we did not detect the presence of Ikaros/STAT3 complexes in TFH-like cells. Whether this was due to the absence of such complexes or was a limitation of the Co-IP analysis itself remains unresolved. Still, these findings support the existence of a novel Aiolos/STAT3 protein complex in TFH-like cells.

To examine whether Aiolos and STAT3 may cooperate to induce Bcl6 expression, we overexpressed STAT3CA alone, Aiolos alone, or Aiolos in combination with increasing amounts of STAT3CA (Figure 3.6C). Interestingly, expression of Aiolos or STAT3CA alone resulted in only slight increases in the Bcl6 transcript. However, when Aiolos was expressed in combination with increasing amounts of STAT3CA, we observed significant increases in Bcl6 expression compared with the control sample or the samples in which Aiolos or STAT3CA were expressed individually

(Figure 3.6C).

Figure 3.6. Aiolos and STAT3 physically interact and regulate Bcl6 expression. (A) Co-immunoprecipitation

(Co-IP) of endogenously expressed proteins in TFH-like cells with control antibody (α-V5) or α-Ikaros, followed by immunoblot analysis (IB) with α-Aiolos. Shown is a representative blot of four independent immunoprecipitation experiments performed. (B) Co-IP of endogenously expressed proteins in TFH-like cells with control antibody (α-V5) or α-STAT3, followed by immunoblot analysis (IB) with α-Aiolos. Shown is a representative blot of three independent immunoprecipitation experiments performed. (C) EL4 T cells were CA CA transfected with Aiolos alone, STAT3 alone, Aiolos and STAT3 (increasing amounts indicated by wedge) in combination, or empty vector control. Immunoblot with an α-V5 antibody was performed to assess relative CA abundance of overexpressed Aiolos and STAT3 . Following a 24 hr time period, RNA was isolated and Bcl6 expression was measured by qRT-PCR. Data were normalized to Rps18 as a control and the results are represented as fold change in expression relative to the control (ctrl.) sample (mean of n = 3 ± s.e.m.). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey multiple comparison test).

N- and C-terminal ZF domains of Aiolos are required for induction of Bcl-6

In addition to the N-terminal ZF DNA-binding domain discussed previously, members of the IkZF family contain a C-terminal ZF domain that mediates homo- or heterodimerization with other IkZF proteins36, 56. To determine whether either ZF domain may be required for interaction between

Aiolos and STAT3, we co-expressed STAT3CA with wild-type Aiolos or with Aiolos mutants harboring disruptions to the N- or C-terminal ZF domains and performed Co-IP analysis (Figure

3.7A). As with the TFH-like cells, wild-type Aiolos and STAT3 interactions were readily detected.

Similarly, we also detected interactions between STAT3 and the AiΔZF1, 2 mutant. However,

STAT3 was unable to interact with the Aiolos mutant lacking the C-terminal ZF dimerization domain (AiolosΔC), suggesting that this domain is required for the interaction between Aiolos and

STAT3 (Figure 3.7A). To determine the functional impact of the C-terminal mutation, we overexpressed STAT3CA with wild-type Aiolos or AiolosΔC and assessed the impact on Bcl6 expression. Indeed, compared with co-expression of wild-type Aiolos and STAT3CA, Bcl6 expression was significantly diminished when STAT3CA was co-expressed with the AiolosΔC mutant (Figure 3.7B).

Although the AiΔZF1, 2 mutant was able to interact with STAT3, the Bcl6-reporter data suggested that the N-terminal ZF DNA-binding domain was required to induce promoter activity. To determine the functional impact of the N-terminal mutation, we overexpressed STAT3CA with wild-type Aiolos or AiΔZF1, 2 and assessed the impact on Bcl6 expression. Indeed, similar to the results observed for the AiolosΔC mutant, the combination of STAT3CA and AiΔZF1,2 was unable to induce Bcl6 expression, suggesting that both the N- and C-terminal ZF domains are required for Aiolos-dependent activation of Bcl-6 expression(Figure 3.7C). Taken together, these data suggest that STAT3 and Aiolos form a transcription factor complex via the Aiolos C-terminal

74 protein– protein interaction domain and that this novel complex is capable of promoting Bcl6 expression. It is important to note that, although interactions between Ikaros and STAT3 were not detected, our data do not preclude the possibility that such interactions, or interactions between some combination of STAT3, Aiolos, and Ikaros, may play important roles in regulating Bcl-6 expression.

Figure 3.7. Functional ZF domains are required for Aiolos-dependent induction of Bcl6 expression (A) Co- CA IP of overexpressed wildtype Aiolos or indicated mutants (V5-tagged) and tagless STAT3 in EL4 cells. Lysates were immune-precipitated with α-STAT3, followed by immunoblot analysis with α-V5 (for detecting Aiolos proteins). Shown is a representative blot of three independent immunoprecipitation experiments performed. (B) CA CA EL4 T cells were transfected with Aiolos and STAT3 , AiolosΔC and STAT3 , or empty vector control. Immunoblot with an α-V5 antibody was performed to assess relative abundance of overexpressed proteins. Following a 24 hr time period, RNA was isolated and Bcl6 expression was measured by qRT-PCR. Data were normalized to Rps18 as a control and the results are represented as fold change in expression relative to the empty CA vector control sample (mean of n = 3 ± s.e.m.). (C) EL4 T cells were transfected with Aiolos and STAT3 , CA AiolosΔZF1,2 and STAT3 , or empty vector control. Data were obtained, normalized, and represented as in ‘B’ (mean of n = 6 ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey multiple comparison test).

Aiolos and STAT3 cooperatively regulate cytokine receptor expression

Our findings suggest that Aiolos and Ikaros are direct regulators of Bcl6 expression in Th cells. As discussed previously, signals from environmental cytokines are key determinants of Th cell differentiation. These include IL-6 and IL-2, which have been shown to positively and negatively influence TFH development, respectively 21, 57, 58. Therefore, we wanted to assess whether Aiolos or Ikaros may play a broader role in promoting TFH cell differentiation by regulating the expression of the receptors for these cytokines. We began by performing Aiolos (Ikzf3) and Ikaros (Ikzf1) siRNA-knockdown experiments to assess the effect on Il6ra and Il2ra expression. Indeed, the expression of Il6ra decreased significantly upon Ikzf3 and Ikzf1 knockdown (Figure 3.8A).

Importantly, this decrease in expression was specific to Il6ra, because the expression of Il2ra was unaffected (Figure 3.8A). To determine whether Aiolos and STAT3 may cooperate to induce Il6ra

(as with Bcl6), we overexpressed STAT3CA alone, Aiolos alone, or Aiolos in combination with increasing amounts of STAT3CA and examined Il6ra expression. Importantly, the co-expression of Aiolos and STAT3CA resulted in a significant increase in Il6ra expression, whereas the expression of Il2ra was unchanged (Figure 3.8B). Collectively, these data suggest that the interplay between Aiolos, Ikaros, and STAT3 may play a broader role in regulating TFH differentiation, perhaps through induction of the cytokine receptor Il6ra.

Figure 3.8. STAT3/Aiolos differentially regulate Il6rαand Il2rα expression. (A) TFH-like cells were nucleofected with either siRNA specific to Ikaros (siIkzf1), Aiolos (siIkzf3), both (siIkzf1/siIkzf3), or a control siRNA. Following a 48-hour time period, RNA was harvested and expression of Il6ra or Il2ra was assessed by qRT-PCR. The data are normalized to Rps18 and presented as fold change in expression relative to the control CA sample (mean of n = 4 ± s.e.m.). (B) EL4 T cells were transfected with Aiolos alone, STAT3 alone, Aiolos and CA STAT3 (increasing amounts indicated by wedge) in combination, or empty vector control. Following a 24 hr time period, RNA was isolated and Il6ra or Il2ra expression was measured by qRT-PCR. Data were normalized to Rps18 as a control and the results are represented as fold change in expression relative to the control sample (mean of n = 3 ± s.e.m.). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey multiple comparison test).

Aiolos expression is increased in Ag-specific TFH cells after influenza infection

Our mechanistic findings implicate Aiolos in the positive regulation of Bcl6, and possibly the TFH differentiation program, via a cooperative mechanism with the known TFH regulator STAT3. To extend these findings, we sought to determine whether Aiolos was preferentially expressed in in vivo generated TFH cells, as opposed to non-TFH effector Th (TEFF) cells, in response to infection.

To this end, we infected mice with influenza and assessed Aiolos expression in Ag-specific TFH

hi hi lo lo (Bcl-6 Cxcr5 ) and TEFF (Bcl-6 Cxcr5 ) populations (Figure 3.9A). Importantly, at the peak of infection (12 days after infection), nucleoprotein (NP)-specific TFH cells expressed significantly

lo more Aiolos than that observed in the Bcl-6 TEFF population (Figure 3.9B). Collectively, these in vivo data, in combination with our in vitro findings, are supportive of a role for Aiolos in promoting Bcl-6 expression and TFH cell differentiation.

Figure 3.9. Aiolos expression is increased in antigen-specific TFH cells post-influenza infection. Mice were + - - infected with influenza (PR8) and NP-specific CD4 CD19 Foxp3 T cells from the mLNs were analyzed on day + - - 12 after infection by flow cytometry. (A) Expression of Bcl-6 and Cxcr5 in NP-specific CD4 CD19 Foxp3 T cells lo lo hi hi + - - (B) Expression of Aiolos in Bcl6 CXCR5 (T ) and Bcl6 CXCR5 (T ) NP-specific CD4 CD19 Foxp3 T cell EFF FH populations. Histogram overlay and mean fluorescence intensity (MFI) in NP-specific TFH and TEFF populations are shown (data are shown as the mean ± s.d. (n=5 mice/group). *P < 0.05 (P values were determined using a two-tailed Student´s t-test).

Discussion

Bcl-6 has well-established roles in the development of a multitude of immune cell types, including

TFH cells, memory T cell populations, and B cells. As such, there has been an ongoing interest in identifying the molecular mechanisms involved in the transcriptional regulation of Bcl-6 expression. In this article, we describe previously unidentified roles for the IkZF family members

Aiolos and Ikaros in the induction of Bcl-6. Perhaps most intriguingly, our data have begun to define a novel cooperative relationship between STAT3, a known positive regulator of Bcl-6 expression, and the IkZF factor Aiolos.

The interplay between opposing STAT factors (e.g., STAT3, STAT5) at the Bcl6 promoter is an important contributor to the regulation of Bcl-6 expression21, 57, 58. Unexpectedly, our findings demonstrate that STAT3 physically interacts with Aiolos. Thus, our data support the possibility that a primary role for STAT3 may be to recruit Aiolos to the Bcl6 locus. Taken together with the increase in Aiolos expression observed in in vitro– and in vivo–derived TFH cell populations, these data suggest that this novel IkZF/ STAT protein complex may be an important driver of Bcl-6 expression and, perhaps, TFH cell fate. Indeed, it will be of interest to establish whether the

Aiolos/STAT3 complex regulates additional TFH genes beyond Bcl6 and Il6ra. Likewise, given that Aiolos and STAT3 are members of transcription factor families that are both widely expressed and highly conserved, it will be of considerable interest to determine whether STAT and IkZF interactions regulate the differentiation and function of other immune cell populations, including additional Th cell subsets. For example, Aiolos has been shown to influence TH17 differentiation through the direct repression of IL-2 expression (37). Interestingly, TH17 and TFH cells share a number of regulatory features during their development, including sensitivity to the IL-2/STAT5 signaling axis and dependence on STAT3 activity26, 29, 30, 59, 60. Thus, an intriguing possibility is

81 that the STAT3/Aiolos complex identified in this study may also play a role in TH17 differentiation.

The precise molecular mechanisms by which STAT3, Aiolos, and Ikaros cooperate to regulate

Bcl-6 expression remain unclear. The activity of IkZF factors has been attributed primarily to their association with chromatin-modifying enzymes, including the switch/sucrose nonfermenting and

Mi-2/nucleosome remodeling and deacetylase complexes56, 61. Indeed, our results demonstrate that the association of Aiolos and Ikaros with the Bcl6 promoter correlates with alterations to the chromatin structure surrounding this region, including increased histone acetylation and methylation. These chromatin modifications are indicative of an accessible chromatin structure and consistent with an actively transcribing gene. Our observations are not without precedence, because IkZF factors have been implicated in gene activation prior to the current study36, 56, 62, 63.

It is also possible that Aiolos and Ikaros may act to remodel the chromatin structure of regulatory regions located proximal to the Bcl6 promoter. Intriguingly, there are predicted CCCTC-binding factor binding sites surrounding this region, suggesting the presence of insulator or silencing elements. Another, non-mutually exclusive, possibility is that Aiolos and Ikaros association with the Bcl6 promoter near the TSS contributes to the assembly and/or activation of the transcriptional initiation complex. In this regard, Ikaros has been shown to physically interact with, and alter the activity of, the RNA Pol II complex64 (64). It is also possible that Aiolos and Ikaros may be required to mediate interactions between the Bcl6 promoter and distal enhancers. In support of this possibility, Ikaros has been implicated in promoting long-range chromatin interactions between regulatory elements at other genetic loci65, 66. Further experimentation will be required to address these possibilities and to understand whether established IkZF-remodeling activities are involved or whether a novel mechanism may be responsible for promoting Bcl-6 expression.

Future studies will also be necessary to comprehensively assess the distinct contributions of Aiolos and Ikaros to the regulation of Bcl-6 expression. Thus far, our data support the existence of

STAT3/Aiolos complexes but not those made up of STAT3 and Ikaros, because we could not detect the latter. Still, ChIP analysis of the Bcl6 promoter clearly demonstrates an increase in

Aiolos and Ikaros association at the Bcl6 locus, and we detect the presence of Aiolos/Ikaros complexes in TFH-like cells. Therefore, it is possible that Ikaros and Aiolos could be recruited independently of STAT3 to the Bcl6 locus. However, the signals responsible for Aiolos/Ikaros recruitment to the Bcl6 promoter remain unclear. Based upon our observation that Ikaros is expressed at moderately high levels in naive and TH1 cells, we propose a model in which a basal level of Ikaros is bound to the Bcl6 locus, perhaps allowing this gene to remain in a poised state during Th cell differentiation. Indeed, this would be consistent with the established role of Ikaros as a broad regulator of T cell differentiation and our observation that Ikaros is expressed at

36, 61 moderately high levels in naive and TH1 cells prior to the transition to the TFH-like cell state .

In our proposed model, we hypothesize that the association of Aiolos/STAT3 complexes with the

Bcl6 promoter, in the absence of IL-2/STAT5 signaling, leads to chromatin-remodeling activities that result in the activation of Bcl-6 expression. Additionally, because IkZF factors are known to homo- and heterodimerize upon binding to DNA, we also propose that IkZF proteins could mediate interactions between distal regulatory elements65, 66. Further elucidation of the exact contribution of Aiolos and Ikaros to the induction of Bcl-6 will be of interest and may serve to shed light on how STAT3, Aiolos, and Ikaros cooperate to regulate the expression of additional target genes involved in TFH cell differentiation.

The appropriate temporal expression of Bcl-6 is a molecular linchpin that regulates the differentiation and function of many cell types that are critical to the promotion of an effective immune response. The importance of understanding Bcl-6 regulation is further highlighted when considering the numerous human diseases that have been linked to aberrant expression and function of this transcriptional repressor14, 67. Our findings identify novel regulators and provide insight into the mechanisms by which they promote Bcl-6. Future work will be required to fully elucidate the complex network of signals and factors that regulate Bcl-6 expression. In doing so, we may enhance the potential to design more efficacious vaccines and develop novel immunotherapeutic approaches as a result of the wide-ranging importance of this transcriptional regulator.

Acknowledgements

The authors would like to acknowledge Sheryl Coutermarsh-Ott and Daniel Rothschild for technical assistance with the in vivo studies. The authors would also like to thank Dr. James Smyth and members of the Oestreich lab for insightful discussions.

Disclosures

The authors declare no competing financial interests.

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Chapter 5

Conclusions and future directions

Studies over the years have shown the importance of cytokines in mediating T-helper cell plasticity

1. While significant progress has been made with regards to discovering the molecular mechanisms that regulate flexibility, the complete range of transcriptional factors that integrate extracellular signals and cell intrinsic changes are unknown. Observations made in this dissertation hope to address this issue by understanding additional transcriptional factors influenced by IL-2 that mediate flexibility between TH1 and TFH cells. Here we show three IkZF transcription factors, Eos,

Aiolos and Ikaros are regulated by IL-2 and additionally show that these proteins are required for cytokine-mediated TH1 and TFH development. The research presented here broadens our understanding of the novel transcriptional factors and regulatory mechanisms through which cytokines influence T-helper cell flexibility.

Summary of findings

Data has shown that IL-2 positively regulates development of TH1 cells but impedes TFH

2, 3 differentiation . This regulation of TH1 and TFH cells could be physiologically relevant, as recent studies have shown that upon viral infection TFH cells isolated within the lymph nodes shared characteristics with TH1 cells, such as co-expression of T-bet and Bcl6 and dual production of

4 IFNγ and IL-21 (thus termed as TFH1 cells) . Although circulating forms of TFH1 cells are thought to be beneficial during HIV infection, current data also suggests these cells are detrimental in the advent of other infections such as malaria and autoimmune diseases such as lupus 4, 5, 6, 7, 8, 9. While

130 significant effort has gone into characterizing and understanding functionality, the molecular mechanisms involved in the origins of such cells remain unknown. Given the importance of cytokines in promoting TH1 over TFH development, one possible mechanism for the development

10 of TFH1 cells could be the transitioning of polarized TH1 cells to exhibit a TFH –like phenotype .

Therefore such flexible populations could ultimately be influenced by the spatiotemporal presence of cytokines (within the microenvironment), and the regulatory networks activated as a consequence 3,11, 12.

In the presence IL-2, we observed that TH1 cells expressed the IkZF member, Eos. This transcriptional factor was shown to be important, as loss of Eos disrupted TH1 development and effector functions by diminishing Blimp-1 levels, reducing IFNγ production and attenuating IL-2 receptor subunit levels respectively. In addition to parsing the signaling pathway involved in Eos induction, we observed that Eos was able to form a novel protein complex with STAT5 and capable of promoting Prdm1 expression. Another interesting observation was that Eos could regulate

STAT5 activation and this consequently led to enrichment of STAT5 within regulatory regions of signature TH1 genes such Prdm1, Ifng and Il2ra.

However, upon IL-2 depletion from the microenvironment, we found TH1 cells upregulating other members of the IkZF family, Aiolos and Ikaros. In addition to enrichment histone modifications reflective of active gene transcription, both these factors, along with STAT3 were enriched within the Bcl6 gene locus. Like Eos and STAT5 in TH1 cells, Aiolos and STAT3 physically interacted to form a protein complex in cells and were capable of inducing signature TFH genes such as Bcl6 and Il6ra. Furthermore, the importance of Aiolos in promoting TFH development was observed in vivo, as Aiolos levels were significantly higher in TFH cells when compared to other effector T- helper cells when mice were infected with influenza.

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Taken together we show a mechanism by which IL-2 can regulate plasticity between in TH1 and

TFH cells by not just modulating STAT factors but also regulating expression of IkZF members, with absence of these members severely impacting TH1 and TFH development. While our observations are exciting, several questions remain unanswered, including how IkZF members could mediate T helper cell development.

Insights into the regulatory mechanisms by which IkZF and STAT factors modulate T-cell development and plasticity.

As with cytokine regulation of STAT activation, our data show that IL-2 within the microenvironment influences IkZF expression within T-cell subsets, which consequently play an important role in mediating T-helper cell development and plasticity. As mentioned in Chapter 2,

IkZF factors, much like STAT factors, are known to regulate gene expression by associating with remodeling and transcriptional complexes 13, 14, 15.

During the advent of next generation sequencing, an important observation was made while investigating the role of STAT factors in mediating gene expression epigenetically. It was noted that there could be four cases; 1) STAT factors were responsible for local epigenetic status and transcription by associating with histone modifying complexes 2) Transcription was STAT- dependent but local epigenetic status was not 3) Local epigenetic status was STAT-dependent, but transcription was not and 4) Neither transcription nor local epigenetic status was STAT-dependent

16. This hypothesis was generated by data obtained through Chromatin Immunoprecipitation sequencing (ChIP seq.), wherein regions of the DNA bound by STAT factors were sequenced 17.

To further complement this, enrichment of histone marks and transcriptional complexes was also

132 evaluated, with loss of STAT factors significantly de-regulating histone modifications and association of certain transcriptional complexes within the genome 18, 19. Intriguingly, over time,

IkZF proteins were also shown to associate with similar transcriptional complexes.

Therefore, does epigenetic regulation (by IkZF factors) of TH1 and TFH cells rely on the complex formation between IkZF and STAT factors, with IkZF/STAT modules necessary to modify histone composition and recruitment of transcriptional and remodeling complexes, or can such changes be exerted by IkZF factors independent of STAT proteins?

As such, by looking at enrichment for STAT and IkZF factors in TH1 and TFH cells, along with permissive and repressive histone marks, global transcriptional regulators and remodeling complexes would show us the mechanisms by which IkZF and STAT modules influence flexibility

13, 14, 20, 21, 22, 23 . Performing similar ChIP seq. assays on TH1 and TFH cells lacking Eos and Aiolos would inform us the dependency of these factors in regulating gene expression epigenetically. It’s also important to note that STAT factors can promote development by negatively regulating gene expression 19. Therefore, overlaying ChIP seq. data with data showing differential gene expression between TH1 and TFH cells (obtained via. RNA sequencing) would inform as to whether STAT and IkZF factors promote or impede gene expression.

Although Eos deficiency impacted TH1 development significantly, we observed only modest effects in genome accessibility. While further studies are required to assess the epigenetic role Eos has on TH1 development, this would suggests that other mechanisms might exist by which IkZF factors modulate T helper cell development.

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Non-transcriptional mechanisms of IkZF factors mediating T-helper cell flexibility

Interestingly, the presence of Eos enhanced STAT5 activation in TH1 cells. In addition, enrichment of STAT5 binding at signature TH1 gene such as Prdm1, Ifng and Il2ra was greater in the presence of Eos. Thus an additional mechanism by which Eos modulates STAT5 activity in TH1 cells is through enhancing or maintaining STAT5 activity. There are potentially two ways in which Eos could achieve this; Eos could be repressing the induction of phosphatases, such as the Suppressor

Of Cytokine Signaling (SOCS) protein family or Dual Specificity Phosphatases (DUSPs), which are known to regulate STAT activity in T-helper cells 24, 25. Secondly, as with Aiolos and STAT3, we observed a similar complex protein complex forming between Eos and STAT5. Thus, this complex formation might play a crucial role in STAT5 activity, as Eos/STAT5 complexes could physically prevent phosphatases from binding to and attenuating STAT5 activity 26, 27. One method by which this hypothesis could be tested is by treating TH1 cells with known phosphatase inhibitors and evaluating levels of STAT5 activation in Eos deficient cells.

Finally, since we have observed Eos modulating STAT5 activity in TH1 cells, and given the conserved nature within members of IkZF and STAT factors, another exciting area for investigation is whether Aiolos has similar effects on STAT3 activity in TFH cells and if such modulation follows similar regulatory mechanisms as seen with Eos and STAT5.

134

IkZF/STAT factors modulating flexibility of other T-helper cell subsets

STAT factors are crucial for T-cell development and play major role in regulating plasticity among

T-helper cell subsets. Based on environmental cues, activated members within the STAT family

(STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6) translocate into the nucleus where they can regulate gene profiles within T-helper cells 28. Our data so far show that IkZF proteins form unique protein complexes with STAT factors to significantly regulate cytokine induced T-helper cell development. Given the conserved nature of members within the STAT and

IkZF family of transcription factors, it would be prudent to evaluate whether additional

IkZF/STAT complexes mediate cytokine dependent plasticity not only in T-helper cell subsets but also in other cells that exist within the immune system (Figure 5.1). In conclusion, investigating the molecular and epigenetic mechanisms by which IkZF/STAT complexes regulate T-cell plasticity would allow us to devise better therapeutic strategies in the advent of autoimmune diseases.

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IL-6 IL-21 IL-2 IL-12 IL-2 IL-4 IL-6 TGFβ IL-2 TGFβ

STAT5 STAT4 STAT5 STAT6 STAT3 STAT5 STAT3

SMAD2/3

STAT6 STAT3 STAT5 STAT5 STAT4 STAT3 STAT5 SMAD2/3

Eos Ikaros Helios Ikaros Aiolos Eos Helios Aiolos Ikaros

TH1 TH2 TH17 TREG TFH

Figure 5.1. STAT and IkZF regulatory networks underlying the expression of T helper cell gene programs. This schematic depicts representative cytokines implicated in the development of murine T helper cell populations, their downstream STAT signaling pathways, and the Ikaros zinc finger transcription factors recently associated with the differentiation and functions of each subset. A recent study demonstrated that the IkZF factor Aiolos cooperates with STAT3 to induce the expression of Bcl6, the lineage-defining transcription factor for the TFH cell type. The differential expression and/or activity of IkZF and STAT factors across T helper cell subsets is suggestive of the intriguing possibility that additional, cell type-specific STAT/IkZF regulatory modules may regulate T helper cell differentiation decisions across subsets.

136

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