BACTERIAL EXPOSURE AND IMMUNE HOMEOSTASIS:

A MECHANISTIC VIEW OF THE HYGIENE HYPOTHESIS

By

JENNY LYNN JOHNSON

Submitted in fulfillment of the requirements for the

Degree of Doctor of Philosophy

Thesis Advisor: Dr. Brian A. Cobb, Ph.D.

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

May 2015 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

Jenny Lynn Johnson

Candidate for the degree of Doctor of Philosophy*

Committee Chair

Eric Pearlman

Committee Member

George Dubyak

Committee Member

Kristie Ross

Committee Member

Brian Cobb

Committee Member

Clive Hamlin

Date of Defense

March 16, 2015

*We also certify that written approval has been obtained for any proprietary material contained therein

2 Dedication

I would like to dedicate this work to those who have supported me through the six years

I have spent at CWRU, and in particular my parents, Mark and Sharon Johnson, and my cousins, Janice, David and Henry Graves.

3 Table of Contents

List of Tables ...... 7

List of Figures ...... 8

Acknowledgements...... 10

List of Abbreviations ...... 12

Abstract ...... 15

Chapter 1: An Introduction to Bacteroides fragilis, PSA, and Asthma ...... 17

1.1 Hygiene Hypothesis ...... 18

1.2 Intro to Asthma ...... 21

1.3 Currently available therapeutics ...... 22

1.4 Intro to Bacteroides fragilis ...... 24

1.5 Polysaccharide A ...... 25

1.6: Immunomodulation by Bacteroides fragilis and Polysaccharide A ...... 26

Chapter 2: Clonal T cell Expansion Induced by PSA Treatment ...... 28

2.1: Summary ...... 29

2.2: Introduction ...... 30

2.3: Methods ...... 31

2.3.1: Mice and Bacteria ...... 31

2.3.2: Airway Inflammation Model ...... 31

2.3.3: T cell Flow Cytometry...... 32

2.3.4: Histology ...... 32

4 2.3.5: Deep Sequencing ...... 33

2.3.6: General Data Analyses ...... 33

2.4: Results ...... 33

2.4.1: Phenotypic Description of PSA Responding T cells ...... 33

2.4.2: Analysis of TCR from PSA responding T cells via Deep Sequencing ...... 34

2.4.3: Clonal T cell Expansion by PSA ...... 36

2.4.4: Asthma Induction is Inhibited by PSA-expanded T Cells...... 39

Chapter 3: Polysaccharide A from Bacteroides fragilis Inhibits Asthma Induction ...... 42

3.1: Summary ...... 43

3.2: Introduction ...... 44

3.3: Methods ...... 46

3.3.1: Mice and bacteria ...... 46

3.3.2: Asthma models ...... 46

3.3.3: Sterile T cell sorting ...... 47

3.3.4: Histology ...... 47

3.3.5: General data analysis ...... 48

3.4: Results ...... 48

3.4.1: Oral PSA exposure protects against OVA-induced airway inflammation ...... 48

3.4.2: CD4+ T cells drive PSA-mediated asthma protection ...... 51

3.4.3: PSA responding T cells are not traditional Foxp3+ regulatory T cells ...... 55

5 3.4.4: PSA mediated asthma inhibition is IL-10 dependent ...... 56

Chapter 4: Novel T cell-T cell Interaction Promotes Immune Homeostasis ...... 58

4.1: Summary ...... 59

4.2: Introduction ...... 59

4.3: Methods ...... 61

4.3.1: Mice and Bacteria ...... 61

4.3.2: Asthma model ...... 61

4.3.3: Sterile T cell sorting ...... 62

4.3.4: Cell Culture ...... 62

4.3.5: Histology ...... 62

4.3.6: Flow analysis ...... 63

4.3.6: General data analysis ...... 63

4.4: Results ...... 63

4.4.1: Suppressive IL-10 Production is External to PSA Experienced T cells ...... 63

4.4.2: PSA Experienced T cells induce IL-10 Production in Foxp3+ T cells ...... 64

4.4.3: A Soluble Molecule Mediates Effector Memory T cell-Regulatory T cell Interaction .. 68

4.4.4: Soluble Mediator from Activated TEM Cells Induces IL-10 Production in vivo ...... 69

4.4.5: Activated TEM cells Inhibit Asthma Induction ...... 70

Chapter 5: Discussion and Future Directions ...... 72

6 List of Tables Chapter 3: Clonal T cell Expansion Induced by PSA Treatment

Table 1: Sequencing information generated through next generation sequencing...35

Table 2: Compilation of TCRβ CDR3 zwitterionic sequences...... 40

7 List of Figures

Chapter 2: Clonal T cell Expansion Induced by PSA Treatment

Figure 1: PSA immunization promotes an anti-inflammatory phenotype in CD4+ T

cells...... 34

Figure 2: Vβ and J segment usage does not vary with PSA immunization...... 36

Figure 3: CDR3 loop length remains consistent with PSA immunization...... 37

Figure 4: PSA immunization induces clonal proliferation of CD4+ T cells...... 38

Figure 5: PSA and OVA clonally expand CD4+ T cells with a similar sequence

frequency...... 39

Figure 6: PSA immunization increases zwitterionic motifs within the TCRβ CDR3

loops...... 39

Figure 7: PSA clonally expanded T cells inhibit asthma induction...... 41

Chapter 3: Polysaccharide A from Bacteroides fragilis Inhibits Asthma Induction

Figure 8: Oral PSA inhibits asthma induction...... 50

Figure 9: CD4+ T cells drive PSA suppressive activity...... 52

Figure 10: PSA inhibits multiple etiologies of asthma...... 53

Figure 11: PSA suppression is not driven by Foxp3+ Tregs...... 54

Figure 12: Foxp3 is not induced by oral PSA treatment...... 55

8 Figure 13: IL-10 is required for PSA mediated suppression...... 57

Chapter 4: Novel T cell-T cell Interaction Promotes Immune Homeostasis

Figure 14: IN-10n PSA experienced T cells inhibit asthma induction...... 64

Figure 15: WT PSA experienced T cells fail to inhibit asthma induction in IL-10n

recipients...... 65

Figure 16: PSA experienced T cells induce IL-10 production in Foxp3+ Tregs...... 66

Figure 17: TEM cells promote synergistic IL-10 production in vitro...... 67

Figure 18: TEM-Treg interaction is facilitated through a soluble mediator...... 68

Figure 19: Conditioned TEM media promotes IL-10 production in vivo...... 69

Figure 20: TEM cells activated in vitro inhibit asthma induction...... 71

9 Acknowledgements

I could not have accomplished all that I have during my time in Cleveland without the immense level of support I received from family, friends, lab members, and my faith community.

To start, I would like to thank my advisor Dr. Brian Cobb for all of the support and guidance he has given me. I entered your lab as an excited, naïve student, and your training and perseverance have helped to shape me into a skilled and inquisitive researcher ready to take on new questions and new problems. I can’t thank you enough for the time spent in your lab.

All past and present members of the Cobb lab have contributed to the completion of my thesis through their support, teaching, and cooperatively. You were all a sounding board, teacher, and coworker at times, and I can’t thank you enough for it. Thank you so much Sean, Kari, Fan, Colleen, Lili, Lori, Amruth, Mark, Janice, and Doug for all your help.

I would also like to add a large thank you to my thesis committee, Dr. Eric Pearlman, Dr.

George Dubyak, Dr. Kristie Ross, and Dr. Clive Hamlin, for your continued support and guidance.

Additional thanks to fellow graduate students, both within the Pathology department and around the School of Medicine. The many social events outside of lab helped to keep me sane and keep my priorities straight. I would like to particularly thank Jessica and Lana for all the dinners and gatherings we have had in the last six years. I’m glad we

10 got to experience it all together. Also, a big thank you goes out to members of the

Abbott lab for all of the social interactions and making sure our lab was never quiet for too long.

The wonderful faith community I became a part of in 2009 at Grace Lutheran Church has made my journey toward a PhD a blessed one. There are too many individuals to thank specifically, but I loved being a part of this congregation and I thank them for their support.

Lastly, I would like to thank my family for their continued support of my academic and research endeavors. My love of science clearly came from my father, Mark Johnson, and my mother, Sharon Johnson, has always supported my decision and passion for answering the interesting questions. My brothers, Mark Johnson and Stuart

Schoonover, may be located far away, but I know that I have their support, and my sister from another mother, Erika Popp, has been there whenever I need to vent or celebrate. My large extended family has continuously been behind me, especially

Janice, David, and Henry.

I could not have finished this incredible journey without any of you. Thank you.

11 List of Abbreviations

APC: antigen presenting cell

BAL: bronchoalveolar lavage

CDR3: complementarity determining region 3

CLIP: class-II associated invariant chain peptide

CRA: cockroach antigen

EAE: experimental autoimmune encephalomyelitis

ELISA: enzyme-linked immunosorbent assay

FEV1: forced expiratory volume

GlyAg: glycoantigen

H&E: hematoxylin and eosin

HDM: house dust mite

HLA-DR2: human leukocyte antigen-DR2

IBD: inflammatory bowel disease

IHC: immunohistochemistry

Ii: Invariant chain

IL-10n: IL-10 deficient mice

12 IN: intranasal iNKT: invariant natural killer T cells

IPEX: immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

LPS: lipopolysaccharide

MHCII: major histocompatibility complex 2

MPO: myeloperoxidase

NO: nitric oxide

OVA: ovalbumin

PFA: paraformaldehyde

PSA: Polysaccharide A

RSV: respiratory syncytial virus

SPF: specific pathogen free

TCM: central memory T cells

TCR: T cell receptor

TEM: effector memory T cells

Th1: T helper cell type 1

Th2: T helper cell type 2

13 TLR2: Toll like receptor 2

TN: naïve T cells

Treg: regulatory T cells

ZPS: zwitterionic capsular polysaccharides

14 Bacterial Exposure and Immune Homeostasis:

A Mechanistic View of the Hygiene Hypothesis

Abstract

by

JENNY LYNN JOHNSON

Recently, drastic increases in the prevalence of allergic diseases and asthma have been observed in western societies. This dramatic increase has been tied to an increased emphasis on sterility and a lack of early bacterial exposure, a phenomenon known as the hygiene hypothesis. Bacteria colonization or bacterial antigens to modulate the immune response has been clearly shown; however, the mechanism behind these changes is not understood, and previously published work has little connection with human diseases known to be influenced by bacterial exposure. Utilizing the capsular polysaccharide PSA from the commensal bacterium Bacteroides fragilis, we strove to understand the alterations to the immune system following bacterial exposure, how that affects peripheral immune homeostasis, and identify potential mechanisms for the hygiene hypothesis.

Prior work has identified CD4+ T cells as the responding population to Bacteroides fragilis and PSA, we examined responding T cells following PSA treatment. Through phenotypic and functional analysis we identified this population as effector memory

Low (TEM) CD45Rb cells with increased IL-10 production. Next generation sequencing

15 novelly demonstrated that polysaccharide antigens are capable of clonally expanding T cells, similarly to a protein antigen. To study these cells in the context of a condition associated with the hygiene hypothesis, we utilized a murine model of asthma. We showed PSA capable of suppressing multiple etiologies of asthma through CD4+ T cells induced by PSA in an IL-10 dependent manner; however, further analysis revealed the source of suppressive IL-10 to be tissue resident Foxp3+ regulatory T cells (Tregs), and not the PSA induced T cells. Analysis of in vitro and in vivo data showed PSA expanded

TEM cells interact with Tregs through a soluble mediator.

Therein, we show one potential mechanism for the hygiene hypothesis in which T cells activated by a bacterial polysaccharide in the gut microenvironment inhibit peripheral inflammation. Additionally, we show the first evidence that polysaccharide A from

Bacteroides fragilis induces clonal proliferation of T cells, similar to conventional protein antigens. Understanding how T cell interactions can modulate immune homeostasis yields yet another platform for developing novel therapeutics for multiple inflammatory conditions, including asthma.

16 Chapter 1: An Introduction to Bacteroides fragilis, PSA, and Asthma

17 1.1 Hygiene Hypothesis

The impact of bacterial exposure on health and disease prognosis has become a hot topic for research in the last decade; however, work on understanding the mechanism, or mechanisms, by which the bacteria interacts with our immune system has been lacking. The idea that bacterial exposure can promote good health and decrease inappropriate immune activation was proposed in 1989 as the hygiene hypothesis (1).

This seminal paper described instances of hay fever decreasing in families with older siblings, and therefore, increased bacterial exposure. This finding has been expanded in the intervening years to show a reduced incidence of allergic disease in adulthood following early exposure to various bacterial sources.

Perhaps the most widely reported of these correlations is the comparison of children born vaginally versus those delivered by cesarean section. Children who experience an auto-colonizing event through vaginal delivery, as opposed to a sterile delivery through cesarean section, are shown to have higher serum levels of IL-10 and colonic flora that closely resembled the maternal flora (2). Sterilely delivered counterparts had higher levels of IFNγ and IL-13 following antigen challenge, indicating a pro-inflammatory basal state. Additionally, cesarean delivered children are more susceptible to respiratory syncytial virus (RSV) related hospitalization, a common precursor to a diagnosis of asthma (3).

After birth, bacterial exposure at a young age continues to be important for immune development and overall health. Antibodies generated in response to bacterial antigens

18 have been shown to have protective effects against developing asthma (4), and have been implicated in heritable non-chromosomal modifications (5). Early exposure to bacteria, in particular bacteria found in a farm environment, promotes a similar altered pattern of methylation in genes associated with asthma and other allergic conditions

(6). Exposure to a farm with livestock and raw milk has also been frequently associated with protection from allergic diseases (7-9), but the most beneficial antigen, or set of antigens, has yet to be identified. Early colonization, from vaginal delivery, to farm exposure, and even to the method of cleaning a pacifier (10) appears to be key in gaining any beneficial immune responses; however, the cellular mechanism of this interaction has yet to be completely defined.

While mechanistic studies in mouse models have lagged behind correlative human work, studies comparing germ-free mice to specific pathogen free (SPF) mice are beginning to show evidence of the impact of bacterial exposure on immune development and homeostasis. In one of these studies, it was found that specific pathogen free (SPF) mice have lower numbers of invariant natural killer T (iNKT) cells within the colon, lung, and liver than their germ-free counterparts (11). The accumulation of these cells can lead to increased susceptibility to inflammatory models, suggesting that by decreasing the number of iNKT cells within these tissues, the microflora is helping to suppress inappropriate immune activation. Interestingly, the numbers of iNKT cells were only able to be restored to normal SPF levels when germ- free mice were colonized as neonates. Exposing germ-free adult mice to SPF conditions

19 resulted in no change (11), illustrating the same importance of early exposure for immune benefit as seen in human studies.

Following this early period of colonization, manipulation of microbiota has proven to be detrimental. For example, even one course of oral antibiotics can cause drastic changes within an individual’s microbiome which are not completely reversed back to the flora composition seen prior to antibiotic treatment (12-14). While studies have shown a rise in antibiotic resistant bacteria, the changes seen in patient flora following antibiotic use is another concerning factor when studying the effects of antibiotics on long term immune homeostasis, and has yet to be addressed in mouse models with colonization following antibiotic use.

It is important to note that bacterial antigens are not the only antigens of importance in immune development and immune suppression. Infections with viruses or helminths have both been shown to be beneficial in the induction of regulatory T cells (Tregs) and resistance to additional viral infections (15) or inflammatory disorders such as diabetes

(16), a concept known as concomitant immunity (15). In addition to their own antigens, helminths promote generation of regulatory T and B cells, modulate CD11cLoCD103- dendritic cells, and modify signaling from epithelial cells (17,18). While the distinct mechanism, or set of mechanisms, by which bacterial exposure can promote immune homeostasis remains unclear, the wealth of existing studies highlights the importance of this phenomenon and a better understanding of the cellular mechanisms involved in the

20 hygiene hypothesis could lead to novel therapeutics and better preventative measures for immune mediated conditions.

1.2 Intro to Asthma

One condition that has most frequently been associated with hygiene hypothesis studies is asthma. Historically, asthma has been characterized as a chronic inflammatory disorder with activation of Th2 (T helper type 2) cells causing reversible airway obstruction and a tightening of the smooth muscles of the airway. The cytokines released by Th2 cells, IL-4, IL-5, IL-9, and IL-13, play a critical role in the development of asthma, influencing the generation of antigen-specific Th2 cells, IgE class switching, infiltration of eosinophils, and production of mucus (19,20). Peripherally, the percentage of B cells expressing CD23, the low affinity IgE receptor, is increased in asthmatic patients, and soluble fragments of CD23 have been correlated with increases in serum IgE levels, as well as severity of lung inflammation (21). Structural changes, such as increased smooth muscle mass, deposition of extracellular matrix proteins, mucus production, and epithelial hyperplasia are also seen in asthmatic patients.

Various genetic and environmental factors have been linked to the onset of asthma. A genetic predisposition to atopy and/or hyperresponsiveness, obesity, and gender have been shown to play a roll in addition to environmental factors such as allergens, viruses, occupational hazards, tobacco smoking, air pollution, and diet (22).

More recently, asthma is being recognized as a heterogeneous condition. In addition to the traditional, allergic manifestation of asthma, female patients with a more mild

21 disease have reduced eosinophil counts but increased neutrophil counts in sputum and respond better to inhaled steroids when compared to atopic patients (23). Patients with asthmatic symptoms despite no detectible increase in eosinophils were first described in 1995 (24), and the lack of eosinophilic infiltration has been shown to be consistent for at least 5 years, indicating that the lack of an allergic response is not simply a reduction at the initial time of measurement (25). Neutrophilic asthma is also accompanied by an increase in sputum IL-8 levels over healthy controls and eosinophilic asthma patients (26), who do not have a comparable elevation in IL-5 or eotaxin, implying that the increase in IL-8 may not be solely responsible for the infiltrating neutrophils (27). Additionally, severe asthmatics, patients that are resistant to corticosteroid therapy, can be separated into two main groups based on eosinophil infiltration. Patients without eosinophilic infiltration have less subbasement membrane thickening, lower number of cells infiltrating into the sputum, and fewer required intubations; however, these patients had slightly higher airway obstruction as measured by forced expiratory volume (FEV1) (28). The variation seen in exacerbation triggers, cellular involvement, and extracellular remodeling makes diagnosis and treatment extremely complex, and further work is needed to develop a treatment option that would benefit multiple subtypes of asthma.

1.3 Currently available therapeutics

Approximately 300 million people worldwide are thought to have asthma, and the

World Health Organization has calculated asthma to be 1% of global disease burden

(22). Asthma has been cited as the most common reason for school absences and

22 missed work days. While medical costs for treating asthma continue to be high, not managing asthma results in more emergency room visits and more missed work days, leading to a higher cost (22).

Current treatments for asthma include quick acting reliever medications, such as bronchodilators, and long term controller medications, such as corticosteroids. These treatments cannot cure the disorder, do not prevent a decrease in lung function over time, and up to 30% of patients, severe asthmatics, never reach optimum control with these medications (29). Much of the recent research has been toward novel treatment options for severe asthmatics. Initial treatment of severe asthmatic patients consists of inhaled corticosteroids in combination with a long-acting β-agonist, which has been shown to reduce the needed dose of inhaled corticosteroids. If symptoms worsen, or if these therapies are ineffective, patients are then given oral corticosteroids, which have more potent side effects and few available alternatives. Researchers have examined additional treatment options for these patients, including anti-IgE and anti-cytokine therapies, but few have shown effectiveness in large populations. Bronchial thermoplasty, a procedure recently approved by the FDA, involves the application of radial heat during a bronchoscopy to reduce smooth muscle mass which accumulates during the remodeling phase of asthma. While this therapy has shown improvement in quality of life and a reduction in the severity of symptoms, it does not address the cellular causes of inflammation. In recent years, work has been done to further understand the cause of asthma and then to treat the cause with biological, rather than pharmacological, methods, such as stem cell therapy (30).

23 1.4 Intro to Bacteroides fragilis

One commensal bacterium with a proven immunomodulatory effect, and as of yet no direct connection with the hygiene hypothesis, is the gram negative organism

Bacteroides fragilis, which can comprise up to 1% of the colonic flora of colonized individuals. Composed of 8 different polysaccharides, the bacterial capsule can vary greatly between individual bacteria, with the potential of 256 unique combinations (31).

Each polysaccharide stems from a separate locus, which are under the control of a single promoter and contains between 11 and 21 genes. The regulation of the majority of these loci, seven of the eight, is maintained by the reversible inversion of the DNA containing the promoter, creating a phenotype known as phase variation. A 1975 report by Dennis L. Kasper and Marcel W. Seiler was the first to suggest that large polysaccharides, along with a smaller sugar, probably lipopolysaccharide (LPS), were responsible for the antigenicity of B. fragilis (32), and later work showed circulating antibodies specific for capsular polysaccharides (33). More recent work has shown that the expression of more than one polysaccharide better enables colonization of the intestine, which corrects the skewed Th1/Th2 ratio among CD4+ T cells in germ-free mice (31), although the impact of B. fragilis on T cells was realized much earlier.

Bacteroides fragilis was identified as the most common anaerobic isolate found in abscesses in humans with bacteremia, and immunization with the capsular polysaccharide was found to protect rats against abscess formation (34). This protection was able to be transferred through splenic T cells from immunized rats

(35,36), and further work confirmed that CD4+ T cells were responsible for the

24 prevention of abscesses (37) and surgical adhesions (38,39) following immunization with

B. fragilis capsular polysaccharide or colonization with B. fragilis.

1.5 Polysaccharide A

Of the 8 capsular polysaccharides on B. fragilis, one has been more intensively studied due to its immunomodulatory capacity. Polysaccharide A, or PSA, is comprised of a tetrasaccharide repeating unit consisting of 2,4-dideoxy-4-amino-D-N-acetylfucose, D-N- acetylgalactosamine, D-galactopyranose, and D-galactofuranose with a 4,6-pyruvate attached to the galactopyranose (40). These repeating units form an extended right- handed helix with positive and negative charges alternating on the outer surface, created by the zwitterionic motif of the carboxyl group of the 4,6-pyruvate and the amino group of the 2,4-dideoxy-4-amino-D-N-acetylfucose (31,40,41). Several other zwitterionic polysaccharide antigens have been identified, and this class of molecules has been shown to activate CD4+ T cells through MHCII presentation, which was specifically shown for PSA in 2004. Antigen presenting cells (APCs) endocytose PSA and upon external ligation of toll like receptor 2 (TLR2) by PSA or other TLR2 ligand, a nitric oxide (NO) burst processes the polysaccharide from an initial size of greater than

100kDa to 5-10kDa. These endosomes containing cleaved PSA then fuse with lysosomes, which then fuse with exocytic vessels containing the MHCII machinery, including HLA-DR, HLA-DM, and invariant chain (Ii). From this point, PSA is loaded into

MHCII similar to protein antigens, whereby CLIP (class-II associated invariant chain peptide) is displaced by the polysaccharide and the PSA-MHCII complex is then trafficked to the cell surface for presentation (42).

25 In addition to being dependent on TLR2 engagement and NO production for processing, presentation of PSA is dependent on the glycosylation status of MHCII. N-linked glycans are thought to be important in protecting MHCII complexes from degradation, but for zwitterionic polysaccharide antigens these glycans have been shown to be important in presentation and binding to MHCII. MHCII HLA-DR2 (human leukocyte antigen-DR2) contains three N-linked glycosylation sites, with one, N78, being associated with the peptide binding groove. These glycosylation sites had been previously shown to be irrelevant in peptide presentation; however, when these sites were altered to change their glycosylation status, whether through chemical manipulation or genetic mutation, glycoantigen presentation, but not peptide presentation, was impeded (43,44).

1.6: Immunomodulation by Bacteroides fragilis and Polysaccharide A

B. fragilis and purified PSA have been studied as an agent of immunomodulation for 30 years. B. fragilis, has been shown to protect against induction of experimental autoimmune encephalomyelitis, EAE, (45), and PSA was shown to be critical for this protection (45). Similarly, B. fragilis was able to protect against the induction of, as well as the treatment of, experimental colitis (46), and was able to prevent abscess formation (39). Furthermore, anti-inflammatory responses were seen in vitro when human CD4+ T cells were treated with PSA (47). This connection between altering immune conditions in mice and modulating T cell responses in cultures of human cells suggests that perhaps PSA could be a potential therapeutic, but even more fundamental, could be one of the extrinsic molecules involved in immune homeostasis.

26 PSA is part of a larger family of carbohydrate antigens referred to as zwitterionic capsular polysaccharides, or ZPS (40). Although multiple polysaccharides, such as most

Streptococcus pneumonia capsular polysaccharides, Vi antigen from Salmonella typhi, and the capsule from group B Streptococcus, contain negative charges within their repeating unit (48-50), the presence of alternating positive charges, such as those seen in PSA, CP1 from Streptococcus pneumoniae, and types 5 and 8 polysaccharides from

Staphylococcus aureus, are necessary for T cell dependent responses (39,51). While PSA has been the most widely studied, other ZPS family members have shown in vivo efficacy. For example, CP1 was shown to prevent formation of surgical adhesions (39).

Further analysis of the interactions between ZPS family members and the immune system could yield potential mechanisms of the hygiene hypothesis, leading to novel therapeutics for a wide range of inflammatory conditions.

27 Chapter 2: Clonal T cell Expansion Induced by PSA Treatment

28 2.1: Summary

Despite the description of MHCII presentation of processed fragments of zwitterionic polysaccharides to T cells, the view of MHCII-dependent antigen presentation has been completely dominated by peptide antigens. Additional studies have established polysaccharide A (PSA) from the capsule of Bacteroides fragilis as a potent activator of

CD4+ T cells which participate in key biological processes, including the maintenance of immunological homeostasis. The phenotype of these responding T cells and the nature of the TCR-MHCII complex remains poorly defined. In this study we utilize next generation sequencing of the TCR of CD4 T cells from PSA treated mice compared with protein antigen and untreated controls. We found that oral treatment with PSA induced clonal expansion of a subset of potently suppressive CD4+CD45RbLow effector memory T cells. Additionally, analysis of the complementarity determining region 3 (CDR3) revealed a lack of specific variable and joining region use with average CDR3 loop length. An increased percentage of CDR3 loop sequences containing a zwitterionic motif were found in PSA expanded clones, which correlates with the requirement for a similar motif within PSA to enable presentation and T cell activation. Together these data frame a model in which PSA, and potentially other T cell dependent glycoantigens, clonally expands specific CD4+ T cells with an increased preference for dual-charged

CDR3 loop sequences interacting with dual-charged PSA.

*Work featured in this chapter was published in The Journal of Biological Chemistry

(Johnson JL, Jones MB, and Cobb BA; 2015, 290(8): 5007-5014 (ref# 52))

29 2.2: Introduction

Cellular responses to polysaccharide antigens remain controversial, even with data documenting T cell responses and immunomodulation in multiple model systems since

1985 (39,45,46,53). The gram negative commensal Bacteroides fragilis, and in particular the capsular Polysaccharide A (PSA), has been shown to exert immunosuppressive effects in vivo in various murine models as well as within in vitro human studies through

CD4+ T cells (39,45-47,54). While the PSA responding T cells are known to regulate inflammation, little is known about phenotypic markers and TCR-MHCII complex within this population.

Similarly, the current understanding of regulatory or suppressive T cell populations is limited. Surface phenotypic analysis of Treg populations and utilizing Foxp3 reporter mice have given information about the origin of these cells and their potential to regulate inflammation (55); additionally, the detrimental effects resulting from the lack of Foxp3+ cells in immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) patients confirms the necessity of regulatory populations (56).

However, the amount of antigen recognition and specificity of regulatory cells is still not well understood. Traditional techniques used to isolate and expand clonal populations of T cells have proven futile when the responding cell population exhibits a suppressive phenotype. In this work, we show that oral PSA treatment induces a population of CD4+

T cells that phenotypically match previously published human data (47). Furthermore, for the first time we show that a polysaccharide antigen is capable of clonally expanding

T cells in a manner comparable to canonical protein antigens.

30 2.3: Methods

2.3.1: Mice and Bacteria

Breeding pairs for WT C57Bl/6 mice were obtained from Jackson labs, and housed in specific pathogen free (and B. fragilis free) conditions as stipulated by the guidelines of the Institutional Animal Care and Use Committee of Case Western Reserve University in

Cleveland, OH. Experimental mice were 7-12 weeks of age. B. fragilis was grown in anaerobic conditions, and PSA was purified as previously described (42,57). Similar to previously published studies (45), all oral treatments were given as oral gavages over 12 days (100 μg/dose in saline every 3 days). Negative controls were given saline only.

OVA immunized mice were given 100 μg/dose OVA (Sigma) in alum intraperitoneally on days 0 and 7 with splenocytes collected on day 14.

2.3.2: Airway Inflammation Model

WT mice were sensitized to OVA by intraperitoneal doses of 100 μg of OVA (Sigma) in alum on days 0 and 7. On day 14, the mice received intranasal OVA (40 μg/dose in PBS

(Sigma) for 6 consecutive days before being killed on day 7. Mice were anesthetized for intranasal challenge using a tabletop anesthesia system (VetEquip) with 3% isoflurane

(Baxter). To asses T cell function in vivo, splenic CD4+ T cells were purified from PSA- or saline-treated mice with magnetic resin (Miltenyi Biotec), and 2x106 were adoptively transferred via intravenous tail vein injection 24h prior to intranasal OVA administration. Following intranasal challenges, mice were euthanized using a mixture of 8.6% ketamine (Fort Dodge Animal Health), 1.7% xylazine (AnaSed), and 2.9%

31 acepromazine (Boehringer Ingelheim) diluted in sterline saline at 0.006 cc/g. Next mice were tracheotomized, and lungs were flushed three times with a 1 mL of PBS containing

0.6 mM EDTA. Cells from these washes were pooled and differentials were obtained through Cytospin and microscopic counting. Right lungs were inflated with O.C.T.

(Tissue-Tek) and fixed in 10% formalin prior to paraffin embedding and sectioning at the

Case Western Reserve University Tissue Procurement and Histology Core Facility.

2.3.3: T cell Flow Cytometry

CD4+ splenocytes were purified as described above and stained with CD4, CD25,

CD45Rb, CD62L, and CD44 (eBiosciences). For in vitro stimulations, CD4+ T cells were stimulated for 3 days with anti-CD3/CD28 resin (eBiosceince). Cytokine levels were assessed by standard sandwich ELISA (BioLegend). Flow cytometric analysis was performed on a BD Accuri C6 flow cytometer using FCS Express software (De Novo

Software).

2.3.4: Histology

H&E staining of lung sections was performed by the Case Western Reserve University

Tissue Procurement and Histology Core Facility. Unstained slides were stained with antibodies specific for EpCAM (epithelial cell adhesion molecule, 6 μg/mL, eBioscience) and myeloperoxidase (1:100 dilution, Abcam; anti-rabbit secondary, 1:1000 dilution,

Invitrogen).

32 2.3.5: Deep Sequencing

CD4+ splenocytes (106) were purified as before from PSA-treated, OVA-treated, or untreated (saline) mice and sent to Adaptive Biotechnologies for RNA extraction and deep sequencing of the TCR-β chain using their proprietary assay platform, as reported elsewhere (58). Data analysis was performed using Adaptive Biotechnologies immunoSEQ web tools and graphed using GraphPad Prism (version 5.0).

2.3.6: General Data Analyses

All data are shown as mean ± SEM. Mice included a minimum of four animals per group per replicate experiment. Graphs and statistical measures were generated with

GraphPad Prism (version 5.0). For comparisons between multiple groups, analysis of variance was used, whereas for comparisons between two groups (where appropriate) the Student’s t test was used.

2.4: Results

2.4.1: Phenotypic Description of PSA Responding T cells

In order to analyze the phenotype of PSA responding T cells, WT C57Bl/6 mice were orally immunized with 100 μg of PSA five times over 12 days, as reported previously

(59). CD4+ T cells were harvested from both the spleen and mesenteric lymph nodes for flow cytometric analysis or restimulated with anti-CD3/CD28 resin to examine cytokine output. The bulk CD4+ T cell populations from PSA-treated mice were skewed away from IFNγ production and toward IL-10 production when compared to saline-treated controls (Figure 1A). Analysis of surface phenotypic markers indicated that PSA

33 treatment expanded a population of CD45RbLow T cells, with many of these also expressing the IL-2 receptor CD25, which are markers commonly found on regulatory T cells (Figure 1B). There was no change observed in CD25 among CD45RbHi cells (Figure

1B and D). Additionally, the majority of the CD4+CD45RbLow T cells (66.5%) exhibited an effector/memory phenotype (CD62LLowCD44Hi) (Figure 1C and D). Collectively these data show expansion of CD4+CD45RbLowCD62LLowCD44Hi T cells following murine oral

PSA exposure, which skews cytokine responses toward IL-10 production, consistent with our prior discoveries in humans (47).

Figure 1: PSA immunization promotes an anti-inflammatory phenotype in CD4+ T cells. WT mice were treated with PSA as before. CD4+ splenocytes were isolated, stimulated with ant-CD3 for 72hr, and cytokines were measured by ELISA (A). CD4+ splenocytes were stained for CD25, CD45Rb, CD44, and CD62L, and were analyzed by flow cytometry (B-D).

2.4.2: Analysis of TCR from PSA responding T cells via Deep Sequencing

To better define the TCR of PSA responding T cells, CD4+ T cells were purified from spleens of WT mice treated with either PSA, ovalbumin, as a conventional protein control, or saline, as a negative control. As described within the methods section, total

34 RNA was isolated from 106 CD4+ T cells, a library for deep sequencing was created for each mouse, and the β chain of the TCR was analyzed using targets sequencing. In each sample, over 700,000 total and 65,000 unique productive sequences were found (Table

1).

Naïve 1 Naïve 2 OVA 1 OVA 2 OVA 3 OVA 4 PSA 1 PSA 2 PSA 3 All Sequences 760,822 706,728 815,408 668,174 781,977 780,581 693,759 731,230 843,518 Unique Sequences 66,451 67,785 69,433 66,732 64,843 60,728 63,745 69,838 67,605 All Productive Sequences 755,927 702,065 810,186 633,761 777,296 774,838 689,321 726,419 837,352 Unique Productive 65,909 67,234 68,857 66,131 64,313 60,234 63,209 69,264 66,989 All Sequences with Stops 4,895 4,663 5,222 4,413 4,681 5,743 4,438 4,811 6,166 Sequences Unique Sequences with Stops 542 551 576 601 530 494 536 574 616

Table 1: Sequence information generated through next generation deep sequencing.

Analysis of the individual sequences showed no substantial shift in the use of a particular V or J segment regardless of stimulation (Figure 2), which aligns with the idea that, unlike super antigens, PSA and OVA, do not activate T cells based exclusively on germ-line sequences. Additionally, the length of CDR3 loops among all sequences in PSA and OVA immunized groups were identical to those in the saline group (Figure 3). This suggests that although MHCII presented PSA is considerably larger (5-10 kDa, (41,42)) than a traditional peptide (1-2 kDa, (60)), the CDR3 loop length remains a conventional length for PSA recognition.

35 Figure 2: Vβ and J segment usage dose not vary with PSA immunization. Sequences obtained through next generation deep sequencing were analyzed for Vβ and J segment usage. Mice were immunized with Saline (A; vehicle control), OVA (B), or PSA (C). N=2-4 mice per group

2.4.3: Clonal T cell Expansion by PSA

The ability of PSA (41,42,61) and other zwitterionic polysaccharides (62,63) to activate

CD4+ T cells through MHCII presentation has been well characterized; however, the degree of T cell clonal expansion and recognition of polysaccharide antigens remains controversial. To more clearly illustrate the clonality of the response against PSA, CDR3

36 Figure 3: CDR3 loop length remains consistent with PSA immunization. Sequences obtained through next generation deep sequencing were analyzed for CDR3 loop length. Data is shown as percent of total sequences and percent of unique sequences. Mice were immunized with Saline (A; vehicle control), OVA (B), or PSA (C). N=2-4 mice per group loop sequences from each sample were compared to distinguish those common to both naïve and immunized mice and those unique to each sample. A comparison of the two naïve samples (saline 1 versus saline 2) found 52,528 sequences unique to saline 1,

54,718 sequences unique to saline 2, and 13, 527 sequences found in both samples

(Figure 4A). Similarly, a comparison of a naïve sample (saline 1) with an ovalbumin- immunized sample (OVA) showed 55,830 sequences unique to saline 1, 48,181 sequences unique to OVA, and 12,415 sequences shared (Figure 4B). Lastly, a comparison of naïve with PSA-immunized samples (PSA 1, PSA 2, and PSA 3) revealed

37 55,295 sequences unique to saline 1, 50,340 sequences unique to PSA 1, and 12,950 sequences in common (Figure 4C). Equivalent numbers of sequences were seen with

Figure 4: PSA immunization includes clonal proliferation of CD4+ T cells. Individual clones were identified for each treatment group from sequences obtained through next generation deep sequencing. Each plot is comparing two individual mice. the other saline and PSA sample comparisons. To attain a better understanding of the most abundant clones, the top 50 sequences of the combined cohorts (saline, OVA, and

PSA) were examined. For both of the immunized groups, a small number of clones were significantly increased above the background in the saline group, suggesting specific clonal expansion (Figure 5). The CDR3 loop sequences of the top clones is consistent with the lack of significant differences in total CDR3 loop length (Figure 3). Further analysis of the amino acid sequences of the top 50 clones interestingly showed a significantly higher proportion of zwitterionic motifs containing both negatively charged acidic residues and positively charged basic residues in the PSA samples as compared to both the OVA samples and the saline samples (Figure 6 and Table 2). This suggests that

38 electrostatic interactions may play a role in

the TCR recognition of PSA since a similar

charge motif is required on PSA for T cell

activation.

Figure 5: PSA and OVA clonally expand CD4+ T cells with a similar sequence frequency. Individual clones were identified for each treatment group from sequences obtained through next generation deep sequencing, and the percent sequence frequency for each clone was determined. N=2-4 mice per group.

2.4.4: Asthma Induction is Inhibited by

PSA-expanded T Cells

To show in vivo activity of PSA clonally expanded T cells, WT mice were immunized with PSA as before, splenic

CD4+ T cells were isolated from these or saline treated mice, and adoptively transferred into OVA-sensitized recipient mice. Mice were then given intranasal Figure 6: PSA immunization increases zwitterionic motifs with the TCRβ CDR3 loops. OVA challenges daily to induce asthma. Sequences obtained through next generation deep sequencing were probed for charge motifs. On day 7, the mice were sacrificed and N=2-4 mice per group. lungs were lavaged to assess cellular

39 infiltration followed by sectioning for histological analysis through H&E staining and immunohistochemistry.

Table 2: Compilation of TCRβ CDR3 zwitterionic sequences.

Table 2: Compilation of TCRβ CDR3 zwitterionic sequences.

Mice that received CD4+ T cells from PSA-treated mice showed significant reductions in leukocyte infiltration into the airway when compared with recipients of T cells from saline-treated mice (Figure 7A-D). Additionally, PSA-expanded T cells inhibited the

40 presence of inflammatory leukocytes around the airways as detected by both H&E

(Figure 7E) as well as IHC (Figure 7F). These data illustrate the suppressive potential of

CD4+ T cells clonally expanded by PSA, and together with the sequencing data establish a connection between a T cell response to a commensal bacterial polysaccharide and maintaining peripheral immune homeostasis.

Figure 7: PSA clonally expanded T cells inhibit asthma induction. WT mice were treated orally with PSA or saline as before. CD4+ T cells were purified and transferred into WT mice who were sensitized with OVA/Alum. Following IN challenges, cellular infiltration was assessed as before (A-D). Histological changes were visualized as before (E-F). 41 Chapter 3: Polysaccharide A from Bacteroides fragilis Inhibits Asthma Induction

42 3.1: Summary

Changes in bacterial exposure over the last several decades have been linked to increased incidence of allergic and other immune mediated diseases in the Western world. A plethora of studies have shown that normal microbiota in the gut has a profound effect on immune homeostasis and the susceptibility to a multitude of disease, including asthma. The mechanisms underlying the influence of microbes on peripheral immune health remains poorly defined. Here we show that oral exposure to

PSA, a capsular polysaccharide derived from the commensal bacterium Bacteroides fragilis, inhibits the induction of experimental asthma. Direct treatment of mice with

PSA generates protection from asthma, and this effect can be transferred to naïve recipients upon adoptive transfer of CD4+ T cells from PSA-exposed mice. Surprisingly, we found that the PSA-induced T cells lack the canonical regulatory T cell transcription factor Foxp3, but were still able to potently inhibit both Th1 and Th2 models of asthma in an IL-10 dependent fashion. Through these findings, we show that bacterial polysaccharides link microbiota with the peripheral immune system via activated

CD4+Foxp3- T cells following gastric exposure, which then inhibits unnecessary inflammation through IL-10 production.

*Work featured in this chapter was published in Glycobiology (Johnson JL, Jones MB, and Cobb BA, 2015, 25(4): 368-375 (ref#:64))

43 3.2: Introduction

A dramatic increase in the incidence of allergy and asthma has been seen in Western societies, as compared with developing and agrarian societies, over the past several decades. Commensal bacterial, parasitic helminthes, and the molecules they produce, among other factors, have been shown to play a significant role in immune system development (65) and promoting homeostasis (66-68). The profound effects of these components on the immune system were first predicted in 1989, as an idea now known as the hygiene hypothesis (1,69), which suggested that decreased exposure to microbes may lead to greater susceptibility to allergic disease.

Studies have shown connections between microbes and susceptibility to airway inflammation, showing a potential key role for antigens of gut-resident commensal flora in maintaining homeostasis within the lung environment (70). Studies beginning in the

1990’s on abscess responses (71,72), postsurgical adhesion formation (38,39) and more recently inflammatory bowel disease (IBD) (73) illustrate the potent immunomodulatory and anti-inflammatory properties of the polysaccharide PSA from the commensal anaerobic bacterium Bacteroides fragilis; however, none of these previous models utilized diseases characteristically associated with the hygiene hypothesis. It is reasonable to propose that since B. fragilis and PSA have proven to profoundly impact localized immune environment that it could also have an effect on more peripheral tissues and the susceptibility to immune-mediated diseases in keeping with the hygiene hypothesis.

44 Commensal polysaccharide antigens, like PSA, exert their protective functions through their ability to activate CD4+ T cells (38,74). Following their uptake by professional antigen presenting cells, glycoantigens are processed to a low molecular weight by a nitric oxide burst promoted by TLR2 activation (75). They are then loaded onto MHC II for recognition by αβ T-cell receptors (42). The interaction between MHCII and TCR is high affinity (61), promoted by the classical antigen exchange factor human leukocyte antigen DM (42,61,62), and is dependent on a select glycosylation pattern of MHCII

(43,44); however, the nature of the PSA-responding T cells has not been fully established. Previous work has identified them as T helper type 1 (Th1) cells based on their expression of interferon gamma (IFNγ) (75,76) and their pro-inflammatory impact

(71), but additional work has also identified them as potent regulatory T cells (Tregs) which can suppress lymphocyte activation via IL-10 (39,47,73,74). Even within the work identifying the responders as Tregs, there is discrepancy as to the expression of the transcription factor Foxp3. Some studies suggest that PSA-responding T cells are canonical Tregs expressing Foxp3, but the changes in Foxp3+ T cells within in these murine models are rarely over 2% overall (46,54). Contrary to these studies, we have previously reported a lack of Foxp3 expression in PSA-responding T cells through our human studies (47). It is still unclear which population of T cells, Foxp3+ or Foxp3-, is directly responding to PSA in existing models.

In this current study, using two variations of an ovalbumin (OVA)-induced model of asthma, we examined the effect of gastric exposure to a commensal glycoantigen, PSA, on peripheral immune homeostasis. We found that oral exposure to PSA inhibits

45 asthma induction in an IL-10 and T cell dependent mechanism, without expression of

Foxp3 in the PSA-responding T cells. This demonstrates the direct influence of a commensally derived glycoantigen on immune homeostasis in peripheral tissues.

3.3: Methods

3.3.1: Mice and bacteria

Breeding pairs for WT C57Bl/6, OTII, and IL-10n mice were obtained from Jackson labs, and Foxp3-eGFP mice were a kind gift from Dr. A. Rudenskey. All mice were on the

C57Bl/6 background. Mice were housed in specific pathogen free (and B. fragilis free) conditions as stipulated by the guidelines of the Institutional Animal Care and Use

Committee of Case Western Reserve University in Cleveland, OH. Experimental mice were 7-12 weeks of age. B. fragilis was grown in anaerobic conditions, and PSA was purified as previously described. (42,57). Similar to previously published studies (45), all oral treatments were given as oral gavages over 12 days (100 μg/dose in saline every 3 days). Negative controls were given saline only.

3.3.2: Asthma models

Splenic CD4+ T cells were isolated from OTII mice using magnetic bead separation

(Miltenyi Biotec). Cells were transferred intravenously, 2x106 per mouse, 24 hours prior to the initial intranasal OVA administration. For co-transfer experiments, CD4+ splenocytes were isolated as above, and 2x106 cells from PSA treated mice were transferred along with OTII CD4+ T cells as above. Mice were given OVA for six consecutive days (40 μg/dose in saline; Sigma), with the negative controls getting saline

46 alone. For the intranasal challenges, mice were anesthetized using a table top anesthesia system (VetEquip) with 3% isoflurane (Baxter). On the seventh day, mice were euthanized using a cocktail of ketamine (8.6%; Fort Dodge), xylazine(1.7%;

Anased), and acepromazine(2.9%; Boehringer Ingelheim) in sterile saline at 0.006 cc/g.

Blood was taken via cardiac puncture and centrifuged in Microtainer serum collection tubes (BD Biosciences). Mice were then given a tracheotomy, and lungs were flushed with 1 mL PBS containing 0.6mM EDTA three times. Cells from all three washes were combined, resuspended in 50 μL PBS with 0.6 mM EDTA, and automated differentials were generated using a Hemavet 950 Hematology Analyzer. Right lungs were inflated with OCT (Tissue-Tek) and fixed in 10% formalin for 24 hours prior to paraffin embedding and sectioning at the Case Western Reserve University Tissue Procurement and Histology Core Facility. Left lungs were homogenized and incubated in 500 μL supplemented RPMI (Gibco) for 24hours. Supernatant samples (serum, lung homogenate supernatant) were frozen at -80C until analyzed. Cytokine levels were measured by standard sandwich ELISA (BioLegend).

3.3.3: Sterile T cell sorting

Sterile flowsorting of cell populations was performed in the Department of Pathology

Flow Cytometry Core on a FACSAria (BD Biosciences).

3.3.4: Histology

The H&E staining of lung sections was performed by the CWRU Tissue Procurement and

Histology Core Facility. Unstained slides were stained with antibodies specific for EpCAM

47 (6 μg/mL; eBioscience) and MPO (1:100, Abcam; anti-rabbit secondary: 1:1000,

Invitrogen).

3.3.5: General data analysis

All data are shown as mean ± SEM. Mice included a minimum of four animals per group per replicate experiment. Graphs and statistical measures were generated with

GraphPad Prism v5.0 graphing software. Comparisons between multiple groups were done using an ANOVA test, whereas comparisons between two groups (where appropriate) were done using a Student’s t-test.

3.4: Results

3.4.1: Oral PSA exposure protects against OVA-induced airway inflammation

In order to understand the impact of bacterial antigens on airway inflammation, we developed a Th1-driven OVA-induced model of asthma in C57Bl/6 mice where a positive example of asthma is characterized by pathological changes in and around the airways, cellular infiltration into the alveolar space, proinflammatory cytokine production, and T cell activation within the lung draining mediastinal lymph nodes. To begin understanding the potential impacts of bacterial antigens on peripheral immunity, mice were orally treated with the capsular polysaccharide PSA from Bacteroides fragilis prior to induction of asthma. There were significant reductions in infiltration of leukocytes

(white blood cells; WBC; Figure 8A), neutrophils (Neuts; Figure 8B), lymphocytes

(Lymph; Figure 8C), and monocytes/macrophages (Macs; Figure 8D) into the bronchoalveolar lavage (BAL) samples of mice exposed to PSA as compared to positive

48 asthmatic controls. As expected, no changes were seen in eosinophil numbers in this

Th1 driven model (data not shown).

Analysis of lung tissue homogenates showed reduced IFNγ levels in the mice treated with PSA when compared with asthmatic mice (Figure 8E). Additionally, anti-CD3/CD28 stimulated CD4+ T cells purified from mediastinal lymph nodes also showed basal levels of IFNγ in PSA treated mice as compared to the 3 fold induction seen in asthmatic mice

(Figure 8F). Consistent with previously published work (47) and a global shift toward an anti-inflammatory state, re-activation of CD4+ splenocytes from PSA treated mice by anti-CD3 shows a marked increase in IL-10 production (Figure 8G). Lastly, while myeloperoxidase-positive (MPO+) leukocytes are present around the airways in saline- treated mice, they are notibly absent in mice treated with PSA, in both hematoxylin and eosin (H&E) and confocal immunohistochemistry (IHC) staining (Figure 8H). Together, these data illustrate the prevention of airway inflammation by microbial products following gastric exposure.

49 A B C D

E F

G i.n. OVA i.n. saline

GlyAg

Saline

Figure 8: Oral PSA inhibits asthma induction. WT mice were given PSA (GlyAg) or saline orally prior to adoptive transfer of CD4+ T cells from OTII mice. Recipients were then given IN challenges of OVA or saline for 6 consecutive days, and sacrificed on day 7. BALs were performed, and cellular infiltration into the airway was assessed in combined BALs for each mouse (A-D). White bars represent unchallenged or unstimulated controls. Cytokine production was measured directly in lung tissue (E) and indirectly in a recall assay through restimulated CD4+ T cells isolated from mediastinal lymph nodes (F). Histology was examined through both H&E sections and IHC (G). IHC sections were stained with Ep-CAM (green) and MPO (red). Data represents 4-6 mice per group (*p,0.05; #p>0.05)

50 3.4.2: CD4+ T cells drive PSA-mediated asthma protection

The immunomodulatory properties of glycoantigens have previously been tied to CD4+ T cells (38,74). This has predominantly been shown in a gut localized context, and the influence of these glycoantigen experienced T cells on peripheral inflammation remains unclear. To determine if PSA was inhibiting asthma induction through T cells, WT mice were treated with PSA as before, and splenic CD4+ T cells were adoptively transferred 24 hours prior to asthma induction into naïve WT recipients. The transferred PSA- experienced T cells were able to prevent the cellular infiltration into the airways (Figure

9A-D) to a similar extent as direct treatment. Again, eosinophil numbers remained unchanged from baseline in this model (data not shown). Significant reductions were also observed in pro-inflammatory cytokines within the lung tissue (Figure 9E and F) as well as lung histology and IHC (Figure 9G) in PSA-experienced T cell recipients.

Collectively these data show that the prevention of asthma induction mediated by PSA is T cell dependent.

In order to verify the effectiveness of PSA experienced T cells in protecting against multiple etiologies of asthma, an allergic or T helper type 2(Th2)-skewed model of asthma was utilized. WT mice were treated with PSA as before, and CD4+ splenocytes were adoptively transferred into the transgenic OTII mice, which carry a germ-line encoded T cell receptor (TCR) specific for OVA. This produced a more severe phenotype and inflammatory state with eosinophil infiltration and IgE production; however, mice that received T cells from PSA treated mice were still protected from asthma induction

(Figure 10A-G). The T cells from PSA treated mice were able to block eosinophil

51 A B C

D E F

G

-

CD4

GlyAg

CD4

- Saline

Figure 9: CD4+ T cells drive PSA suppressive activity. WT mice were treated with PSA (GlyAg) or saline as before, and CD4+ splenocytes from treated mice were transferred along with OTII CD4+ into naïve WT recipients. Following IN OVA challenges, cellular infiltration was assessed as before (A- D). Cytokine production within the lung was detected through ELISA (E-F), and histological changes were visualized as before (G). Data represents 4-6 mice per group. (*p<0.05; #p>0.05)

52 A B C

D E F

G

-

CD4

GlyAg

CD4

- Saline

Figure 10: PSA inhibits multiple etiologies of asthma. WT mice were treated with PSA (GlyAg) or saline as before, and CD4+ splenocytes were purified and adoptively transferred into OTII mice. Following IN challenges, cellular infiltration was assessed as before (A-E). Total serum IgE was quantified by ELISA (F), and histological changes were visualized as before (G). Data represents 4-6 mice per group. (*p<0.05; #p>0.05)

53 infiltration as well as elevated serum IgE (Figure 10E and F). Additionally, a significant reduction in inflammatory cells around both airways and blood vessels were seen in both H&E and confocal imagine of lung sections (Figure 10G). These data collectively illustrate the potent efficacy and T cell dependency of oral PSA treatment in two distinct murine models of asthma.

A B C D

E

GlyAg

Saline

Figure 11: PSA suppression is not driven by Foxp3+ Tregs. Foxp3-GFP mice were treated with PSA (GlyAg) or saline as before, and CD4+ splenocytes were purified and transferred along with CD4+ OTII splenocytes into naïve WT recipients. Cellular infiltration was assessed as before (A-D), and histological changes were visualized as before (E). Data represents 4-6 mice per group. (*p<0.05; #p>0.05)

54 3.4.3: PSA responding T cells are not traditional Foxp3+ regulatory T cells

Within the literature, there are conflicting reports as to the role of canonical CD4+Foxp3+ regulatory T cells (Tregs) in PSA mediated suppression. Data from in vitro human trials clearly identifies the PSA responding population as Foxp3- (47), but data from mouse models has been less clear (45,54,73). In order to determine the requirement for Foxp3 in PSA mediated asthma inhibition, Foxp3-GFP reporter mice were orally treated with

PSA as before. CD4+Foxp3- T cells were isolated using sterile flow sorting, and these were adoptively transferred into naïve WT recipients. Cellular infiltration, both into alveolar spaces as well as around airways and blood vessels, was inhibited (Figure 11A-

E) similarly to the transfer of bulk CD4+ T cells (Figures 9 and 10). This demonstrates that the T cells directly responding to PSA do not require Foxp3 expression to exert their suppressive activity.

To further support the conclusion of a lack of induced Foxp3 expression in PSA responding cells, CD4+ T cells were analyzed by flow cytometry following PSA treatment.

In keeping with the depletion experiment shown in Figure 11, there was no significant

A B Saline GlyAg Saline GlyAg

CD25+ FoxP3+ p>0.05

+

FoxP3 p>0.05

0.0 0.5 1.0 1.5 2.0 CD25 fold CD4+ T cells FoxP3 Figure 12: Foxp3 is not induced by oral PSA treatment. WT mice were treated with PSA (GlyAg) or saline as before. CD4+ splenocytes were profiled for expression of CD25 and Foxp3 (A and B). Fold change represents experimental replicates.

55 change in CD25+Foxp3-, CD25+Foxp3+, or CD25-Foxp3+ populations following PSA treatment (Figure 12), although induced expression of Foxp3 at later time points cannot be fully ruled out.

3.4.4: PSA mediated asthma inhibition is IL-10 dependent

Our previous work suggests that PSA can suppress proinflammatory T cell activation in an IL-10 mediated fashion in humans (47). To determine whether that mechanism plays a role in the ability of PSA T cells to suppress asthma, IL-10 deficient mice (IL-10n) were sensitized with OVA-Alum, thereby enriching for OVA specific CD4+ T cells. These enriched cells were adoptively transferred into IL-10n recipients which had been treated with PSA as before. The ability to inhibit asthma induction was ablated in this IL-10 deficient model (Figure 13A-E). Finally, to confirm a lack of PSA mediated regulation induced in the OVA specific T cells, IL-10n mice were treated with PSA as before followed by transfer of IL-10 sufficient OTII T cells prior to asthma induction. Even though the OTII cells are IL-10 sufficient, oral PSA treatment was unable to suppress asthma induction in the absence of IL-10 in the treated mice (Figure 13F-J). The data presented here illustrate that oral PSA treatment induces a population of suppressive T cells that lack Foxp3 expression that can protect against asthma induction through IL-10 production.

56 A B C D

E

g

GlyA

Saline

F G H I

J

g

GlyA

Saline

Figure 13: IL-10 is required for PSA mediated suppression. IL-10n mice were immunized with OVA/Alum, and OVA-enriched CD4+ splenocytes were purified and transferred into IL-10n mice which had been treated with PSA (GlyAg) or saline as before. Following IN challenges, cellular infiltration and histological changes were assessed as before (A-E). Additionally, IL-10n mice were treated with PSA as before, and CD4+ OTII splenocytes were transferred into treated mice followed by IN challenges. Cellular infiltration and histological changes were assessed as before (F-J). Data represents 4-6 mice per group. (*p<0.05; #p>0.05)

57 Chapter 4: Novel T cell-T cell Interaction Promotes Immune Homeostasis

58 4.1: Summary

Following up on our previous work with asthma, we sought to better understand the suppressive activity shown by PSA experienced T cells. Contrary to expected results, transfer of IL-10n PSA experienced T cells into WT recipients was able to inhibit asthma induction to a similar extent as WT cells, despite our earlier data showing IL-10 was required for PSA mediated suppression (52). Further data shows that the cellular source of the suppressive IL-10 was tissue resident Foxp3+ cells in the recipient mouse. Utilizing an in vitro co-culture system, we further confirmed that TEM cells were able to induce IL-

10 production synergistically in Foxp3+ Treg cells regardless of prior PSA. Additionally, we show that this promotion of IL-10 production is prompted by a soluble mediator secreted by the TEM cells. Returning this data back in vivo, we show that both treatment with conditioned media samples or transfer of in vitro activated cells induces suppressive IL-10 production within the context of asthma. This previously unidentified

T cell-T cell interaction has large implications for the understanding of immune regulation and homeostasis, including a potential to commandeer the mediator as a novel therapeutic for a multitude of inflammatory conditions.

4.2: Introduction

Previous work, including work presented here, has shown immunomodulation through

Bacteroides fragilis colonization or purified PSA treatment, and there is a consensus that this is mediated through an expanded population of CD4+ T cells (37-

39,45,46,52,54,59,73,74). Additional work from our lab has conclusively shown that the

T cells directly responding to PSA do not upregulate or require expression of the

59 canonical regulatory transcription factor, Foxp3, to inhibit inflammatory responses

(47,64). However, beyond the knowledge that IL-10 production is critical in their suppressive activity (47,64), little work has been done to understand the cellular mechanisms that promote homeostatic or anti-inflammatory environments.

Additionally, while a large collection of studies have focused on interactions of B. fragilis, PSA, or PSA induced T cells on peripheral immune homeostasis (37-

39,45,46,52,54,59,73,74), isolating the effector populations for a more specific analysis in vitro/ex vivo has yet to be accomplished. Our phenotypic characterization of PSA responding T cells as CD4+Foxp3-CD45RbLowCD62LLowCD44Hi (47,52) has provided a stepping stone to isolate/enrich for PSA responding T cells by sterile flowsorting without knowing the specific TCR or generating PSA-loaded MHCII tetramers. In order to utilize the potent immunomodulatory properties of PSA and the suppressive capacity of PSA generated TEM cells, it is essential to understand the mechanism of action at a cellular level both in vivo and in vitro. Here we show that PSA experienced T cells promote IL-10 production in tissue resident Foxp3+ T cells, as well as purified Foxp3+ T cells in vitro. We further show that this subset of TEM cells can induce IL-10 production irrespective of prior PSA exposure, and it does so through a soluble mediator. Conditioned media from activated TEM cells is capable of inducing IL-10 in vivo, and a transfer of activated TEM cells can suppress asthma induction to a similar degree as seen in a bulk CD4+ T cell transfer from PSA treated mice. These results indicate that the potent immune suppression seen in PSA treated mice is merely expanding an already existing subset of

T cells capable of interacting with Foxp3+ T cells to promote immune homeostasis.

60 Identification of this novel T cell-T cell interaction opens up a new avenue of immune regulation research, including multiple potentials for drugable targets.

4.3: Methods

4.3.1: Mice and Bacteria

Breeding pairs for WT C57Bl/6 mice were obtained from Jackson labs, and housed in specific pathogen free (and B. fragilis free) conditions as stipulated by the guidelines of the Institutional Animal Care and Use Committee of Case Western Reserve University in

Cleveland, OH. Experimental mice were 7-12 weeks of age. B. fragilis was grown in anaerobic conditions, and PSA was purified as previously described (42,57). Similar to previously published studies (45), all oral treatments were given as oral gavages over 12 days (100 μg/dose in saline every 3 days).

4.3.2: Asthma model

Splenic CD4+ T cells were isolated using magnetic bead separation (Miltenyi Biotec).

Cells were transferred intravenously, 2x106 per mouse unless indicated, 24 hours prior to the initial intranasal OVA administration. For co-transfer experiments, CD4+ splenocytes were isolated as above, and 2x106 cells from PSA treated mice were transferred along with OTII CD4+ T cells as above. Mice were given OVA for six consecutive days (40 μg/dose in saline; Sigma), with the negative controls getting saline alone. For the intranasal challenges, mice were anesthetized using a table top anesthesia system (VetEquip) with 3% isoflurane (Baxter). On the seventh day, mice were euthanized using a cocktail of ketamine (8.6%; Fort Dodge), xylazine(1.7%;

61 Anased), and acepromazine(2.9%; Boehringer Ingelheim) in sterile saline at 0.006 cc/g.

Mice were given a tracheotomy, and lungs were flushed with 1 mL PBS containing

0.6mM EDTA three times. Cells from all three washes were combined, resuspended in

50 μL PBS with 0.6 mM EDTA, and automated differentials were generated using a

Hemavet 950 Hematology Analyzer. Right lungs were inflated with OCT (Tissue-Tek) and fixed in 10% formalin for 24 hours prior to paraffin embedding and sectioning at the

Case Western Reserve University Tissue Procurement and Histology Core Facility.

4.3.3: Sterile T cell sorting

Sterile flowsorting of cell populations was performed in the Department of Pathology

Flow Cytometry Core on a FACSAria (BD Biosciences) utilizing monoclonal antibodies to

CD45Rb, CD44, and CD62L (eBioscience).

4.3.4: Cell Culture

CD4+ T cells were purified using magnetic bead separation (Miltenyi Biotec), and further separated using sterile flowsorting as described above. Cells were then resuspended in supplemented RPMI (Gibco) and plated on U-bottom 96 well plates (Costar). Cells were activated for 72 hours by plate bound anti-CD3 (eBioscience), and cytokine levels were assessed by ELISA (Biolegend).

4.3.5: Histology

The H&E staining of lung sections was performed by the CWRU Tissue Procurement and

Histology Core Facility. Unstained slides were stained with antibodies specific for EpCAM

(6 μg/mL; eBioscience), MPO (1:100, Abcam; anti-rabbit secondary: 1:1000, Invitrogen),

62 and Collagen II (10ug/mL, R&D Biosystems; goat anti-sheep secondary: 1:1000,

Invitrogen).

4.3.6: Flow analysis

Expression of GFP was detected using an Accuri C6 cytometer (BD Biosciences), and analyzed using either CFlow software (BD Biosciences) or FlowJo software (FlowJo).

4.3.6: General data analysis

All data are shown as mean ± SD. Mice included a minimum of four animals per group per replicate experiment. Graphs and statistical measures were generated with

GraphPad Prism v5.0 graphing software. Comparisons between multiple groups were done using an ANOVA test, whereas comparisons between two groups (where appropriate) were done using a Student’s t-test.

4.4: Results

4.4.1: Suppressive IL-10 Production is External to PSA Experienced T cells

While IL-10 production has been shown to be critical for PSA mediated suppression of asthma induction, the requirement of IL-10 from the PSA experienced cells has not yet been directly shown. In order to address this issue, IL-10n mice were treated with PSA as before (64) and CD4+ splenocytes from treated mice were adoptively transferred into

WT recipients along with OTII CD4+ splenocytes followed by six days of intranasal OVA challenges. Unexpectedly, the transferred T cells from the PSA treated mice were able to inhibit asthma to a similar extent as WT cells despite the lack of IL-10 (Figure 14 A-E)

(64).

63

A B E

CD4

- GlyAg

C D

CD4

- Saline

Figure 14: IL-10n PSA experienced T cells inhibit asthma induction. IL-10n mice were treated orally with PSA or saline as before. CD4+ T cells were purified and transferred into naïve WT mice along with OTII CD4+ T cells. Following IN challenges, cellular infiltration was assessed as before (A-D). Histological changes were visualized as before (E).

Since prior work has shown IL-10 to be required in PSA mediated suppression of asthma induction (64), we next performed the reciprocal experiment to look at the requirement of IL-10 in the recipient, or asthmatic, mouse. WT mice were treated with PSA as before, and CD4+ splenocytes were transferred along with OTII CD4+ splenocytes into IL-

10n recipients. The cells from the PSA treated WT mice were unable to protect the IL-

10n recipient against asthma induction (Figure 15 A-E). Together these experiments show that while IL-10 is required for PSA mediated suppression, the cells that are actually producing the IL-10 do not need to see PSA for this effect.

4.4.2: PSA Experienced T cells induce IL-10 Production in Foxp3+ T cells

Since IL-10 has proven to be critical in the suppression of asthma induction, we next wanted to examine which cell type within the recipient mice were responsible for IL-10

64 production. IL-10n mice were treated with PSA and CD4+ splenocytes from treated mice

along with OTII CD4+ splenocytes were adoptively transferred as before into IL-10GFP

reporter mice. After six days of intranasal OVA challenges, cells from bronchoalveolar

lavages were analyzed by flow for the presence of IL-10 (GFP). Mice that received T cells

from PSA treated mice showed an 8.7% increase in GFP+ CD4+ T cells as compared to

mice that received T cells from PBS treated mice (Figure 16 A). This increased

percentage corresponded to a 2 fold increase in the total number of GFP+ CD4+ T cells

(Figure 16 B). No change was seen in MFI within macrophages (Figure 16 C) or dendritic

cells (data not shown). Further analysis of GFP+ T cells revealed an increased percentage of

Foxp3+ T cells (Figure 16 A), indicating that T cells from a PSA experienced mouse may induce IL-

10 production within the regulatory T cells of a recipient mouse. A 1000 B 100 E 800 80 i.n. OVA 600 60

400 40

WBCs (x1000)

200 Neuts (x1000) 20

CD4 - 0 0

GlyAg CD4 + - + - + - + - GlyAg Saline CD4 - + - + - + - + i.n. OVA + + - - + + - -

800 30 C D 600

20

400

CD4 - 10

200

Macs (x1000)

Lymph (x1000)Lymph Saline

0 0 GlyAg CD4 + - + - + - + - Saline CD4 - + - + - + - + i.n. OVA + + - - + + - -

Figure 15: WT PSA experienced T cells fail to inhibit asthma induction in IL-10n recipients. OTII mice were treated orally with PSA or saline as before. CD4+ T cells were purified and transferred into naïve IL-10n mice along with OTII CD4+ T cells. Following IN challenges, cellular infiltration was assessed as before (A-D). Histological changes were visualized as before (E).

65 To verify the interaction between PSA experienced T cells and Foxp3+ T cells, CD4+ splenocytes were obtained from PSA treated IL-10n mice and sterilely sorted into

+ Hi - Hi populations of naïve (TN; CD4 CD45Rb CD44 CD62L ), central memory (TCM;

+ Low + Hi CD4 CD45Rb CD44 CD62L ), and effector memory (TEM;

CD4+CD45RbLowCD44+CD62LLow) cells. These were then co-cultured with Foxp3+ Tregs and activated by anti-CD3 for 72 hours. Effector memory cells, and to some extent

Figure 16: PSA experienced T cells induce IL-10 production in Foxp3+ Tregs. IL-10n mice were treated orally with PSA or saline as before. CD4+ T cells were purified and transferred into IL-10-GFP mice along with OTII CD4+ T cells. Following IN challenges, cellular infiltration was stained for CD4, CD25, and Foxp3, and assessed by flow cytometry (A). Fold change of CD4+GFP+ was calculated (B), and MFI of F4/80+GFP+ cells was assessed by flow (C).

66 central memory cells, from IL-10n mice promoted IL-10 production in Foxp3+ T cells.

Effector memory cells promoted this IL-10 production regardless of the expression of

+ CD25 by the Foxp3 population (Figure 17 A-B). TEM cells from PSA treated mice showed reduced IFNγ production (Figure 17 C), which recapitulates previously published data

(52). Since PSA is known to expand a subset of T cells with a TEM phenotype (52), similar co-culture assays were performed with cells from mice which had never seen PSA.

Synergistic IL-10 production was seen in co-cultures of TEM and Treg cells (Figure 17 D-E), indicating that this response is not dependent on PSA immunization, but rather represents a novel T cell-T cell interaction which promotes immune regulation or

C A B

D E 2000 * 2000 * 1.6x 1.8x 1500 1500

1000 1000

500

500 IL-10 mL pg/ pg / mL IL-10 mL / pg

0 0 + - CD25 T T T TCM TEM TN CD25 T T T TCM TEM TN Treg CM EM N Treg CM EM N +CD25+Treg +CD25-Treg + Figure 17: TEM cells promote synergistic IL-10 production in Foxp3 Tregs in vitro. IL-10n mice were treated orally with PSA or saline as before. CD4+ T cells were purified, sterilely sorted based on CD44 and CD62L, and co-cultured with Foxp3+ Tregs stimulated by anti-CD3 for 72hr. Cytokines were measured by ELISA (A-C). T cells were isolated from naïve WT mice, sorted as before, and cultured as above. Cytokines were measured by ELISA (D, E).

67 suppression.

4.4.3: A Soluble Molecule Mediates Effector Memory T cell-Regulatory T cell Interaction

To identify if this T cell-T cell interaction was mediated by contact dependent or

+ - independent mechanisms CD4 Foxp3 cells were separated into TN and TEM populations which were then cultured in monocultures to generate conditioned media. This conditioned media was then applied to CD4+Foxp3+ Treg cells for 72 hours. The production of IL-10 seen in the Tregs with TEM conditioned media was similar to that seen in co-cultures with both Tregs and TEM cells, indicating that the mechanism of interaction between the two cell types is a soluble mediator (Figure 18 A).

A B

Figure 18: TEM –Treg interaction is facilitated through a soluble mediator. CD4+ splenocytes from Foxp3-GFP mice were stained with CD44 and CD62L and sorted as before. Conditioned media was collected after 72hrs and added to fresh cultures of the designated cell type (A). Sorted cells were fixed after anti-CD3 stimulation for 72hr and added to fresh Tregs (B). Cytokines were determined by ELISA. *p<0.05

68 To verify that the action was mediated by a soluble molecule and not a surface bound

molecule, activated monocultures of TN and TEM were fixed with 2% paraformaldehyde

(PFA) and incubated with live Tregs. IL-10 production was only seen in the co-cultures

of live TEM and Treg cells, not in cultures containing either of the fixed populations

(Figure 18 B). This data confirms the need for secreted molecules to mediate the

interaction between effector memory cells and regulatory T cells.

4.4.4: Soluble Mediator from Activated TEM Cells Induces IL-10 Production in vivo

To connect these in vitro findings with

40 our in vivo asthma studies OTII CD4+

+ * T 17%

N 30 splenocytes were adoptively transferred

IL-10 + GFP 20 into IL-10 reporters to induce asthma.

10 Mice were given conditioned media from

T %BALF CD4 33% either TEM or TN monocultures alongside EM 0 Hi Lo RbT RbT N EM their intranasal OVA challenges. Analysis

of CD4+ T cells within the

Figure 19: Conditioned TEM media promotes IL-10 production in vivo. CD4+ bronchoalveolar fluid showed an splenocytes from Foxp3-GFP mice were stained with CD44 and CD62L and sorted as increase in the percentage of IL-10 before. Conditioned media was collected after 72hrs. CD4+ T cells from OTII mice producing cells (Figure 19) similar to the were purified and transferred into naïve IL- increase previously shown with the 10-GFP mice. Following IN challenges, GFP was measured in CD4+ cells from BALs by transfer of cells from PSA treated mice flow cytometry. *p<0.05 (Figure 16).

69 4.4.5: Activated TEM cells Inhibit Asthma Induction

To further show that a molecule secreted from TEM cells promotes suppression of

- asthma in vivo, purified Foxp3 TEM or TN cells were activated with anti-CD3 for 24 hours in vitro and then adoptively transferred into WT recipients along with CD4+ OTII splenocytes. Following six days of intranasal OVA challenges, mice were assessed for cellular infiltration and pathology. Within the airway space, transfer of only 60,000 TEM cells was able to significantly reduce the number of infiltrating cells, whereas the transfer of 160,000 TN cells slightly increased the number of infiltrating cells as compared to mice that received neither TN or TEM cells prior to OVA challenges (Figure

20 A-D). Similarly, histology showed decreased pathology with activated TEM transfer in both H&E sections and confocal images (Figure 20 E-F).

70 E

IN PBS IN OVA

+T cells N +T cells IN OVA EM IN OVA

Figure 20: TEM cells activated F in vitro inhibit asthma induction. CD4+ splenocytes from Foxp3-GFP mice were IN PBS IN OVA stained with CD44 and CD62L and sorted as before. Cells were activated for 24hr with anti-CD3 before being transferred along with OTII CD4+ T cells. Following IN challenges, cellular +T cells +T cells infiltration was assessed as N EM IN OVA IN OVA before (A-D). Histological changes were assessed through H&E (E) and IHC (F) with Ep-CAM in green, MPO in red, and Collagen II in blue. *p<0.05

71 Chapter 5: Discussion and Future Directions

72 More than 20 years of research has clearly shown that zwitterionic polysaccharides like polysaccharide A from the commensal bacterium Bacteroides fragilis can activate CD4+ T cells (71,76). This T cell activation results from the processing of polysaccharide antigens into smaller units (5-10 kDa; (41,42))through nitric oxide mediated oxidation and presentation by MHCII molecules (42,62,77). While the binding and presentation of polysaccharides on MHCII has been intensely studied (41,42,61-63,75,77), the corresponding T cell recognition remains poorly defined.

One factor that has hindered dissecting the nature of T cell recognition of PSA is the inability to clone PSA-specific T cells. Through our studies with human T cells, we discovered that PSA-responding T cells become highly anergic upon re-stimulation with

PSA, which cannot be fully broken with the addition of growth factors including IL-2 (47).

Additionally, T cells from PSA-immunized mice fused to create hybridomas fail to proliferate, preventing traditional cloning and establishment of a clonal cell line (data not shown). In the work presented in Chapter 2, we used next generation deep RNA sequencing of the CDR3 loop of TCRβ in CD4+ T cells isolated from saline treated, PSA- immunized, or OVA-immunized mice to begin to understand the nature of PSA responding T cells. TCRβ was chosen due to the expression of a single TCRβ allele within a mature T cell, as compared to TCRα which does not have the same allelic exclusion.

Through analysis of the sequencing data we found a limited subset of clones expanded in response to PSA stimulation, which is similar to expansion seen with traditional protein antigens. The response to PSA is characterized by a lack of specific Vβ and J segment usage and changes in average TCRβ CDR3 loop length. The use and distribution

73 of Vβ segments within the responding T cell population can be used to differentiate between a response to a conventional antigen and a superantigen. Superantigens activate T cells by cross-linking MHCII and Vβ domains independently of CDR3 loop rearrangements; therefore, this type of stimulation activates the majority of T cells containing particular Vβ segments. Traditional peptide antigens are recognized through the rearranged CDR loops, and not through particular Vβ or J segments. Since the resulting CDR3 sequences from PSA immunized mice revealed no selection of specific Vβ or J segments, this suggests that the T cell response to PSA is antigen-specific, and not driven by a superantigen-like nonspecific interaction.

An interesting difference seen in PSA immunized samples was the increased percentage of CDR3 loop sequences containing zwitterionic motifs. It has been well established that the zwitterionic motif within the repeating unit of PSA is required for PSA mediated T cell activation (37,71), and previous work from our lab has shown that loss of positive, negative, or both charges changes the conformation of PSA and precludes any interactions with MHCII (41). This presence of alternating charges within the repeating unit is the key feature of T cell dependent polysaccharide antigens

(41,42,51,62,63,71,72,76). Identifying an increased percentage of zwitterionic TCRβ

CDR3 loop sequences within the PSA immunized samples indicates the potential of electrostatic interactions during the recognition of zwitterionic polysaccharide antigens by T cells. Further studies need to be performed to address what other characteristics could be important in polysaccharide recognition, such as optimum length of PSA,

74 glycosylation state of the responding TCRs, and co-stimulatory molecules that are actively engaged during MHCII-TCR binding.

Through the work included in Chapter 3, we show that the commensal polysaccharide

PSA from Bacteroides fragilis clonally expands a population of CD4+CD45RbLow effector memory cells that are skewed toward IL-10 production and are capable of suppressing

OVA induced airway inflammation. This is the first evidence of a non-peptide MHCII- dependent antigen being specifically recognized by and clonally expanding CD4+ T cells, altering the long held peptide centric view of T cell specificity to include polysaccharide antigens. Additionally, the ability of T cells expanded by PSA immunization to suppress inflammation induced by intranasal OVA challenges shows potent bystander suppression, which is consistent with our previous findings using human cells (47).

Further information can be gleaned from this data set to further understand PSA responding T cells. By focusing on the zwitterionic CDR3 loop sequences specifically and examining which Vβ or J segments those particular T cells use, T cells could be separated for those specific segments, allowing for a more detailed focus on PSA responding T cells. This population would likely be enriched for PSA responding T cells, and more directed questions about protein expression, antigen specificity, and T cell subsets could be answered with more certainty than when analyzing bulk CD4+ T cells. These questions could in part be approached through activation assays, flow cytometry, and immunohistochemistry, but additional information could be found by performing

RNAseq on the purified or enriched populations. Production of particular cytokines,

75 receptors, or transcription factors found through this approach could then be verified at the protein level through a western blot or ELISA. Understanding the phenotype, functionality, and downstream signaling mechanisms involved in PSA responding T cells will enable future work in the importance of this bacterial polysaccharide in immune conditions and homeostasis.

In order to begin to understand the functionality of PSA expanded cells, we next examined the effect of PSA treatment in a murine model of asthma. Through the second portion of my data presented here, we show that oral treatment with PSA inhibits asthma induction in a T cell dependent manner. Additionally we established that this protection is mediated through IL-10 production, and, in keeping with our previous observations in human cells (47), we showed that the PSA-responding T cell population lacks expression of the canonical regulatory transcription factor Foxp3.

Notably, we also saw inhibition of asthma for both Th1 and Th2 skewed asthma, demonstrating the potent ability of PSA to induce bystander suppression.

Contrary to previously published data on the immunomodulatory properties of PSA, here we show a direct connection between exposure to a commensal bacterial product and a condition with a direct link to the hygiene hypothesis. While it is highly unlikely that a single pathway is responsible for all potential effects of microbial exposure on the immune system, our findings illustrate one mechanism to further our understanding of the hygiene hypothesis. This model system that replicates findings of the hygiene hypothesis with a relevant disease model can promote additional inquiries into the role

76 of T cell responses driven by the microflora within the mucosa and how they can interact with peripheral immune systems. More specifically, it could allow future studies to understand why certain bacterial and environmental exposures protect from allergic diseases (7,78), while exposure to other antigens, such as cockroaches, pet dander, and house dust mite increases likelihood of a diagnosis of asthma (79,80).

Another important factor in studying impacts of microbial antigens is the timing of exposure, with many studies showing that earlier exposure tends to be more protective than later exposure. For example, an initial colonizing event during vaginal delivery, as compared with a cesarean section, impacts basal cytokine levels (2)as well as the potential to develop asthma (3,81,82). Additionally, exposure to prebiotics, including non-digestible oligosaccharides, early in life can reduce wheezing episodes two years after treatment (83); however, there is still little evidence to show if this protection extends into adulthood. Furthermore, conflicting evidence exists surrounding the effectiveness of probiotics in the protection from allergic diseases (reviewed in: 84), with the lack of reproducible responses suggesting that perhaps exposure to bacterial antigens needs to occur during immune development in order to achieve maximal results. Although a reduced susceptibility to asthma has been associated with owning dogs, this protection only occurs if the dog is present in the home during the first two years of the child’s life (85). Once this developmental window has passed, the presence or absence of a dog has no significant effect on the development of asthma. Utilizing

PSA as a model bacterial antigen, multiple aspects of bacterial exposure leading to

77 immune modulation could be better understood, such as the most beneficial window of exposure, critical dosage, and essential components of the antigen.

Crucial to further studies of bacterial exposure, and in particular capsular polysaccharide exposure, is clarifying the cellular mechanism of PSA action. Many studies have been performed focusing on Bacteroides fragilis and PSA as a model for the effect of commensal bacteria on overall health (38,39,42,43,47,61,62,73-75), but a duality of proinflammatory versus anti-inflammatory consequences of microbial exposure is seen among these studies. Several decades ago, work with B. fragilis centered around a proinflammatory response generated by activating CD4+ T cells, leading to the formation of intra-abdominal abscesses (38,57,71,75), and further work identified the same population of cells as causative agents in a model of surgical adhesions (74). Contrary to those studies, treatment with purified PSA or colonization with B. fragilis without any adjuvant can promote anti-inflammatory conditions limiting or inhibiting the induction of IBD (73), intra-abdominal abscesses (37,59), and postsurgical adhesions (74). This duality of response to PSA and B. fragilis must be resolved before live bacteria or bacterial products are translated into the clinic as treatments for inflammatory diseases.

It appears that the route of delivery may be critically important for the downstream response to polysaccharide or bacterial exposure, as following exposure in the peritoneal cavity abscesses are generated, whereas exposure in a more physiological setting, such as the gut, results in protection against inflammatory agonists. Another possibly confounding factor is the presence of an adjuvant, which appears to promote a pro-inflammatory response, although this has only been examined in intraperitoneal

78 delivery (38,71). One way to further examine these two aspects would be to vary the method of PSA delivery, with or without an adjuvant, in mice that are then subjected to an inflammatory protocol, such as asthma, IBD, or EAE.

While there is recent evidence of Treg specific antigens playing a role in the response to gut localized bacteria (86), there are holes that remain in understanding responses to bacteria which are foreign to the host as compared to bacteria already present in the microflora. While we are now beginning to understand the role of specific cell subsets in the suppressive response to PSA and B. fragilis, there is still a lack of knowledge in signaling pathways and kinetics that generate such a potent response. While all of the

PSA studies described here utilize the same 12 day treatment protocol, the transfer of cells activated in vitro indicates that such a long period of exposure to PSA may not be necessary. By comparing mice treated for a shorter period of time, or a reduced dosage, we could better understand the activation requirement needed, both in terms of amount of PSA required as well as the timing of exposure, to generate the potent anti-inflammatory response.

Additionally, the work presented here and in other studies have illustrated the significant immunomodulatory properties of PSA and the ability of PSA to prevent inflammation (37,45,46,54,59,64,73,74), but very little work has been done to date examining the ability of PSA to reduce or eliminate existing inflammation.

Heterogeneous inflammatory conditions, such as asthma, necessitate the generation of a multitude of treatment options, particularly for steroid resistant, severe asthmatics,

79 which make up approximately 3.6-10 % of adult asthmatics (87,88). These patients are often on several medications for over 50% of the year, yet do not achieve full control of their asthma symptoms (87). Treating mice with PSA following asthma induction would begin to establish the ability of PSA to be used as a treatment option. While the oral route of treatment and cellular transfer methods mentioned here would be used initially to validate the efficacy of PSA as a therapy, other delivery methods would be more readily accessible to asthma patients, such as intranasal or aerosolized treatments similar to inhalers patients would be comfortable with. With these additional delivery methods, other factors will have to be examined, such as ability of PSA to get to all needed areas of the lung, rate of antigen uptake within the inflamed tissues, and potential for degradation of PSA prior to antigen uptake by proteases, which can be upregulated in of asthma (89).

Furthermore, translating the preventative efficacy of PSA treatment into models with physiological relevance is needed. The house dust mite (HDM) allergen Der p 1 has been well established as a causative agent for developing asthma, with children exposed to higher concentrations of Der p 1 having earlier wheezing episodes than those exposed to lower allergen concentrations (79). Mouse models have been developed to mimic human responses to HDM, including cellular infiltration, pathology, and airway hyperresponsiveness (90,91). Utilizing this model system, the effectiveness of oral PSA treatment on preventing, as well as treating, will be assessed. Another more physiologically relevant antigen that is used is the cockroach antigen (CRA) (92); however, this model uses an adjuvant during the sensitization phase, which may impede

80 the effects of PSA treatment. The other aspect to using these physiologically relevant antigens is to study a chronic setting of asthma as both of these model systems can be extended to a chronic study and exhibit remodeling similar to that seen within human patients following exacerbations (93). Additionally, these antigens can be combined, or added to the traditional OVA model, to create another model representing a more inner city type of environment, and, as expected, the inflammation seen with dual-antigen challenge is increased over either antigen alone (94,95).

Although multiple studies have been completed examining the impact of bacteria or bacterial products on immune homeostasis, the understanding of a clear mechanism through which the beneficial effects are realized has been lacking. In the work presented in chapter 4, we show that TEM cells expanded in response to the capsular polysaccharide PSA from the gram negative commensal Bacteroides fragilis induce IL-10 production by Foxp3+ T cells within the lung. We were able to recapitulate this interaction in vivo, as well as establish that TEM cells from mice without any previous PSA exposure are also capable of inducing IL-10 in vitro. Unlike other T cell-T cell interactions that have recently been reported for enhancing stability or promoting differentiation (96,97), we show the interaction between TEM cells and Tregs which promotes suppressive IL-10 is accomplished through a soluble mediator. Furthermore, we were able to verify this soluble mediator produced by activated TEM cells is able to induce IL-10 production in vivo, and activated TEM cells are able to suppress asthma induction to a similar extent as transfer of a bulk CD4+ population (52,64).

81 Additional work needs to be completed to identify the molecule or molecules responsible for the synergistic IL-10 production in Foxp3+ Tregs. To generate a list of molecules to test, RNAseq will be performed on activated populations of TEM cells, TN cells, and Tregs, as well as unstimulated populations. We can then shorten the long list of genes by eliminating genes that are already highly expressed in unstimulated cells, as well as those that are also present in high amounts in TN cells, since they do not promote the synergistic IL-10 production. Some logical deduction can then be applied to the remaining hits by separating them based on cellular location and known impacts on inflammatory models. Hits from this narrowed list will then be tested in the synergy assays by blocking antibodies in co-cultures or supplementing with recombinant protein in mono-cultured Tregs. Once candidates have proven effective in vitro, mice will be treated with recombinant protein or blocking antibodies in combination with established asthma protocols to verify functionality in vivo. Identifying the soluble mediator, and confirming its functionality in vitro and in vivo facilitates the development of a novel therapeutic for a number of inflammatory conditions.

In developing a therapy with the soluble mediator, there are several factors which need to be optimized, in a similar fashion to those discussed earlier with the PSA therapeutic studies. These include delivery method, timing of delivery, accessibility of the molecule to the needed tissues, and protecting the molecule from protease cleavage. In this case, if a recombinant protein is used, then manipulations can be made to remove common protease cleavage sites within the protein.

82 Overall, the work presented here illustrates the importance of exposure to bacterial antigens on immune health and homeostasis. The hygiene hypothesis, while being widely accepted within the medical community, still lacks mechanistic details, and the studies shown here develop models to study this phenomenon and begin to understand one potential mechanism of immune homeostasis influenced by this hypothesis. The expansion of TEM cells by PSA is undoubtedly not the sole mechanism responsible for the positive benefits of microbial exposure, and more work is still needed to understand how this exposure affects other immune cell types, such as B cells, NKT cells, and dendritic cells, and humoral immunity components, such as secreted antibodies and complement. It has already been shown that polysaccharide specific antibodies are not sufficient to inhibit abscess formation (35), but since they can prevent bacteremia, they may have a supporting role in immune homeostasis or suppressing inflammation.

Although more work needs to be done to better understand the cellular impacts of bacterial products on immune health, the work presented here gives one potential mechanism of the hygiene hypothesis by which gastric exposure to a bacterial polysaccharide can impact immune regulation in the periphery.

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