CD4 T Cells in the Protection and Pathogenesis of Persistent Salmonella Infection

CD4 T cells in the protection and pathogenesis of persistent Salmonella infection

A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY

Tanner Michael Johanns

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Advisor: Sing Sing Way

October 2010

Acknowledgements

I would first like to acknowledge my advisor, Dr. Sing Sing Way, and thank him for his continued support over the past three years. I am extremely appreciative and grateful for the emotional, mental, and financial resources he has provided me during my graduate training. He has provided me with a firm foundation that will be applicable to all stages of my future education. Additionally, I would like to acknowledge all members of the Way lab, in particular Jim, Jared, Joe, and Calvin. Thank you for making the past few years so memorable. It was a real pleasure “to do science” with all of you. Also, I would like to acknowledge Dr. John Schreiber (Tufts Medical School) who supported me throughout my first year in graduate school.

Thank you to everyone who served on my thesis committee: Drs. Kris Hogquist

(chair), Stephen McSorley, Dave Masopust, and Marc Jenkins, and a special thank you to

Vaiva Vezys for her support and help in lab meetings. I appreciate all your helpful advice and continual support. Also, thank you to the MICaB graduate program, in particular

Louise Shand and all my colleagues, for all your patience and encouragement. I would not have been able to navigate my way through the last four years without your assistance. Similarly, I thank the MD/PhD program, especially Dr. Tucker LeBien, Susan

Shurson, and Nick Berg for supporting me over the past six years. Thank you for taking a chance on me.

I would also like to thank everyone who helped me with the work presented in this thesis, which includes Jim Ertelt, Jared Rowe, Joe Lai, Calvin Law, Ross Avant, and

Hope O’Donnell. In addition, thank you to Drs. Stephen McSorley and Marc Jenkins for graciously providing me with crucial reagents for these experiments. Thank you to the

Department of Microbiology for endowing me with the Dennis W. Watson Fellowship that has financially supported my training over the past year.

Lastly, and most importantly, I have to thank my family: my mom and dad, my sisters, Shannon and Janna, my beautiful kids, Taylor, Keira, and Trezdon, and my wonderful wife, Carrie. While I would not have made it through the past four years without your emotional support, I am particularly grateful and indebted to Carrie who has been my rock and solace. She has handled and put up with my absence and distractions with grace. You are an amazing woman and I am forever thankful to have you.

Dedication

This thesis is dedicated to my children, Taylor, Keira, and Trezdon. You will always be my greatest contribution to this world. I am so proud to be your dad.

iii

Abstract

CD4 T cells contribute a diverse and non-redundant role to host defense against infections by orchestrating the activation and quality of the innate and adaptive immune responses. The diversity of CD4 T cell function is accomplished by differentiating into a plethora of distinct effector and regulatory lineages that dictate the kinetics and extent of immune activation. However, due to the range and breadth of CD4 T cell function, the precise role and mechanism of these various effector and regulatory subsets in host immunity remains incompletely understood. As persistent infections represent a significant source of morbidity and mortality worldwide, and CD4 T cells play a critical role in protection against this class of pathogens, we sought to elucidate the relative contribution of effector and regulatory CD4 T cell subsets in the pathogenesis and protection of this class of pathogens. Using a murine model of persistent Salmonella infection, we demonstrate that CD4 T cells are required for protection during primary infection but dispensable for secondary immunity. Moreover, both host and pathogen factors limit the generation of a protective effector CD4 T cell response during primary disease including increased regulatory CD4 T cell suppressive function and Salmonella- associated virulence genes, respectively, that enables establishment and persistence of disease. Together, these findings provide novel insight into disease process of persistent

Salmonella infection that will aid in the design of future therapeutic and prevention strategies.

Table of Contents

Acknowledgements ...... i Dedications ...... iii Abstract ...... iv Table of Contents ...... v List of Tables ...... vii List of Figures ...... viii

Chapter 1: Introduction ...... 1

I. General overview of infection and the importance of CD4 T cells ...... 2 Burden of persistent infections ...... 3 The immune response during persistent infection ...... 4 CD4 T cells in host defense ...... 5

II. CD4 T cell effector lineages – host defense ...... 8 Th1 CD4 T cells ...... 10 Th2 CD4 T cells ...... 15 Th17 CD4 T cells ...... 16

III. CD4 T cell effector lineages – differentiation ...... 20

IV. Regulatory CD4 T cells ...... 26

V. Salmonella infection ...... 36 Epidemiology ...... 37 Emerging problems – Antimicrobial resistance ...... 43 Emerging problems – Vaccine efficacy ...... 44 Pathogenesis ...... 49 Experimental mouse models of Salmonella infection . . . . . 51

VI. Protection to Salmonella – Primary infection ...... 54 T cells ...... 54 B cells ...... 60

VII. Protection to Salmonella – Secondary immunity ...... 63

VIII. Thesis statement ...... 67

Chapter 2: Naturally-occurring altered peptide ligands control Salmonella-specific CD4+ T cell proliferation, IFN-γ production, and protective potency ...... 69

Introduction ...... 72 Results ...... 75 Discussion ...... 85 Materials and Methods ...... 89

Chapter 3: Regulatory T Cell Suppressive Potency Dictates the Balance between Bacterial Proliferation and Clearance during Persistent Salmonella Infection ...... 99

Introduction ...... 101 Results ...... 104 Discussion ...... 118 Materials and Methods ...... 125

Chapter 4: Activated monoclonal and endogenous antigen-specific CD4 T cells display differences in survival during Salmonella infection . . . . . 141

Introduction ...... 143 Results ...... 146 Discussion ...... 155 Materials and Methods ...... 160

Chapter 5: Early eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection ...... 168

Introduction ...... 170 Results ...... 173 Discussion ...... 177 Materials and Methods ...... 181

Chapter 6: Concluding Statements ...... 189

References ...... 198

List of Tables

Chapter 2

Table 1 ...... 92

vii

List of Figures

Chapter 2 Figure 1 ...... 93 Figure 2 ...... 94 Figure 3 ...... 95 Figure 4 ...... 96 Figure 5 ...... 97 Figure 6 ...... 98

Chapter 3 Figure 7 ...... 129 Figure 8 ...... 130 Figure 9 ...... 131 Figure 10 ...... 132 Figure 11 ...... 133 Figure 12 ...... 134 Figure 13 ...... 135 Figure 14 ...... 136 Figure 15 ...... 137 Figure 16 ...... 139 Figure 17 ...... 140

Chapter 4 Figure 18 ...... 162 Figure 19 ...... 163 Figure 20 ...... 164 Figure 21 ...... 165 Figure 22 ...... 167

Chapter 5 Figure 23 ...... 183 Figure 24 ...... 184 Figure 25 ...... 185 Figure 26 ...... 186 Figure 27 ...... 187 Figure 28 ...... 188

viii

Chapter 1

Introduction

I. GENERAL OVERVIEW OF INFECTION AND THE IMPORTANCE OF CD4 T CELLS

Infection results from a potentially pathogenic microbe invading a susceptible host, and the outcome of a given infection is determined by the characteristics of the respective host-pathogen interaction [1]. In other words, the kinetics, severity, and resolution for a given infectious disease are dependent upon the dynamics of the host- pathogen relationship. Therefore, the study of the host-pathogen interaction can provide insight into both the pathogenesis of and protection against infectious diseases. For example, by investigating the pathogen side of this relationship, one can identify the factors involved in the establishment and progression of disease. Conversely, examination of the host aspect of this interface can provide information into the requirements for host defense and/or prevention of disease.

In general, infection results in either an acute or persistent disease state. An acute infection by definition refers to a relatively brief disease course and occurs when a pathogen invades an incompatible host, which means the host and pathogen are unable to exist symbiotically. As a result, this antagonistic interaction favors a one-sided outcome that results in either host or pathogen survival – but not both. Conversely, a persistent infection refers to a protracted or indefinite disease state where the host and pathogen are able to establish an equilibrium in which both the host and pathogen can mutually co- exist. This type of disease state requires that not only the pathogen has adapted means of surviving long-term within the host but the host, in turn, is able to survive despite the continued presence of the pathogen.

Burden of persistent infections

While the global pandemic caused by influenza virus in 1918 or the bacterium,

Yersinia pestis (the causative agent of the bubonic plague, or “Black Death,” of the 14th century), both illustrate the impact that a single acute infection can have on the human population, persistent infections are currently the most significant source of morbidity and mortality worldwide. In fact, the greatest causes of human death due to infectious disease currently are attributed to persistent disease. The World Health Organization’s

“big three,” which includes HIV, Mycobacterium tuberculosis, and Plasmodium falciparum the causative agents of AIDS, tuberculosis, and malaria, respectively, are estimated to cause a combined 4.4 million deaths, annually [2]. Moreover, more than 250 million people were estimated to be persistently infected with one of these pathogens in

2004 [2]. In addition, the newly-coined “Neglected Tropical Diseases,” which largely comprises helminth or parasitic worm infections, are estimated to be persistently prevalent in more than 1 billion people globally and contribute to an additional half million deaths annually [3]. Together, persistent infection is truly a devastating public health concern and the lack of highly effective, widely accessible prevention methods indicates that these pathogens will remain a major reservoir of disease burden. Therefore, the overall basis of this work is to gain deeper insight into the mechanisms that drive the pathogenesis of and mediate protection to persistent infection that can aid the future development of more effective therapeutic or preventative treatment strategies, such as vaccines, against these diseases.

The immune response during persistent infection

During persistent infection, the host-pathogen relationship is a highly dynamic interchange where the balance between the pathogen and host is largely dictated by the quality of the host immune response. For example, if a host becomes immunocompromised, there is the potential for this interaction to favor pathogen growth, which can then lead to reactivation and/or progressive disease. This is the case in patients given high dose immunosuppression following allogeneic hematopoietic stem cell transplantation or solid organ transplantation that frequently suffer from reactivation of latent Epstein-Barr virus (EBV), cytomegalovirus (CMV) or Mycobacterium tuberculosis infections [4-6]. In contrast to a host becoming immunocompromised, the converse can happen as well where the host immune response becomes reinvigorated and can clear a previously persistent infection. For example, chronic hepatitis B virus is estimated to afflict approximately 400 million people worldwide [7] and approximately 1-5% of these patients annually will undergo spontaneous clearance of their persistent infections associated with an enhanced antiviral immune response [8]. Based on these observations, the quality of the host immune response clearly impacts the balance of the host-pathogen relationship in persistent disease, and therefore, determining the factors that influence the quality of the host immune response will further our knowledge of elements that drive the pathogenesis and host control of persistent infection.

CD4 T cells in host defense

CD4 T cells are referred to as helper cells as they secrete a wide range of cytokines that are involved in coordinating all aspects of the host immune response, which includes modulating the effector functions of cells from both the innate and adaptive systems. CD4 T cells play an important, non-redundant role in host defense, and the essential role of this cell population is most evident in a number of naturally-arising human conditions in which the CD4 T cell population is selectively absent. The most prominent of these CD4 T cell deficiency disorders is HIV-associated AIDS [9,10], however similar clinical manifestations have also been observed in patients with idiopathic CD4 T cell lymphocytopenia [11,12], drug-induced CD4 T cell lymphocytopenia following chemotherapy with cytotoxic agents or long-term corticosteroid use [13,14], and bare lymphocyte syndrome (a MHC class II deficiency)

[15]. Patients with a CD4 T cell lymphocytopenia typically suffer from a wide range of recurrent infections that can be severe, disseminated, and often fatal [10,16-18]. The most common of these manifestations include bacterial or fungal pneumonias caused by

Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenza b and

Pneumocystis jiroveci; reactivation of latent Herpes family viral infections (EBV, CMV, and varicella zoster virus [VZV]) or Mycobacteria tuberculosis; disseminated or invasive bacteremia or fungemia caused by Salmonella, Mycobacteria, Candida, Histoplasma,

Aspergillus, and Cryptococcus. Additionally, protozoan infections with Cryptosporidium,

Toxoplasma, or Entamoeba histolytica are frequent causes of chronic diarrhea in these patients. The increased susceptibility of these patients to infectious complications caused by this wide array of microbial species suggests that CD4 T cells are important for host

5 defense to many classes of pathogens. Moreover, the increased risk of developing not only newly-acquired infections, but reactivation of pre-existing latent infections such as the Herpes family of viruses and Mycobacteria tuberculosis further implicate CD4 T cells as an important component in controlling the balance of the host-pathogen interaction in both acute and persistent infections.

Perhaps an even more interesting observation is that while patients with CD4 T cell lymphocytopenias are highly-susceptible to developing systemic infections with strains of the intracellular bacterial pathogens, Salmonella and Mycobacteria, that are weakly virulent and typically do not cause disease in immunocompetent hosts, which supports the general role for CD4 T cells in the control of these pathogens, there exists strains within each of these species, specifically Salmonella typhi and Mycobacteria tuberculosis, that are capable of causing a persistent clinical disease in immunocompetent hosts. This observation suggests that these strains of pathogens have adapted mechanisms that allow them to effectively evade the normal host CD4 T cell response in order to invade and persist within a human host. Thus, these pathogens represent ideal models in which to study the contribution of CD4 T cells to the overall balance between host and pathogen in a persistent infection as well as the role of this T cell subset in disease progression and host defense.

Therefore, based on the observations that 1) persistent infections are a major global health concern against which no effective treatment strategies currently exist, 2) the host-pathogen relationship is a dynamic interchange that dictates disease outcome and is largely influenced by the quality of the host immune response, 3) that CD4 T cells are

6 important for host defense against a large number of pathogens, including those that cause persistent infections, and 4) evasion of the host CD4 T cell response represents a potential mechanism by which pathogens are able to establish a persistent infection, the goal of this thesis work is to identify the factors that impede or promote the development of a protective CD4 T cell response during a persistent infection as well as establish the relative contribution of CD4 T cells in host defense against persistent pathogens during primary and secondary infection. The work presented herein was conducted using a murine model of systemic, persistent Salmonella infection that is highly comparable to the human disease state. Below, a general overview of the role of CD4 T cells in host protection against various pathogens as well as the factors that mediate their protective functions and drive their development will be discussed. Following the discussion on

CD4 T cells in infection, the function of CD4 T cells will be further discussed as it directly relates to their role in protection against Salmonella.

II. CD4 T CELL EFFECTOR LINEAGES – HOST DEFENSE

CD4 T cells are important for host defense against a broad range of pathogens. As such, CD4 T cells must execute an equally diverse range of effector functions, which they accomplish by differentiating into a number of distinct lineages. Each effector subset is defined by the cytokines they produce as these lineage-specific cytokines drive the function, and for some cytokines, the differentiation (discussed later), of each CD4 T cell subset. For instance, the Th1 CD4 T cell subset is characterized by the production of the type 1 cytokines – IFN-γ, IL-2, and lymphotoxin-α (also known as TNF-β), whereas the production of type 2 cytokines – IL-4, IL-5, IL-9, and IL-13 defines the Th2 CD4 T cell subset [19,20]. However, as neither of these subsets express the full range of cytokines under all contexts examined [21], each subset has since become associated with a single lineage-defining cytokine – IFN-γ for Th1, and IL-4 for Th2. In addition to Th1 and Th2 effector subsets, Th17 CD4 T cells represent a third effector lineage that is so-named based on the preferential secretion of the IL-17 cytokine family members, IL-17A and IL-

17F, however, production of IL-21 and IL-22 is also associated with this lineage of CD4

T cells [22]. Importantly, while each of these distinct effector lineages were initially identified and characterized in mice, these same subsets have since been demonstrated to possess similar effector properties and functions in humans as well [23-28].

The diverse and distinct repertoire of effector cytokines produced by these various

CD4 T cell effector subsets are critical for determining the qualitative properties of the subsequent host immune response. This is accomplished by coordinating the recruitment

8 and activation of additional immune components that are more directly involved in the actual control and/or elimination of the offending pathogen. In general, type 1 cytokines, in particular IFN-γ, are associated with the activation of monocytes and macrophages, whereas the Th2 CD4 T cell-associated cytokines are involved in the activation of basophils, eosinophils and mast cells. Additionally, the Th17 subset predominantly mediates its effects by modulating the extravasation and activation of neutrophils. In addition to modulating various cellular components of the innate immune system, CD4 T cells are also intimately involved in regulating the properties of the adaptive immune response such as isotype class switching by B cells or priming and sustaining a cytotoxic

CD8 T cell response.

Thus, one can imagine the distinct properties of each CD4 T cell subset necessitates that not only is the appropriate differentiation of CD4 T cells critical for proper host defense but an inappropriate CD4 T cell response could be detrimental.

Therefore, determining the precise role of each CD4 T cell subset in host defense against various pathogens has important implications for the design of therapeutic and prophylactic therapies to treat or prevent infections. This has been best exemplified in the development of an effective vaccine against respiratory syncytical virus (RSV). In patients administered a formalin-inactivated RSV vaccine, there was a reported increase in both incidence and severity of disease compared to unvaccinated individuals [29]. It was later demonstrated in relevant mouse models of the disease that this loss of protection was associated with a Th2 type CD4 T cell response and the presence of IL-4 rather than a protective IFN-γ producing Th1 CD4 T cell response [30]. Similar to RSV

9 immunization, vaccination of IFN-γ deficient mice with formalin-inactivated Borrelia burgdorferi, the spirochete responsible for human Lyme disease, results in a Th17 dominant response that results in a severe destructive arthritis upon secondary infection with Borrelia burgdorferi [31]. Importantly, synovial fluid from patients with Lyme disease-associated arthritis contains a prominent population of CD4 T cells that readily express IL-17 upon ex vivo restimulation [32] suggesting a skewed Th17 response leads to more severe disease outcomes in humans as well. Thus, a thorough knowledge of both the mediators of protection and the factors involved in generating an appropriate CD4 T cell response is required before progress can be made in designing more effective vaccines against a broader range of pathogens.

Th1 CD4 T cells

T cell-mediated immunity (CMI) is absolutely required for protection against intracellular pathogens since components of the humoral immune response, which includes antibodies and complement, are unable to access microbes within this protected niche [21,33]. While both CD4 and CD8 T cells are involved in CMI and contribute broadly to protection against this class of pathogen, IFN-γ producing Th1 CD4 T cells are considered the primary mediators of protection against a subset of these pathogens, which comprise microbes that are capable of invading, replicating and residing within host macrophages [34]. Indeed, the importance of Th1 CD4 T cells in protection and impact of appropriate Th1 CD4 T cell differentiation on host defense against such intracellular pathogens was first described in the murine model of Leishmania infection, an obligate

10 intramacrophage protozoan pathogen [35,36]. The requirement for Th1 CD4 T cells has since been expanded to include additional classes of pathogens that have also evolved evasion mechanisms enabling them to survive within the intracellular environment of macrophages. These pathogens include: bacteria (i.e. Salmonella [34], Mycobacterium

[34], and Chlamydia [37]); fungi (i.e. Histoplasma [38] and Cryptococcus [39,40]); and other protozoa (i.e. Trypanosoma [41]). Importantly, several of these pathogens, in particular Salmonella, Mycobacterium, and Leishmania, are able to establish persistent infections in the host suggesting the generation of an ineffective Th1 type response leading to incomplete clearance of these pathogens.

Leishmania infection can result in either a self-limiting cutaneous disease or fatal visceral disease, and in mice, the type or extent of the disease that occurs is dependent upon the genetic background of the individual mouse strain infected [42,43]. For example, resistant strains of mice such as C57BL/6 and C3H are able to restrict the dissemination of the pathogen resulting in a localized skin lesion that will eventually heal without need for therapeutic intervention. Conversely, susceptible mice, which include the BALB/c strain, develop a disseminated visceral disease that is fatal if not treated.

The differences in susceptibility and disease progression between these various strains of mice are directly related to the quality of the CD4 T cell response generated.

For instance, restriction and resolution of the primary lesion is critically dependent upon the development of a Th1 CD4 T cell response and production of IFN-γ whereas disseminated and fatal disease results from the generation of an IL-4 producing Th2 type response [44-50]. As such, a Th1 T cell line generated against Leishmania-specific

11 antigens is able to transfer protection to susceptible BALB/c mice whereas the transfer of a Leishmania-specific Th2 T cell line results in exacerbated disease [51].

While these transfer experiments demonstrate the ability of Th1 CD4 T cells to confer protection against Leishmania, it is the production of IFN-γ, the primary Th1 effector cytokine, that is primarily responsible for the protective effects of Th1 CD4 T cells against Leishmania infection. For example, the in vivo administration of neutralizing anti-IFN-γ antibody to resistant mice during infection or infection of resistant mice with impaired IFN-γ signaling (IFN-γR1 deficient) results in a progressive form of the disease that is comparable to what is observed in susceptible mice and corresponds to a reciprocal increase in production of Th2-associated cytokines such as IL-4 and IL-5 [44,52].

Likewise, the administration of recombinant IFN-γ to susceptible mice results in protection against Leishmania infection that is associated with the development of a Th1- skewed, IFN-γ producing CD4 T cell phenotype [49].

In contrast to the protection provided by Th1 CD4 T cells and IFN-γ production against Leishmania infection, the generation of a Th2 CD4 T cells actually leads to an exacerbated disease course as IL-4 suppresses macrophage activation and function [53].

In susceptible mice with a deficiency in IL-4 production or IL-4 signaling (STAT6- deficient), an otherwise lethal Leishmania infection is resolved in these mice, however, the increase in protection corresponds to the reciprocal production of IFN-γ by CD4 T cells that is absent in control mice [50,52]. Moreover, the administration of neutralizing anti-IL-4 antibody to susceptible mice during Leishmania infection also reverts the CD4

T cells of these mice to a IFN-γ producing phenotype that results in protection against

12 disseminated disease [54]. Importantly, in addition to mouse models, a similar association between the type of CD4 T cell response generated and clinical disease outcome has also been described in humans infected with Leishmania. Localized, cutaneous leishmaniasis is associated with a Th1 type response and high levels of serum IFN-γ, whereas visceral leishmaniasis, the more severe form of the disease, is associated with a Th2-skewed response and high levels of serum IL-4 and IgE [29,55].

This critical dichotomy between appropriate CD4 T cell differentiation and disease outcome is further demonstrated in patients with leprosy, which is a disease caused by the facultative intracellular bacterial pathogen, Mycobacterium leprae. Patients can present with a broad spectrum of clinical disease manifestations, and relative disease severity is directly correlated to the quality of the CD4 T cell response [33,56]. For example, tuberculoid and lepromatous leprosy represent two distinct poles of disease manifestation where tuberculoid leprosy is a less severe form characterized by relatively few skin lesions that when cultured yield very low numbers of organisms compared to patients with lepromatous leprosy that have multiple, widespread skin lesions containing large numbers of recoverable organisms [56]. Furthermore, the enhanced level of host protection in tuberculoid leprosy is associated with the presence of a strong Th1 type response as CD4 T cells isolated from the lesions of tuberculoid patients produced IFN-γ in response to M. leprae stimulation whereas T cells from lepromatous patients were found to secrete the type 2 cytokines, IL-4 and IL-5 [56,57]. Moreover, naturally- occurring “reversal reactions” have been described in leprosy patients in which lepromatous-type patients upgrade clinically towards the tuberculoid pole and this

13 reversal in disease progression is associated with type 2  type 1 cytokine switch by intra-lesion CD4 T cells [58-61].

These results from experimental Leishmania infection models in mice and clinical studies of leishmaniasis and leprosy in patients support a critical role of Th1 CD4 T cells in protection against intracellular pathogens as well as the need for proper CD4 T cell differentiation to mediate optimal host protection against disease progression. Perhaps more importantly, however, the reversal reaction observed in leprosy patients suggests that the quality of the CD4 T cell response during a persistent infection can directly dictate the dynamic nature of the host-pathogen interaction and implicates a further role for Th1 CD4 T cells and IFN-γ production in mediating long-term control of other intracellular pathogens that cause persistent disease such as Salmonella enterica.

Importantly, however, as Salmonella enterica, Mycobacterium, and Leishmania are all able to establish persistent infections in otherwise immune-competent hosts, it suggests that the development of a protective CD4 T cell response following infection with these pathogens is ineffective or incomplete. Whether this is due to inappropriate differentiation of CD4 T cells into lineages other than Th1 or a result of pathogen- associated evasion strategies that either impair the generation of or evade detection by a

Th1 CD4 T cell response, remains unclear. Therefore, determining the factors that dictate the appropriate development of a protective Th1 CD4 T cell response during such infections is essential for understanding the pathogenesis of these diseases as well as aiding in the design of more effective therapeutic interventions.

Th2 CD4 T cells

While Th2 CD4 T cells are associated with exacerbated disease against intracellular pathogens, this subset of CD4 T cells has been shown to play a crucial role in protection against parasitic worm, or helminth, infections. The protective effects of

Th2 CD4 T cells corresponds to the production of type 2 cytokines (i.e. IL-4, IL-5 and

IL-9) that recruit and activate various cellular components of the innate immune response such as eosinophils, basophils and mast cells that are required for control of such infections as well as IL-4 and/or IL-13 production that induces IgE class switching by B cells which further augments the activation of these various effector cell types.

Similar to Leishmania infection, appropriate CD4 T cell differentiation is crucial for protection against helminth infection and has been well-characterized following infection with the intestinal nematode, Trichuris muris. The development of a Th2 CD4 T cell response in mice results in rapid clearance of the parasite and this protective phenotype is characterized by the presence of the type 2 cytokines IL-4, IL-5, IL-9, and

IL-13, high titers of IgE and mucosal eosinophilia and mastocytosis [62,63]. Conversely, mice that inherently generate an IFN-γ/Th1-skewed CD4 T cell response following

Trichuris muris infection develop chronic disease [64]. Furthermore, the administration of recombinant IL-4 or IL-13 to mice with a predisposition for developing Th1 type responses confers protection to these mice demonstrated by early Trichuris worm expulsion. In contrast, mice that lack IL-4R and IL-13R signaling are unable to expel the pathogen resulting in chronic Trichuris infection [65-67].

In addition to Trichuris muris infection, it is important to note that similar results have been demonstrated in other murine-helminth infection models including

Nippostrongylus braziliensis [68,69]; Trichinella spiralis [70-73], Heligmosomoides polygyrus [69], and Brugia malayi [74]. Moreover, the importance of a Th2 CD4 T cell response in protection against parasitic worm infections has been suggested in humans as well. For example, the development of protective immunity against reinfection with the intestinal trematode, Schistosoma mansoni, the causative agent of schistosomiasis, has been associated with high titers of pathogen-specific IgE and elevated IL-5 levels [75].

Thus, these data not only support the importance of Th2 CD4 T cells in host defense against helminth infections, but also further suggest that the quality of the CD4 T cell response (i.e. Th1 versus Th2) can impact the host-pathogen interaction determining the kinetics of the infection (i.e. acute or chronic disease). Together with the observations obtained from Leishmania and Mycobacteria leprae infections, these data demonstrate that CD4 T cells play a critical role in dictating the outcome of the host-pathogen relationship, which determines host susceptibility, disease severity, and duration of illness of the resulting infection.

Th17 CD4 T cells

Like Th2 CD4 T cells, Th17 cells produce a large number of effector cytokines that play both distinct and redundant roles in host defense. As such, the relative contribution of the various effector cytokines, and by extension the role of Th17 CD4 T cells, to protection may either be overlooked or underestimated if the specific Th17-

16 associated cytokine involved in protection against a specific pathogen is not examined.

However, the current data examining the role of Th17 CD4 T cells in host defense would suggest that the effects are primarily through the recruitment of neutrophils and activated

T cells to sites of infection.

Several clinical diseases have been identified where a deficiency in this particular cell subset exists. These patients have provided some preliminary insight into the role of

Th17 CD4 T cells in host defense. For instance, patients with chronic mucocutaneous candidiasis have been shown to possess reduced numbers of circulating IL-17 producing

T cells [76]. Moreover, it has recently been identified that patients with hyper-IgE syndrome (HIES), a disease characterized by recurrent and/or severe skin infections caused predominantly by Staphylococcus aureus and Candida albicans as well as recurrent bacterial pneumonias (Staphylococcus aureus, Streptococcus pneumoniae, and

Haemophilus influenza b) is due to mutations in the STAT3 gene which leads to a select

Th17 CD4 T cell deficiency [77,78]. Together, these observations have led to the current hypothesis that the primary role of Th17 CD4 T cells in protection is against extracellular bacteria and fungi.

Additional clinical evidence supports this predilection for Th17 CD4 T cell involvement in protection to various bacterial and fungal pathogens but with a preferential involvement at epithelial and mucosal surfaces. For example, human Th17 cells have been shown to express the chemokine receptors CCR2, CCR4, and CCR6

[79,80]. While CCR2 and CCR4 have been shown to be predominantly skin-homing molecules in both normal and diseased states (i.e. psoriasis) [81], the CCR6 ligand,

CCL20, is expressed at steady state by epithelial cells of mucosa-associated lymphoid structures such as Peyer’s patches [79], and can be further induced in inflamed mucosal sites by IL-17 [82]. Additionally, CCR6+ IL-17 producing CD4 T cells were readily isolated from the small bowel of patients with active Crohn’s disease [83], and

CCR6+CCR4+ memory Th17 CD4 T cells specific to Candida albicans-derived antigens were isolated from the peripheral blood of human immune donors [79]. Thus, both the phenotype and antigen-specificity of Th17 cells poises them to be important for protection against specific types of pathogens that preferentially colonize peripheral sites such as skin and mucosal surfaces.

In various mouse models where the differentiation (IL-23p19 deficient mice or anti-IL-23 neutralizing antibody) or effector function (IL-17A, IL-17R deficient mice, or neutralizing antibody to IL-17A or IL-22) of Th17 cells is compromised provide additional insight into the role for these cells in protection. Klebsiella is a gram-negative bacterium that is an important cause of nosocomial infections and pneumonia in humans

[84]. In IL-17R deficient mice, intranasal infection with Klebsiella resulted in increased mortality and disseminated infection compared to wild-type control mice [85]. A similar loss of protection to Klebsiella was observed in IL-17A deficient [86] and IL-23 deficient mice [87]. Interestingly, neutralization of the effector cytokine, IL-22, prior to Klebsiella challenge resulted in higher bacterial burdens and faster rates of death compared to IL-

17A-deficient mice suggesting a preferential role for IL-22 in protection to this pathogen compared to IL-17 [86]. As would be expected, this loss of protection was associated

18 with decreased production of chemokines and neutrophil emigration into infected lung tissue [86].

Aside from Klebsiella, Th17 cells have been implicated to be important for protection against other bacterial species as well. For instance, increased bacterial burden associated with decreased IL-17 production and neutrophil recruitment was also reported following an intranasal infection with Mycoplasma pulmonis in mice treated with anti-IL-

23 antibody prior to infection [88], and intragastric infection with Citrobacter, another gram-negative bacterium, also resulted in decreased survival in IL-23 deficient mice [89].

Thus, Th17 cells do indeed play an important and apparently non-redundant role in protection to a variety of extracellular bacteria.

Moreover, the increased dissemination of Klebsiella and Citrobacter observed in the above mouse models using Th17-deficient mice further suggests an important role for

Th17 cells in limiting the extent of infection at the initial site of colonization. Additional experimental data supporting this role of Th17 cells comes from macaque models of SIV infection. Early after SIV infection, there is a preferential loss of Th17 CD4 T cells in the lamina propria of infected macaques, which leads to increased bacterial dissemination following a subsequent oral challenge with Salmonella [90] suggesting that the normal immune barrier limiting dissemination of gastrointestinal pathogens is lost in the absence of Th17 cells.

Consistent with the clinical data suggesting an important correlation between

Th17 cells and protection against recurrent Candida infection, Th17 has been shown to play an important role in protection against a number of fungal pathogens in various

19 mouse models as well. For example, the depletion of IL-23 or infection of IL-23 deficient mice with Pneumocystis carinii infection results in higher fungal burdens and delayed clearance associated with decreased neutrophil influx [91]. Moreover, IL-23 deficient mice are also highly susceptible to infection with Cryptococcus neoformans infection

[92], and this increased susceptibility was associated with impaired granuloma formation within the liver to contain the primary infection enabling increased pathogen dissemination to the brain ultimately resulting in death. Similarly, IL-17R deficient mice display decreased survival associated with enhanced dissemination of Candida albicans following an intravenous infection that was associated with decreased neutrophilic activity in parenchymal tissues [93].

Together, these results support an important role for IL-17 and Th17 CD4 T cells in controlling certain intracellular and extracellular bacterial and fungal pathogens by restricting the dissemination of these organisms within the host. Furthermore, current data suggests an important role for this cell subset as sentinels at peripheral sites such as mucosal or skin epithelium where the initial host-pathogen interaction occurs. Thus, the locale of these effector CD4 T cells implicates them as potential keys to enhanced vaccine efficacy against a number of pathogens.

III. CD4 T CELL EFFECTOR LINEAGES - DIFFERENTIATION

The proper differentiation of CD4 T cells is absolutely required for effective host defense following infection, and each CD4 T cell effector subset is associated with

20 protection against a specific set of pathogens. Therefore, identifying the factors that drive

CD4 T cell lineage decisions during natural infection and/or immunization conditions is critical for understanding the role of CD4 T cells in determining the outcome of various host-pathogen interactions that result in either protective (i.e. during primary or secondary immunity) or pathogenic (i.e. in the case of inappropriate lineage differentiation) effects. Unfortunately, the current understanding of the factors that dictate the fate decision in CD4 T cell differentiation has been largely limited to in vitro or non- infection in vivo systems. Furthermore, the differentiation of CD4 T cells has been proposed to be influenced by three interrelated signals that include TCR signaling, costimulation and cytokine milieu, yet the differentiation of CD4 T cells has largely been attributed to cytokines. Thus, the relative contribution of these individual signals to the overall lineage choice of CD4 T cells and how changes in these various factors influences host protection following infection has yet to be determined. As such, the current models of Th1, Th2 and Th17 differentiation will be discussed in this section.

For the differentiation of a naïve CD4 T cell into the Th1 lineage, a two-step model based on previous in vitro data and more recent mathematical modeling has been proposed [94]. In the first step, TCR signaling via the NFAT pathway and IFN-γ stimulation via the STAT1 pathway combine to induce the expression of the transcription factor, T-bet. T-bet is considered the master regulator of Th1 differentiation as its expression is both necessary and sufficient for the development of a Th1 phenotype [95].

As would be expected, the lineage-defining transcription factor, T-bet, then binds to the promoter region of the gene encoding the lineage-defining cytokine, IFN-γ, inducing its

21 expression [96]. Interestingly, as IFN-γ and T-bet are both potent inducers of each others expression, it would seem that a IFN-γ  T-bet  IFN-γ autocrine/paracrine feedback loop would be sufficient to sustain and maintain the Th1 phenotype. However, the continued presence of IFN-γ was shown to be insufficient to maintain T-bet expression in

CD4 T cells in vitro [94] suggesting an additional IFN-γ independent step is required for the continued differentiation down the Th1 lineage. This second step in T-bet expression and Th1 differentiation was found to be provided by IL-12 signaling via the STAT4 pathway [94]. Surprisingly, while IFN-γ actually induces the expression of the IL-12 receptor subunit, IL-12Rβ2, its expression is inhibited by continued TCR signaling, which is required for the initial expression of T-bet and IFN-γ [94]. Thus, TCR signaling needs to be terminated before IL-12Rβ2 expression is permitted. Once TCR signaling is abolished, IL-12 is able to induce the upregulation of T-bet as well as other Th1- associated transcription factors such as HLX and RUNX3, and it is the expression of these three transcription factors along with STAT4 that ultimately stabilizes the Th1 lineage [94,97-99].

As for the Th2 lineage, differentiation appears to be much more straightforward.

Along with TCR signaling, the stimulation of a naïve CD4 T cell in the presence of IL-4 activates the transcription factor, GATA3, in a STAT6-dependent manner [100-105].

GATA3 is believed to be the master regulator of Th2 differentiation, and like T-bet in

Th1 differentiation, GATA3 expression is both necessary and sufficient for Th2 development in naïve CD4 T cells [106-108]. IL-4 induced GATA3 then binds to the

“Th2 locus” where it facilitates chromatin remodeling to enable the expression of the

22 type 2-associated cytokines IL-4, IL-5 and IL-13 encoded within the locus [109].

Additionally, IL-4 is able to amplify and maintain its own signal by inducing the expression of the transcription factor c-Maf, which is required for high levels of IL-4 expression [110], and this appears to be sufficient for maintaining a stable Th2 phenotype.

Th17 CD4 T cell differentiation, like Th1 and Th2 differentiation, has also been proposed to progress in a stepwise process that involves 1) an initial differentiation phase followed by 2) an amplification and 3) stabilization phase [111,112]. The early induction signals that determine if a naïve CD4 T cell will differentiate into a Th17 lineage is provided by the cytokines TGF-β and IL-6 [89,113,114]. These cytokines stimulate the upregulation of the transcription factors, RORγt and RORα, which are currently considered the master regulators of Th17 differentiation [115,116]. RORγt and RORα along with IL-6 are required for the initial expression of IL-17 [115-117]. Comparatively, while TGF-β and IL-6 are also required for the induction of RORγt expression in human naïve CD4 T cells [118], the pro-inflammatory cytokine, IL-1β is also required for the production of IL-17 by Th17 CD4 T cells in humans [119-122].

Following the initial deviation of naïve CD4 T cells into the Th17 CD4 T cell lineage, these cells then undergo an amplification step. IL-6, in addition to its role in inducing RORγt and RORα, also signals the production of IL-21 [123]. IL-21, a member of the common gamma chain family of cytokines that also includes IL-2, IL-4, IL-9, IL-

13, and IL-15, triggers the expansion of Th17 cells via an autocrine/paracrine feedback loop [123,124]. Interestingly, both IL-6 and IL-21 mediate their downstream effects

23 through the transcription factor, STAT3. As such, evidence suggests that the presence of

IL-21 along with TGF-β is sufficient to induce Th17 differentiation in the absence of IL-

6 [125]. Such an IL-21-dependent, IL-6-independent alternative Th17 differentiation pathway has also been shown to occur in human CD4 T cells as well [122].

IL-23 is composed of the IL-23p19 subunit and the shared IL-12 subunit, IL-

12p40 [126], and is produced predominantly by dendritic cells and phagocytic cells upon activation [127]. It was originally postulated that IL-23 was the main cytokine involved in the differentiation and expansion of Th17, however, the lack of expression of the IL-

23R on naïve CD4 T cells suggested that the role of IL-23 may, in fact, occur at later stages of differentiation instead [117]. The fact that both IL-6 and IL-21, via STAT3 signaling, are capable of inducing the expression of the IL-23R [123] led to the current hypothesis that the predominant role of IL-23 is to stabilize and maintain the Th17 phenotype in already differentiated cells [111,114]. Consistently, IL-23 has been shown to induce expression of RORγt and together promote the expression of IL-17 [123].

The differentiation of Th1, Th2, and Th17 lineages appear to be tightly associated with the cytokine milieu present at the time of activation. However, as mentioned, other factors have also been implicated to control CD4 T cell fate decisions including TCR signaling. A role for differences in TCR signaling influencing CD4 T cell lineage development is supported by the observation that distinct intracellular calcium flux profiles have been reported among in vitro polarized Th1, Th2 and Th17 CD4 T cells derived from the same monoclonal TCR transgenic mouse following TCR restimulation

[128-130]. This observation suggests that the various CD4 T cell effector lineages have qualitatively different TCR signaling.

The use of altered peptide ligands (APLs) has provided further experimental evidence to support the contribution of TCR signal quality on CD4 T cell lineage development [131]. APLs are defined as variants of CD4 T cell-specific peptides that contain a single amino acid substitution that alters the TCR-peptide interaction.

Stimulation of a population of monoclonal Mycobacterium-specific transgenic CD4 T cells in vitro with the parental cognate antigen stimulates these cell to differentiate solely into IFN-γ producing Th1 effector cells [132]. Conversely, stimulation of this same population of cells with an APL that contains a glycine to alanine substitution in a TCR contact residue produced an exclusive IL-4 secreting Th2 phenotype [132]. In addition, using an in vivo peptide immunization model, it has been demonstrated that an immune- dominant peptide derived from the human collagen IV protein, which is presented by the

MHC class II I-Ab molecule in mice primes IL-4 but not IFN-γ production in the bulk

CD4 T cell population [133]. In contrast, a superagonist APL derived from this parental peptide primes robust IFN-γ but no IL-4 production by bulk CD4 T cells [133]. Together, these results using APLs provides proof-of-concept that TCR signal quality alone can impact CD4 T cell differentiation. However, the impact of changes in TCR signal quality on antigen-specific CD4 T cell differentiation and function in host defense following an in vivo infection is currently unknown.

Thus far, the preceding sections have specifically focused on the role and development of effector CD4 T cell subsets. However, consistent with the tremendous

25 flexibility of CD4 T cell functions, they are also capable of differentiating a subset of cells that possess immunoregulatory properties to limit and control the extent of the host immune response during infection. The regulatory aspect of CD4 T cell function in host defense will be discussed in the next section.

IV. REGULATORY CD4 T CELLS

While CD4 T cells are able to differentiation into a diverse repertoire of effector lineages that are important for protection against pathogens, it has also been appreciated that CD4 T cells have the capacity to acquire immune-regulatory functions that are also critical for the proper development and function of host defense [134]. The best- characterized member of this suppressive subset of CD4 T cells is the Foxp3-expressing regulatory T cell, or Treg. Foxp3 is a transcription factor constitutively expressed by

Tregs and is required for the induction and maintenance of their suppressive phenotype

[135,136]. While Foxp3+ Tregs arise naturally within the thymus (natural Tregs, or nTregs), it has also been recognized that Foxp3 expression can be induced within naïve

CD4 T cells in the periphery resulting in the de novo generation of CD4 T cells with immune suppressive functions (named inducible Tregs, or iTregs) [137-141].

Furthermore, several other subsets of suppressive CD4 T cells, which do not express

Foxp3, but produce various immunosuppressive cytokines have also been described

[138,142]. These include the IL-10 producing Tr1 cells and TGF-β secreting Th3 cells

[134]. However, the lack of lineage-specific markers to identify these cells in vivo has

26 hindered the study of their development and precise function in host defense. As such, only Foxp3+ Tregs will be discussed further as their role in the host-pathogen interaction has been more extensively identified. Furthermore, as there is currently no known marker that can delineate thymic-derived Foxp3+ nTregs from peripherally-induced Foxp3+ iTregs, Foxp3+ Tregs will be discussed as a single lineage subset since there is no clear evidence that these two populations possess functional differences, particularly in the context of an infection.

As mentioned, Tregs comprise an immune-suppressive subset of CD4 T cells that express the transcription factor, forkhead box p3 (Foxp3), and the expression of Foxp3 is both necessary and sufficient to confer the induction and maintenance of suppressive properties in CD4 T cells [136]. The importance of this Foxp3-expressing subset of CD4

T cells is best illustrated in humans and mice with mutations in the Foxp3 gene locus

[143-145]. Both the Scurfy phenotype in mice [146] and IPEX (immune dysregulation, polyendocrinopathy, enteropathy and X-linked) syndrome in humans [147] are due to a lack of functional Foxp3 expression and characterized by the spontaneous development of a lymphoproliferative disorder that results in a destructive multi-organ autoimmune disease with severe disease-associated immunopathology which is fatal within the first three weeks of life for mice and within the first year of life for humans. Based on these genetic deficiencies in Foxp3 expression, it became clear that Foxp3+ Tregs are critical for maintaining immunological self-tolerance and homeostasis.

In addition to maintaining self-tolerance and preventing autoimmune disease during steady state conditions, a role for Foxp3+ Tregs in maintaining an immunological

27 equilibrium during infection has also been suggested. Moreover, the contribution of

Tregs in host defense appears to range from protective to pathologic depending upon the type of infection and stage of infection. As such, Tregs are equally involved as effector

CD4 T cell lineages in determining the dynamics of the host-pathogen interaction and disease outcome. While it is thought that role of Tregs in host defense is to maintain a balance between immune activation to clear the offending pathogen and immune suppression to limit host tissue damage due to excessive immune responses, how Tregs achieve this seemingly complex balance is poorly understood. Several studies have suggested that Tregs display an increased suppressive potency during later stages of persistent infections suggesting changes in Treg function may dictate this balance

[148,149]. However, these studies have limited their analysis of Treg function to a single time point during infection. Unfortunately, as the host-pathogen interaction during an infection is not static but highly dynamic, particularly during a persistent infection, Treg function needs to be addressed throughout an infection to determine how Tregs modulate this equilibrium. Moreover, factors that drive changes in Treg function during infection have yet to be clearly defined. One of the major limitations in the study of Tregs has been the use of CD25 expression to identify and manipulate Tregs. While Foxp3+ Tregs are the predominant population of CD25 expressing T cells under steady state conditions, naive

Foxp3-negative CD4 T cells transiently upregulate the expression of CD25 following activation. Thus, the CD25 population of CD4 T cells during infection contains a heterogeneous population of cells with both suppressive and effector properties. This becomes an increasingly important shortcoming during persistent infections where

Foxp3-negative CD4 T cells are continually being activated. Therefore, additional studies that identify Tregs based on the lineage defining marker, Foxp3, is required to further elucidate the contribution and function of this suppressive CD4 T cell population to host defense. Indeed, the development of mice that co-express the green fluorescent protein or high affinity human diphtheria toxin receptor under the Foxp3 promoter allows the selective tracking and conditional ablation of Foxp3-expressing Tregs, respectively [135].

Future studies using such mice are required to more specifically elucidate the function of

Foxp3-expressing CD4 T cells in host defense.

A role for suppressive CD4 T cells was first demonstrated in C57BL/6 Rag- deficient mice that lack endogenous T and B cells infected subcutaneously with

Leishmania major. The transfer of naïve effector CD4 T cells (CD25 negative) prior to infection resulted in near sterilizing levels of parasite clearance [151]. However, the accompanying lesion was significantly larger than wild-type C57BL/6 mice or control

Rag mice and remained so as long as the pathogen persisted suggesting more robust immune-mediated pathology in Rag mice that were given effector CD4 T cells.

Conversely, the co-transfer of Tregs (CD25 positive CD4 T cells) from a naïve donor inhibited the effective clearance of the parasite by the transferred population of effector

CD4 T cells however mice developed a more mild, localized pathologic lesion that was comparable to wild-type C57BL/6 mice [151]. Tregs have been implicated to mediate similar effects on limiting immune-pathology and restricting pathogen clearance in other persistent infection models including those caused by helminth [148,152,153], viral

[149], bacterial [154,155], and fungal [156,157] pathogens.

Conversely, excessive Treg suppression of an effector response can also be harmful to the host as it may limit the ability of the host immune response to control pathogen dissemination. This has been demonstrated to be the case during a lethal infection with Plasmodium yoelli in mice. Depletion of Tregs with anti-CD25 antibody prior to infection results in a robust pathogen-specific IFN-γ producing CD4 and CD8 T cell response that was able to effectively control the otherwise fatal infection [158].

Interestingly, the absence of Tregs (anti-CD25 depletion) during Plasmodium yoelli has also been associated with a paradoxical decrease in neuroimmunopathology during cerebral malaria in mice. This effect was attributed to the generation of a more effective and robust effector immune response during the early phase of infection that limited dissemination of the parasite to the CNS and subsequent development of a detrimental inflammatory infiltrate that is associated with this disease process [159]. Similarly, Treg- depletion in mice prior to systemic Candida albicans (anti-CD25 depletion) [157] or subcutaneous West Nile virus infections [160] (Foxp3 depletion) resulted in fatal immune-mediated tissue destruction. Together, these data indicate the critical role of

Tregs in maintaining an important equilibrium between generating a robust pathogen- specific immune response to effectively eliminate or control pathogen spread and limiting the immune-mediated pathology associated with prolonged immune activation during pathogen persistence.

Besides maintaining a balance between effector T cell mediated control of infection and associated immunopathology, some infection models have suggested that

Tregs, rather than suppressing the development of protective T cell-mediated immunity,

30 may actually be required for the generation of an effective immune response. For instance, Treg depletion (Foxp3 depletion) prior to a localized infection of HSV-2 in the footpad of mice resulted in increased local pathogen burden as well as more rapid and severe dissemination of the virus into the CNS compared to Treg-sufficient mice, which were able to effectively control early viral spread [161]. This loss of protection was further demonstrated to be due to decreased influx of immune cells (neutrophils, T cells, monocytes) into the site of infection associated with a disruption in normal chemokine production in peripheral and lymphoid tissues. Together, these data suggest a role for

Tregs in coordinating the temporal and spatial homing of the immune response during infection. Whether this seemingly paradoxical role of Tregs in protection is due to the type of infection, stage of infection or use of Foxp3 specific reagents compared to CD25 manipulation is unclear. However, a similar loss of protection was seen in Treg-deficient mice (Foxp3 depletion) following a systemic acute LCMV infection suggesting this role of Tregs in coordinating the homing of immune cells during infection is not associated with the route or localization of infection [161].

In further support of these results suggesting an essential role for Tregs in the development of a protective host immune response during an acute infection, Tregs have been implicated to play a protective role during the later phases of a persistent infection as well. For instance, mice deficient in the Treg-associated immunosuppressive cytokine,

IL-10, were able to control the initial acute phase of Trypanosoma congolense infection, however, subsequent to this initial period of pathogen control, these IL-10 deficient mice rapidly developed a fatal parasitemia that was effectively controlled in wild-type mice

[162]. The inability of IL-10 deficient mice to prevent pathogen dissemination long-term was associated with a loss of tissue integrity within the liver parenchyma, which impaired the effective influx and clearance of the pathogen by the immune response. While additional effector T cells, such as Th1 and Th2 CD4 T cells, are capable of secreting IL-

10 during persistent infections [163], these effects on host defense against Trypanosoma infection cannot be specifically associated with Treg function. However, these results suggest that Tregs and/or their associated immune regulatory functions may be important for maintaining an essential framework that is essential for proper functioning of the host effector immune response. Interestingly, several case reports have also observed an increased susceptibility to various viral and fungal infections in IPEX patients further supporting this protective role of Tregs in pathogen control [147,164]. However, the role of Tregs in preventing infection among this patient population has yet to be determined.

Tregs mediate these critical regulatory functions via a broad arsenal of immunosuppressive effector molecules [165,166]. The precise function of these Treg- associated regulatory molecules have primarily been defined and characterized using in vitro mixed lymphocyte reaction systems and/or in vivo colitis models. In these systems, purified Tregs from specific gene knockout mice and wild-type effector T cells are either co-cultured in vitro or co-transferred into SCID mice that lack endogenous T and B cell with the endpoint being proliferation and acquisition of effector functions by responder T cells and/or the development of colitis-like disease, respectively. Alternatively, blocking antibodies to specific effector molecules have also been used as complementary approaches to gene knockout mice [165,167,168].

Treg-associated effector molecules are generally divided into two categories: 1) cell contact dependent and 2) cell contact independent mechanisms [165]. Regarding cell contact dependent mechanisms, CTLA-4 is clearly the best-characterized molecule as it pertains to Treg function. CTLA-4 expression by Tregs has been shown to be critical for their function and CTLA-4 blockade leads to the development of a fatal autoimmune disease comparable to Foxp3 deficient Scurfy mice [169,170]. CTLA-4 mediates its effects by directly modulating APC function which leads to the induction of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan into the proapoptotic molecule kynurenine [171,172] as well as downregulates the T cell costimulatory molecules, CD80 and CD86 on APCs [173,174] which leads to apoptosis and/or impaired activation of effector T cells, respectively. Importantly, CTLA-4 blockade leads to enhanced resistance but exacerbated immune-pathology in various infection models similar to Treg depletion by anti-CD25 [175-181]. While these results demonstrate a close association between Treg function and CTLA-4 expression, CTLA-4 like CD25 is also transiently upregulated on non-regulatory effector T cells so the exact role of CTLA-

4 expression by Foxp3-expressing Tregs during these various infectious conditions is unclear. Additionally, Tregs have been shown to mediate direct cytolysis of effector T cells in a granzyme B-dependent mechanism [182,183], however these results have been limited to in vivo tumor models and whether this is a mechanism employed by Tregs during infections has yet to be determined. Moreover, membrane bound TGF-β [184] and

LAG-3 [185,186], a MHC class II binding CD4 homolog, have also been suggested to

33 inhibit T cell and DC maturation, respectively, however the in vivo relevance and exact mechanisms of action of these molecules have yet to be defined.

As far as Treg-associated cell contact independent mechanisms, the production of immunosuppressive cytokines such as IL-10 [187], TGF-β [188], and IL-35 [189] have been the best characterized. While IL-10 is clearly involved in limiting immune- pathology during infections, especially in persistent disease, the expression of this cytokine by effector CD4 T cells in addition to suppressive CD4 T cells has led to the idea that IL-10 is an important self-regulatory molecule of CD4 T cells in general thus the relative contribution of IL-10 produced by Foxp3-expressing Tregs in regulating the host-pathogen interaction during host defense is unknown [190]. Conversely, while IL-35 expression is thought to be restricted to the Foxp3-expressing subset of CD4 T cells, its role in Treg function has been limited to an in vivo non-infection colitis model [189].

Additional mechanisms have also been proposed such as expression of the ectoenzymes CD39 and CD73 [191], through the catabolism of AMP into extracellular adenosine, have been shown to inhibit effector T cell activation in vitro. Similarly, because of the constitutive expression of the high affinity IL-2 receptor, CD25, by Tregs,

IL-2 cytokine deprivation has been proposed as a potential mechanism by which Tregs are able to restrict effector T cell expansion [192]. Unfortunately, the in vivo relevance of these mechanisms particularly as they relate to Treg function during host defense has not been examined.

Aside from the various proposed mechanisms and effector molecules involved in

Treg-mediated regulation of the immune response, several recent papers have

34 demonstrated lineage-specific suppression of effector CD4 T cells by Tregs as well.

Through selective depletion of lineage-specific transcription factors in Foxp3-expressing

CD4 T cells, a reciprocal increase in inflammatory cytokines and immunopathology associated with unchecked activation in the corresponding Th effector lineage has been reported. For instance, the transfer of wild-type Tregs into neonatal Foxp3-deficient

Scurfy mice was able to rescue these mice from developing fatal lymphoproliferative, multi-organ autoimmune disease and was associated with a reduction in the number of pathogenic IFN-γ producing Th1 CD4 T cells present in these mice [193]. Interestingly, the transfer of T-bet deficient Tregs did not suppress the development of pathogenic Th1

CD4 T cells leading to a failure of these T-bet deficient Tregs to rescue this phenotype

[193].

Conversely, using a Foxp3-induced Cre recombinase expression system, mice containing floxed IRF-4 or STAT3 alleles were generated with Treg-restricted deletions in these transcription factors, which have been shown to be required for the development of Th2/Th17 or Th17 CD4 T cell differentiation, respectively [194,195]. Interestingly, these mice developed less severe autoimmune disease compared to fully Treg-deficient mice [194,195]. However, the limited disease that did manifest was associated with increases in cytokine expression by the corresponding Th2 and/or Th17 effector lineages.

For instance, mice with STAT3-deficient Tregs developed a gut mucosa-restricted, IBD- like disease that was associated with a select increase in the frequency of Th17 CD4 T cells and production of the Th17-associated cytokines IL-17 and IL-22 within gut- associated lymphoid tissues [194]. Interestingly, suppression of other lineages,

35 specifically IFN-γ production by T-bet-dependent Th1 CD4 T cells, was preserved. The mechanism by which these CD4 T cell lineage-defining transcription factors confer lineage-specific suppressive capabilities to Tregs is unclear. However, it has been proposed that it may be due to co-regulation of associated chemokine receptors, such as

T-bet induced expression of CXCR3 on Th1 CD4 T cells and Th1-specific Tregs, which allows for Tregs to localize to the same inflammatory environment as effectors T cells

[193]. Together, such a diverse repertoire of effector molecules and suppressive sub- lineages displayed by Foxp3+ Tregs implies that there is either a considerable amount of redundancy and/or a context-dependent activity among these molecules. As such, the precise roles and relative contribution of these effector molecules in Treg function in host defense has yet to be clearly defined, particularly in the context of a persistent infection where the balance between immune activation and immune suppression is critical for host protection against excessive pathogen burden or immune-mediated pathology.

To summarize, CD4 T cells are a highly diverse subset of T cells that possess a wide array of effector and regulatory properties that are essential for proper host defense against potential pathogens. The following sections will discuss how these more general characteristics of CD4 T cell effector functions contribute to dictating the host-pathogen relationship during a persistent infection using the intracellular bacterial pathogen,

Salmonella enterica, as a model.

V. SALMONELLA INFECTION

Epidemiology

Salmonella enterica is a rod-shaped, Gram-negative, facultative intracellular bacterium that consists of over 2000 different subspecies, or serovars [196]. Salmonella enterica, in general, infects a broad range of animals but the type of disease that results from Salmonella enterica infection is dependent upon both the specific serovar and host involved. For example, in humans, infection with Salmonella enterica serotype Typhi (S. typhi) results in a systemic, persistent disease whereas infection with Salmonella enterica serotype Typhimurium (S. typhimurium) induces only a mild self-limiting gastroenteritis

[197]. In contrast, mice develop a systemic, persistent disease state following infection with S. typhimurium but S. typhi infection causes only a non-descript, non-invasive disease [197]. Thus, host-adapted strains of Salmonella enterica are able to establish a persistent infection within a specific host population.

Moreover, the ability to evade the host immune response is critical for the establishment of invasive disease by host-adapted strains of Salmonella enterica as infection of immunocompromised patients with non-typhi strains of Salmonella enterica

(i.e. serovars other than S. typhi such as S. typhimurium) results in disseminated systemic infection compared to the mild gastroenteritis observed in immunocompetent individuals.

Indeed, non-typhi strains of Salmonella enterica are the leading cause of bacteremia in

AIDS patients in Africa [198-200]. Thus, infection with host-adapted strains of

Salmonella enterica results in the development of a persistent disease state and represents a highly relevant infection model to study the host-pathogen interaction during persistent infection.

Clinically, patients infected by host-adapted strains of Salmonella enterica develop a systemic persistent disease called typhoid, or enteric, fever. The clinical presentation of typhoid fever is highly heterogeneous but in general is rather insidious upon initial onset and becomes increasingly more severe as the disease progresses.

Following an initial asymptomatic period of 8-14 days (range 3-60 days) [201-203], patients will generally present with a low grade fever and relatively non-specific “flu- like” symptoms after one to two weeks post-infection [202-206]. Symptoms vary among patients but can include: dull frontal headache, malaise, myalgia, abdominal tenderness and cramps, nausea, and a dry non-productive cough [202-206]. The most common sign associated with typhoid fever is eruption of elevated rose red maculopapular rashes on the chest and abdomen that measure about 2-4 mm in diameter but this is observed in less than 50% of cases [202,203,205,206]. Subsequently, the fever rises in patients progressively over the next 7-10 days and becomes highly elevated and sustained (102-

104oF) [202,203,205,206]. If left untreated, the mean duration of fever is approximately one month and dissipation of fever typically follows recovery of disease

[202,203,205,206].

During this “progressive stage” of typhoid fever, disease-related complications or sequelae occurs in over 20 percent of cases and usually is associated with prolonged duration or increased severity of disease symptoms [205,206]. The most frequent is gastrointestinal bleeding occurring in approximately 10-20% of cases and may result in intra-abdominal hemorrhage and/or septicemia [205,206]. Intestinal perforation, on the other hand, only occurs in 1-3% of cases but is frequently fatal and therefore is the most

38 concerning complication associated with typhoid fever [205-208]. Typhoid-related encephalopathy, characterized by a state of agitation or obtunded behavior, delirium, or psychosis, is also frequently reported and associated with a more severe disease course and worse prognosis [209,210]. Importantly, the detection of a T cell-mediated immune response in patients with primary typhoid fever is associated with a reduced risk of developing disease-related complications and a faster rate of recovery [211], which implies that the quality of the host response, in particular the T cell response [212], influences the disease outcome of persistent Salmonella enterica infection.

Relapse or reinfection occurs in up to 15 percent of patients even with proper antimicrobial therapy [201,204-206,213-217]. The disease course, however, among these secondary attacks is generally more mild and of shorter duration [201,205,206].

Furthermore, of the patients who resolve the initial infection, 2 to 4 percent will become chronic carriers and can excrete organisms in their feces or urine for more than one year later [201,205,218,219]. Additionally, a population-based study conducted in an endemic area of Chile estimated the prevalence of chronic carriers to be about 700 per 100,000 people [220]. However, among the patients who become chronic carriers, women over thirty years of age represent the vast majority of this sub-population [218]. Together, these data suggest that while the host response is capable of dictating the severity and duration of primary disease, it is often unable to completely eliminate the pathogen.

Moreover, upon resolution of the primary disease, the development of secondary immunity appears to be insufficient to provide complete protection against rechallenge.

Determining the mediators of both primary and secondary protection against persistent

Salmonella enterica infection will be a critical step in designing more effective strategies to limit the risk of future infections by bolstering the development of protective immunity and eliminating the chronic carrier state which serves as a reservoir for this pathogen to propagate in nature by continually infecting new hosts.

Aside from being a highly conducive model to study the host-pathogen relationship during a persistent infection, invasive Salmonella enterica infection represents a concerning public health problem worldwide. While invasive disease caused by non-typhi strains of Salmonella enterica is a significant source of mortality among immunocompromised patients, systemic disease caused by host-adapted strains of

Salmonella enterica is a significant source of morbidity and mortality among immune- competent individuals particular in areas where Salmonella enterica remains endemic. In

2000, the World Health Organization (WHO) estimated there were over 21.7 million cases of typhoid fever worldwide, of which approximately 1% was fatal [221]. The most common causative agent of typhoid fever in immunocompetent individuals is Salmonella enterica serotype Typhi (S. typhi). In addition, Salmonella enterica serotypes Paratyphi

A, B, and C (S. paratyphi) are becomingly increasingly more prevalent as causes of disease producing an estimated 5.4 million additional illnesses annually [222]. In fact, in some endemic areas of Asia, S. paratyphi was shown to account for ~50% of the reported cases of typhoid fever [223,224]. The actual overall disease burden of typhoid fever, however, remains unknown as most endemic areas consist of low to middle income countries and access to diagnostic reagents such as blood cultures is often unavailable or unaffordable, thus most cases may go undiagnosed therefore drastically underestimating

40 the actual incidence rate [205,222]. Regardless of the actual disease burden, though, it is generally accepted that in the absence of more widely available and effective prevention methods, typhoid fever will continue to be a major public health problem.

Since the 1950’s, the incidence of typhoid fever in the U.S. has remained consistently low with just 0.15 cases per 100,000 people reported in 2008 and a case- fatality rate approaching zero [225]. In contrast, prior to 1950, typhoid fever was an important cause of illness and death in the U.S. with an incidence rate of more than 100 cases per 100,000 people in 1900 [226]. Interestingly, between 1920 and 1950, there was a steady decline in the number of incidences falling from about 40 cases per 100,000 in

1920 to less than 1 case per 100,000 people in 1950 [226]. The dramatic reduction in disease burden is largely attributed to the institution of several public health measures to ensure sanitary food handling protocols, clean public water, and proper sewage treatment.

While these public health initiatives significantly impacted the number of cases, the case- fatality rate remained relatively high at 11-13% [201,206]. It wasn’t until the development and introduction of efficacious antimicrobial drugs in the 1940’s that the mortality rate also dropped and now death due to typhoid related illness is relatively rare in developed countries [201,225,227,228].

Today the predominant source of typhoid fever in developed countries comes from travelers returning from endemic areas. Indeed, a laboratory-based surveillance study conducted by the CDC reported that between 1999-2006, 75% of cases of typhoid fever in the U.S. were contracted from travel to endemic regions [229]. Typhoid endemic areas comprise the overcrowded and impoverished regions of Latin America, Africa, the

Middle East, the Indian subcontinent, China, and Southeast Asia [205,221,222]. Unlike more industrialized countries including the U.S. and Europe, these developing countries maintain high burden of disease due to the lack of adequate clean water and appropriate sewage treatment as well as limited access to adequate medical attention particularly in rural communities. In a recent community-based study conducted by the WHO to estimate the actual burden of disease in 5 endemic areas, the highest incidence was in

India and Pakistan with over 400 cases per 100,000 people. Intermediate rates were reported in Indonesia at 180 cases per 100,000 people and moderate rates of 25-30 cases per 100,000 people in China and Vietnam [230]. Among the cases of typhoid fever reported in this study, children and adolescents (5-15 years old) represent the group with the highest disease burden but very high rates of disease were also reported in children less than 5 years old [230-232]. These estimates are consistent with data reported by a meta-analysis of 10 population-based studies in countries with a low to medium human development index which reported the highest disease burden in India and SE Asia (400-

1000 cases per 100,000 people) predominantly involving children less than 9 years of age

[233].

Similar to the U.S., the median case fatality rate in developing countries was about 11% (range 7.5-19%) in the pre-antibiotic era of typhoid fever (before 1950)

[201,206]. However, unlike what was observed in the U.S. in the post-antibiotic era (after

1950) where the typhoid case-fatality rate has signficiantly dropped, the case fatality rate in endemic areas remains high at an estimated 2.0-6.1% (range 0-41%) [201,233]. This is despite the relatively widespread availability and use of antimicrobials to treat typhoid

42 fever suggesting other factors are contributing to the increased severity of disease burden in these high incidence areas.

Emerging problems – Antimicrobial resistance

In the past 60 years, the treatment of typhoid fever with effective antibiotics has dramatically reduced the morbidity and mortality associated with this disease.

Unfortunately, the widespread use of antibiotics has lead to the selection and emergence of resistance strains of Salmonella enterica. Indeed, since 1989 when the first reported cases of multi-drug resistant (MDR) strains of S. typhi were reported, there has been a worldwide increase in the number of reported clinical isolates that are MDR and has been found at similar frequencies in both S. typhi and S. paratyphi strains [234-236]. MDR is defined as resistance to all traditional first-line antibiotics, which includes chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole. In 1980, ciprofloxacin, a member of the fluoroquinolone class of antibiotics, was introduced, and since the recognition and increasing prevalence of MDR strains of Salmonella enterica, it has become the first-line option for the treatment of typhoid fever [235,237]. Unfortunately, the past ten years has also seen the emergence of decreased sensitivity and full resistance to ciprofloxacin in strains of both S.typhi and S. paratyphi. Unfortunately, resistance to these drugs is associated with the presence of a self-transferable plasmid [235,238,239].

Therefore, MDR can emerge through clonal selection of a strain possessing the associated plasmid or through transfer of the plasmid to other strains [238,239].

Concerningly, typhoid fever caused by antimicrobial resistant strains of Salmonella

43 enterica has been associated with prolonged fever duration and increased rates of treatment failure [240,241].

The frequency of MDR strains varies widely depending upon the region. In the community-based study of 5 Asian countries by Ochiai et al [230], they reported the frequency of MDR strains to range from 0% in China and Indonesia to 65% in Pakistan.

This is consistent with other community-based studies that report a median incidence of

23% (range 0-79%) in developing countries [235]. Moreover, there appears to be a discrepancy in prevalence between community-based and hospital-based studies.

Epidemiological studies done in Vietnam clearly illustrate this point. Between 2002-

2004, a community-based study reported MDR in 22% of isolated strains [230]. During the same timeframe, a hospital-based study in Vietnam reported a MDR frequency of

74% [242]. These differences in frequency between community and hospital isolates support the observation that MDR strains are associated with more severe disease [214].

Currently, frequencies of ciprofloxacin resistance range from <1% - 5% of isolates [243], but these numbers are likely to rise. Therefore, a critical need is emerging for the development of novel therapeutic interventions that can augment antibiotic usage.

Emerging problems – Vaccine efficacy

The emergence of antibiotic resistance has shifted attention to the design of strategies that can effectively prevent infection altogether. As such, the development of more cost-effective and protective vaccines is a potential solution to this problem.

Currently, there are two commercially available vaccines licensed for use against typhoid

44 fever. The Ty21a vaccine is a live, attenuated strain of S. typhi that is administered orally.

The current CDC recommendation is to administer four doses every other day with an additional four doses given every five years thereafter as boosters [244]. The other licensed vaccine is composed of the purified un-denatured Vi capsular polysaccharide from S. typhi. It is delivered as a single dose intramuscularly but needs to be boosted every two years [244]. Surprisingly, neither is routinely being used in typhoid-endemic areas where such vaccines are needed. The reason underlying the scarce use of these vaccines is variable but in general involves a combination of factors such as inconvenience/non-compliance, restricted coverage of at-risk population, and short duration and overall low efficacy of protection.

Several meta-analyses have been done comparing the efficacy of these two vaccines based on available data from randomized-control trials [244,245]. All of the trials included in these analyses were conducted in areas where Salmonella enterica is endemic and the primary endpoint was development of typhoid fever (blood culture positive). The results of these trials demonstrated that there was no overall difference in

3-year efficacy between the two vaccines. Ty21a had a 3-year efficacy of 51% compared to 55% reported for the Vi polysaccharide vaccine.

However, despite the comparable efficacies of the two vaccines reported in these studies, there are some interesting differences to note. First, the development and duration of protection differed slightly between the Vi and Ty21a vaccines. For instance, the Vi polysaccharide vaccine provided peak protection during the first year (68%) but then protection steadily waned over the next two years falling by the second and third

45 years to 60% and 50%, respectively. This rapid fall in protection necessitates a booster immunization every two years to maintain efficacy. Conversely, the Ty21a vaccine was able to provide a more sustained level of protection over four years but did not reach peak efficacy until after the first year post-immunization (49%, 60%, 59%, and 78% for the first four years, respectively). While the duration of protection following Ty21a appears to be enhanced compared to the Vi polysaccharide vaccine, protection following Ty21a vaccination still begins to wane by the fifth year (47%) thus requiring booster immunizations every five years. Regardless, though, of these differences in duration and efficacy of protection by year, the protective immunity generated by either of these vaccines remains poorly protective and relatively short-lived.

The need for less frequent boosters makes the Ty21a vaccine seem like a better candidate. However, the important thing to note regarding the Ty21a vaccine is that the immunization protocol recommends four doses given orally every other day for both the initial and booster regimens to achieve optimum efficacy while the Vi polysaccharide vaccine is a single intramuscular injection every two years. The importance of receiving the recommended number of doses of the Ty21a vaccine was also demonstrated in the meta-analysis. After just one dose of the Ty21a vaccine, the efficacy over three years was

25%, 35%, and 1%, respectively. After two doses, protection comparable to three doses was achieved over the first two years (52% and 71%, respectively), however, efficacy fell dramatically thereafter to around 20% over the next two years and only 7% by the fifth year. Together, the need for frequent boosters with the Vi polysaccharide vaccine and the need for multiple dosages with the Ty21a vaccine makes the logistics of maintaining

46 optimal levels of protection associated with either of these vaccines highly inconvenient particularly in rural areas where access to medical care is limited, which leads to decreased compliance. Additionally, such frequent immunization also increases the associated cost of being vaccinated against typhoid. Thus, neither of these vaccine options provide robust protection against the development of typhoid fever and combined with the need for frequent boosters (every 2 to 5 years) and inconvenient immunization protocols (four doses over 7 days) further emphasizes the need for better vaccination options.

The population range that can be vaccinated is also a major limiting factor in the widespread use of current vaccine options. For instance, while the Vi polysaccharide vaccine is approved for use in children ≥2 years of age, the Ty21a vaccine as it is a live vaccine is only licensed for use in children ≥6 years of age in the United States. With the high disease burden in infants and children between the ages of 0-5 and increasing evidence that infection in this age group is associated with more severe disease and adverse outcomes [232,246], a highly-protective, single dose vaccine that can be given to infants and children that is both safe and effective is an important consideration.

Unfortunately, safety appears to be a significant limitation to the development of more immunogenic and protective vaccines. It is interesting to note that the first licensed vaccine against typhoid fever was approved in 1896. It is an inactivated whole cell vaccine derived from S. typhi. In clinical trials, the 3-year efficacy was shown to be 73%

[245], which is significantly improved compared to the current Vi and Ty21a vaccines.

However, more than 10% of the vaccinees missed either school or work with adverse

47 reactions associated with the vaccination and has thus been deemed unsuitable for usage today [245].

The increasing incidence of S. paratyphi as a causative agent of typhoid fever is another important factor to consider in the development of future vaccines. As there are currently no licensed vaccines against S. paratyphi, a vaccine that is cross protective against both S. typhi and S. paratyphi strains would be an ideal candidate. Vi polysaccharide expression is largely restricted to S. typhi strains and generally not present on S. paratyphi strains [205], thus the Vi polysaccharide vaccine is essentially ineffective at preventing S. paratyphi infection. The Ty21a vaccine on the other hand has been shown in one field trial in Chile to provide some cross-protection against S. paratyphi.

This study reports that the 3-year efficacy of Ty21a in the prevention of typhoid fever due to S. paratyphi was 49%, which unfortunately is comparable to the similarly low efficacy observed against S. typhi [244].

Together, these data indicate that while the morbidity and mortality due to typhoid fever has decreased dramatically since the early 1900’s due to the implementation of various public health initiatives and availability of efficacious antimicrobial drugs, invasive Salmonella enterica infection remains an important cause of illness and death in developing countries. Unfortunately, the identification and emergence of Salmonella enterica strains with multi-drug resistance against current antimicrobial therapies and the present lack of an effective, cheap vaccine, Salmonella enterica infection remains an important global health concern. Thus, vaccines that are able to 1) achieve higher and sustained levels of protection 2) against a wider range of

Salmonella enterica serovars 3) where optimal efficacy can be achieved with fewer doses and 4) can be administered to a broader age group of at-risk individuals is the current goal in vaccine development for prevention of typhoid fever. Moreover, as chronic carriers represent a continuous reservoir of infectious organisms, the availability of a therapeutic vaccine that can enhance the host immune response to effectively eradicate the persistence of this organism within individuals that are or may become chronic carriers has additional value as well particularly in the face of emerging resistance against antimicrobials.

Pathogenesis

Salmonella enterica transmission is via the fecal-oral route. A naïve host ingests water or food contaminated by an individual with active disease or chronic carriage. In healthy adult volunteers, the oral infectious dose has been shown to be >103 CFUs with

7 an ID50 ~10 CFUs [202,203]. Moreover, as would be expected with increasing doses of inocula, there is a corresponding increase in attack rate and decrease in incubation time

[202,203]. Upon traversing the stomach, the pathogen enters the small intestine. Here, they are able to adhere to the mucosal epithelium and gain entry into the mucosa. The typical route of entry is via uptake by M cells, a specialized epithelial cell overlaying the

Peyer’s patches of the small intestine [247]. These cells under steady state conditions play an important role in sampling luminal content and transcytosing antigen to the underlying lymphoid cells [247]. Uptake of Salmonella enterica by M cells appears to be through both passive and active means, the latter involving expression of a type III

49 secretion system (TTSS) and effector proteins encoded by the Salmonella pathogenicity

+ island 1 (SPI-1) virulence gene locus [247]. Alternatively, direct infection of CX3CR1

DCs via intraluminal dendrites has also been described [248] however the true physiological relevance of this pathway is somewhat controversial and may only occur under specific experimental conditions in mice.

After being translocating into the underlying lymphoid follicles, the bacteria migrate through the efferent lymphatics to the draining mesenteric lymph nodes (MLNs), subsequently gaining access to the systemic circulation via the afferent lymphatics and thoracic duct [197,205,249,250]. Circulating bacteria are engulfed by mononuclear phagocytic cells (i.e. macrophages and dendritic cells) residing in the reticuloendothelial networks of lymph nodes, spleen, liver, and bone marrow as well as recirculating into other structures such as the Peyer’s patches of the terminal ileum and gallbladder

[197,205,249,250]. It is within these environments, presumably within macrophages, that the bacteria are able to establish a niche within the host and persist long-term [251].

Utilizing a second TTSS encoded by the SPI-2 gene locus, bacteria are able to inhibit the normal endocytic vesicle trafficking pathway to prevent the fusion of the Salmonella- containing vesicle with lysosomes in unactivated macrophages [252,253]. This allows the pathogen to survive and replicate within the intracellular compartment of macrophages and dendritic cells. Thus, by evading normal host microbicidal mechanisms, the endocytic compartment of phagocytes represents a relatively immune-privileged site where Salmonella enterica is able to avoid detection by antibodies and other immune components and persist within the host.

Experimental mouse models of Salmonella infection

As natural infection with S. typhi is restricted to the human host, study of the pathogenesis and mediators of protection in typhoid fever has been limited. Fortunately, mice develop a comparable systemic disease following infection with S. typhimurium, which has adapted to the murine host environment. The use of the mouse model of typhoid-like disease has allowed for deeper interrogation into the mechanisms that enable

Salmonella enterica to evade the host immune response and establish long-term survival within a host as well as providing a system in which to dissect the contribution of the various immunological components to clearance and protective immunity against this disease.

The mouse models commonly used consist of inherently-susceptible strains, like

C57BL/6 and BALB/c backgrounds, which rapidly succumb to overwhelming bacterial burden following infection with relatively low inoculum of wild-type S. typhimurium

(LD50 <10 CFUs). Alternatively, inherently-resistant strains of mice, which include the

129Sv background, develop a persistent disease following infection with wild-type S.

4 typhimurium (LD50 ~10 CFUs) that closely resembles human typhoid. The genetic determinant between these various strains that confers resistance or susceptibility to S. typhimurium infection is the presence or absence, respectively, of a functional NRAMP1 protein [254]. Natural resistance-associated macrophage protein 1, or NRAMP1, expression is limited to the phagosome of cells of the monocytic/macrocytic lineages and functions as a divalent cation antiporter [255,256]. The exact bactericidal effect of

NRAMP1 is unknown but may involve the sequestration of essential divalent cations (i.e. zinc, magnesium, iron) away from the pathogen effecting the survival and rate of replication [255]. Regardless, mice that do not contain a functional NRAMP1 protein are highly susceptible to infections with various intramacrophage pathogens such as

Salmonella enterica, Mycobacterium and Leishmania [254]. C57BL/6 and BALB/c mice are homozygous for the non-functional Nramp1 allele, which contains a single amino acid substitution (Gly  Asp) at position 169 [257]. The absence of functional NRAMP1 results in an inability of the innate immune system to control the early replication of the bacteria resulting in unchecked and rapid dissemination of the pathogen leading to overwhelming bacterial burdens in these mice. It should be noted that polymorphisms in the gene encoding NRAMP1 in humans has not been associated with disease susceptibility as in the murine model [258] suggesting a potential difference in innate control of S. typhi and S. typhimurium in humans and mice, respectively.

The immune response to S. typhimurium has largely been investigated in inherently-susceptible mice. This may reflect that the fact that the majority of immunological tools such as gene-deficient or conditional knockout mice are more readily available on the C57BL/6 and BALB/c backgrounds. Unfortunately, the fact that these mice are highly sensitive to infection with wild-type strains of S. typhimurium dying within 7-10 days post-infection has limited the use of these mice in studying the immune response during natural infection. However, as death is an easily quantifiable endpoint to assess, the contribution of various virulence factors in Salmonella enterica pathogenesis can be evaluated using these mice since more attenuated strains of S.

52 typhimurium containing deficiencies in specific virulence or survival mechanisms are more readily controlled. Furthermore, death or protection from death in inherently- susceptible mice has also been used to assess vaccine efficacy and determine the mediators of protective immunity to rechallenge with virulent S. typhimurium.

While extremely helpful and insightful, some important limitations need to be kept in mind when interpreting results obtained from these susceptible strains of mice.

One caveat is that these mice are highly sensitive to wild-type S. typhimurium infection and the level of protection afforded by various immunization strategies and/or the immune-related requirements for protection may be skewed based on this inherent lack of innate immunity. In other words, it is possible to artificially overwhelm the immune system of these mice, which would overestimate the requirements for protection and/or underestimate the ability of various immune components to provide minor or incomplete levels of protection. Another caveat is that the requirements for protective immunity may not be the same for protection against primary infection. Thus, the inability of NRAMP1 functionally deficient mice to control wild-type S. typhimurium infection precludes their use in studying the host-pathogen interaction during the various stages of natural persistent Salmonella enterica infection in order to further understand the pathogenesis and immune response required for long-term control of the pathogen.

Fortunately, these limitations can be bypassed through the use of inherently- resistant mice. Infection of these mice with wild-type strains of virulent S. typhimurium results in a persistent infection that exhibits many of the same features of human typhoid and furthermore, it has been shown that these mice are able to establish a long-term

53 carrier state similar to human typhoid characterized by sporadic shedding of bacteria in the feces [251]. Thus, these mice represent an ideal model to study the factors that influence the host-pathogen interaction during natural persistent Salmonella enterica infection and to understand the components of the host immune system required for protection in primary and secondary infections. Unfortunately, a general paucity of data currently exists as it relates to factors influencing the host-pathogen interaction during primary and secondary infections in these resistant mice.

VI. PROTECTION TO SALMONELLA – PRIMARY INFECTION

T cells

As Salmonella enterica is a facultative intracellular pathogen that resides within the endocytic compartment of macrophages, an effective T cell-mediated immune response is required for the control and clearance of systemic Salmonella enterica infection in both mice and humans, like with other intracellular pathogens such as

Leishmania and Mycobacteria [34,259]. Consistently in typhoid patients, the generation of a detectable T cell-mediated immune response is correlated with reduced fever duration and decreased risk of developing disease-related sequelae [211].

In susceptible strains of mice, the absence of all mature CD4 and CD8 T cells

(TCRβ chain deficient and athymic nude mice) confers a loss of protection to even attenuated strains of S. typhimurium and mice ultimately succumb to high pathogen burdens [260-262]. In transfer experiments, splenocytes taken from inherently-

54 susceptible mice with an ongoing attenuated Salmonella enterica infection are able to confer partial protection to naïve susceptible strains of mice against challenge with a virulent strain of Salmonella enterica, and resistance is completely abolished if T cells are depleted prior to transfer further confirming the role of T cells in mediating protection

[261,263].

However, somewhat surprisingly, T cell deficient mice (i.e. TCRβ chain deficient and athymic nude mice) are able to control the initial phase of infection comparably to control mice through the first two to three weeks post-infection suggesting that T cells are not involved in resistance until later phases of infection and that the generation of a protective T cell-mediated immune response is delayed [260-262]. Consistently, the resistance transferred to naïve recipients by splenocytes derived from infected donors described in the preceding paragraph is absent until after the first week of infection and the level of resistance transferred increases progressively as splenocytes are harvested at later time points post-infection [261]. Additionally, similar observations are seen in inherently-resistant strains of mice early after infection with wild-type S. typhimurium where depletion of CD4 and/or CD8 T cells does not significantly impact the kinetics of the infection during the first week as the recoverable bacterial burdens were comparable to control mice [264] further supporting the absence of a protective T cell response generated early after primary S. typhimurium infection. Furthermore, in human typhoid patients, the appearance of T cell-mediated immunity is undetectable until after the first week post-infection and becomes progressively more robust over subsequent weeks

[211,265]. Together, these results suggest that a protective T cell-mediated immune

55 response is slow to develop but ultimately required for the control of invasive Salmonella enterica infection.

Among the T cell subsets, CD4 T cells appear to be the predominant mediators of protection. For example, MHC class II I-Ab deficient mice (on a susceptible strain background) are unable to control pathogen burden beyond the initial three weeks following attenuated S. typhimurium infection comparable to what is observed in T cell deficient mice [260]. However, in contrast to T cell deficient mice that die following attenuated S. typhimurium infection, MHC class II I-Ab deficient mice develop an indefinite state of high bacterial burden [260] suggesting that an additional T cell population that expresses the TCRβ chain, such as CD8 or NKT cells, may be sufficient to prevent mortality due to overwhelming pathogen burden in the absence of CD4 T cells but insufficient to adequately clear the infection. For example, β2 microglobulin deficient mice, which lack CD8 and NKT cells, are able to clear the infection with similar kinetics to T cell sufficient control mice [260]. It should be noted, though, that a slight increase in susceptibility (~30% decrease in survival) has been reported in the same β2 microglobulin deficient mice following infection with a different strain of attenuated S. typhimurium [266] suggesting that CD8 or NKT cells may play a minor role in protection, at least in the absence of CD4 T cells. Additionally, to further address the role of other T cell populations to host protection, mice deficient in gamma-delta T cells

(TCRδ deficient mice) were able to efficiently control infection with attenuated S. typhimurium however the complete clearance of the pathogen seemed impaired or at least delayed [260], thus gamma-delta T cells may play a minor role as well in host protection

56 however CD4 T cells are clearly indispensable regardless. Consistently, a connection between CD4 T cells and human disease outcomes is provided by a study conducted in

Vietnam demonstrating an increased susceptibility to developing typhoid fever in individuals with MHC class II alleles HLA-DRB1*0301/6/8, HLA-DQB1*0201-3 whereas MHC class II alleles HLA-DRB1*04 and HLA-DQB1*0401/2 are associated with disease resistance [267].

Further support for the predominant role of CD4 T cells in protection is demonstrated in transfer experiments, where antibody-mediated depletion of CD4 T cells alone prior to transfer significantly abrogates the ability to confer protection compared to the resistance achieved following transfer of whole splenocytes [261,263]. However, a slight but appreciable loss of protection following CD8 T cell depletion prior to transfer was observed compared to non-depleted splenocytes [261,263]. Furthermore, the combined depletion of CD4 and CD8 T cells did, in fact, result in a further loss of protection compared to CD4 T cell depletion alone [261,263]. Together, the data from these transfer experiments suggest that CD8 T cells, preferentially to NKT cells, partially contribute to pathogen control. Together, these results demonstrate that CD4 T cells are the dominant T cell subset mediating protection against primary Salmonella enterica infection, and while not sufficient for complete control against infection in the absence of

CD4 T cells, other T cell populations may also provide some long-term protection against primary Salmonella enterica infection.

While CD4 T cells are clearly required for clearance of S. typhimurium infection, like other intracellular pathogens, the generation of an IFN-γ producing Th1 lineage is

57 essential for the protective effects of CD4 T cells. Indeed, the importance of Th1 CD4 T cells and IFN-γ in protection against S. typhimurium infection is best demonstrated in patients with an inherited defect in the IL-12 or IFN-γ signaling pathways [268,269]. As described previously, IL-12 is required for the differentiation of CD4 T cells into the Th1 lineage and production of IFN-γ. While IFN-γ is required for the differentiation of Th1

CD4 T cells as well, it is also the primary effector cytokine produced by this lineage.

Thus, patients with a deficiency in either of these pathways are highly susceptible to recurrent and severe, systemic infections caused by weakly virulent strains of Salmonella enterica (non-S. typhi) and environmental strains of Mycobacteria (non-M. tuberculosis) that are typically non-pathogenic in immune-competent individuals [268,269]. In addition, the importance of Th1 CD4 T cell differentiation in protection to Salmonella enterica is further confirmed by the increased susceptibility of T-bet deficient mice (on a susceptible background) to attenuated S. typhimurium infection, as these mice are also unable to differentiate into IFN-γ producing CD4 T cells and, therefore, unable to control pathogen burden leading to increased mortality beyond three weeks post-infection [270].

In contrast, inherently-resistant mice that are chronically exposed to lead in their drinking water preferentially develop an IL-4 producing Th2 CD4 T cell response following wild- type S. typhimurium infection and as a result are unable to clear pathogen load from target organs corresponding to a near 100% mortality rate [271]. Thus, not only are CD4

T cells required for protection but appropriate differentiation into Th1 effectors is critical for their protective function.

Importantly, IFN-γR deficient mice on the inherently-susceptible background begin to die similarly by three weeks post-infection as observed for T-bet deficient and

MHC class II I-Ab deficient mice following attenuated S. typhimurium infection supporting the importance of CD4 T cell derived IFN-γ in controlling S. typhimurium infection [260]. Moreover, the importance of IL-12 in Th1 CD4 T cell differentiation and

IFN-γ production is demonstrated by administration of IL-12 neutralizing antibody to susceptible mice infected with attenuated S. typhimurium which results in a rapid and high rate of mortality comparable to IFN-γR deficient mice, and this loss of protection corresponds to a near complete loss of IFN-γ production by splenocytes [272].

Furthermore, it has been demonstrated in both susceptible and resistant strains of mice that the kinetics of the IFN-γ producing Th1 CD4 T cell response does not peak until around three weeks post-infection following attenuated or wild-type S. typhimurium infection, respectively [273,274]. Thus, the developmental timing of a Th1 CD4 T cell response directly corresponds to the temporal onset of mortality observed in mice with a deficient Th1 CD4 T cell response. Together, these data further corroborate and support the critical role of IFN-γ producing Th1 CD4 T cells in controlling primary Salmonella enterica infection.

As mentioned, host-adapted strains of Salmonella enterica can cause a persistent infection in their respective hosts that is characterized by the development of a chronic carrier state [218,220,251]. Thus far, the data have implicated a critical role for Th1 CD4

T cells and IFN-γ in dictating the outcome of the early host-pathogen interaction to favor host clearance. However, the components involved in maintaining long-term immune

59 control during the chronic carrier state are unclear. One study has suggested that the continued presence of IFN-γ is required. In resistant mice that have developed a chronic carriage state after wild-type S. typhimurium infection, the administration of IFN-γ neutralizing antibody results in a rapid reactivation of S. enterica infection that with prolonged depletion can result in mice becoming severely moribund [251]. Similarly, administration of depleting anti-CD4 T cell antibody to resistant mice persistently infected with wild-type S. typhimurium infection resulted in a transient and partial loss of protection but these results are limited by the fact that there was incomplete depletion of

CD4 T cells as more than 50% of the CD4 T cell population remained. Thus, the role of

CD4 T cells in continued control of persistent Salmonella enterica has yet to be clearly defined.

B cells

In human typhoid patients, the presence of Salmonella-specific antibodies is detectable within the first week after onset of symptoms [275]. Likewise, in both susceptible and resistant strains of mice, pathogen-specific antibodies are also detectable within the first week after infection [276]. While the initial production of antibody in both mice and humans is of the IgM subclass, both IgM and IgG titers rise progressively after the first week of infection [265,276-278]. However, unlike with the emergence of a

T cell-mediated immune response, neither the presence, specificity, titer nor subclass of these pathogen-specific antibodies seem to correlate with disease outcome [265,277].

Moreover, in susceptible strains of B cell deficient (Igµ deficient and Igh-6 deficient) mice, there was no difference observed in the ability of these mice to clear a primary infection with an attenuated strain of S. typhimurium compared to control mice

[278-280]. Interestingly, though, the presence of B cells seems to confer some increased resistance to these mice compared to B cell deficient mice following infection with increasing inoculums of wild-type S. typhimurium [279]. Moreover, these results are consist with observations in the resistant CBA strain of mice where the CBA/N substrain possess a mutation in Bruton’s tyrosine kinase resulting in a defective antibody response characterized by overall low titers of circulating antibody and impaired isotype class- switching [281]. As a result, the LD50 dose of CBA/N mice is approximately 1000-fold less compared to CBA mice following infection with wild-type S. typhimurium [282], but display no difference in susceptibility to attenuated S. typhimurium infection [283].

Furthermore, patients with primary immunodeficiencies in B cell function resulting in hypogammaglobulinemia (i.e. CVID and XHIM) demonstrate an increased risk of invasive disease due to Salmonella enterica compared to immune-competent individuals

[284-286]. Thus, while CD4 T cells appears to be both necessary and sufficient for control of attenuated S. typhimurium infection in susceptible mice, these results suggest a potential role for B cells in controlling infection that is apparent only when the T cell response is overwhelmed as is the case with a wild-type S. typhimurium infection in susceptible mice.

Importantly, it is not clear if the increased susceptibility of these B cell deficient mice is due to a lack of antibodies or secondary to an impaired B cell-T cell interaction

61 resulting in an impaired T cell response. Indeed, B cells are an important antigen- presenting cell and source of polarizing cytokines required for proper T cell expansion and differentiation [287-291]. Moreover, it has recently been demonstrated that B cell deficient mice (Igh-6 deficient) preferentially generate a Th2 CD4 T cell response early after attenuated S. typhimurium infection, and furthermore, fail to develop a robust Th1 response even up to 15 weeks later [279,292]. Thus, it is possible that B cells play an antibody-independent role in controlling primary S. typhimurium infection associated with impaired differentiation and development of a protective Th1 CD4 T cell response.

It is interesting to note that in both patients with typhoid fever and in mouse models of persistent Salmonella enterica infection that the development of a protective

Th1 CD4 T cell response is significantly delayed. In mouse models, the importance of

CD4 T cells in the eventual clearance of primary S. typhimurium infection has been clearly demonstrated and the generation of a protective Th1 CD4 T cell response is clearly essential for determining the outcome of the host-pathogen interaction during persistent S. typhimurium infection. However, as the studies to determine the factors controlling the kinetics of protective Th1 CD4 T cell development have largely been done in susceptible mice with attenuated strains of S. typhimurium where both the host and the pathogen have been manipulated, it is necessary to further address these questions in a model of a natural persistent S. typhimurium infection where the host-pathogen interaction has not been altered. Such studies would provide greater insight into the host- pathogen interactions involved in the pathogenesis and protection against human typhoid fever.

VII. PROTECTION TO SALMONELLA – SECONDARY IMMUNITY

In areas with unsafe sanitation conditions, Salmonella enterica remains endemic, thus reexposure is ultimately inevitable. Unfortunately, the mediators of secondary immunity to prevent recurrent disease following subsequent exposure(s) are incompletely understood. In human studies, natural typhoid disease or immunization (either inactivated whole cell vaccine or live, attenuated Ty21a vaccine) have been shown to generate both

B cell and T cell responses [293-298]. However, the ability of either of these immunization methods to provide significant protection against secondary attacks is a matter of debate. In a population based study where two typhoid outbreaks, caused by separate S. typhi strains, occurred within 5 months of each other within the same community, it was found that individuals who developed typhoid fever after the initial outbreak demonstrated a 40% reduction in attack rate compared to previously uninfected individuals (20.4% versus 34.2%) [299]. Furthermore, in a more controlled trial, 22 volunteers were experimentally infected orally with 105 CFUs of S. typhi (a dose shown experimentally to cause clinical disease in 25% of infected human volunteers, [ID25]

[202,203]) and allowed to recover from the disease. Of these previously infected individuals, 23% of them developed clinical typhoid fever following a secondary challenge with the same ID25 dose one year later [293]. As a 30% attack rate was observed in the control group consisting of 34 healthy naïve volunteers challenged in parallel [293], these results suggest naturally acquired typhoid fever only conferred a

24% reduction in disease incidence when controlling for infectious doses. In comparison, volunteers that were immunized with an inactivated whole cell vaccine demonstrated a similar 30% reduction in disease incidence compared to healthy unimmunized volunteers when challenged with a similar ID25 dose [202,203,293]. Thus, naturally acquired infection or vaccination results in development of incomplete or partial secondary immunity.

Interestingly, this meager level of protection is completely ameliorated when the secondary infecting inoculum is increased. For instance, the same immunized volunteers that demonstrated a 30% reduction in disease occurrence following a challenge dose of

5 10 CFUs (ID25), were not any more protected than unimmunized healthy volunteers

7 when the challenge dose was increased to 10 CFUs (ID50) [202,203]. Thus, the efficacy of acquired protective immunity is dependent upon the challenging inoculum and can apparently be overwhelmed by increasing bacterial burdens.

Therefore, to generate vaccines with increased protective efficacy, it is first essential to understand the immunological components involved in protection before designing methods to boost their respective responses. As mentioned, in humans both B and T cell responses can be generated following vaccination, yet the relative contribution of each to protection is unclear. In vaccine field trials, while the Vi polysaccharide vaccine is thought to generate a T cell independent antibody response and the live attenuated Ty21a vaccine generates both a B and T cell response [296-298,300], both demonstrate comparable three year efficacies of 55% and 51%, respectively [244,245].

Thus, these results would suggest that an effective antibody response is sufficient to

64 provide low level secondary immunity. Moreover, several reports have suggested that post-immunization antibody titers correlate with secondary protection following Ty21a and Vi polysaccharide vaccinations [202,203,295,301,302]. However, these observations do not rule out a potentially protective role for T cells as the overall efficacy of both vaccines is relatively poor and neither is able to confer long-term protection suggesting that antibody-mediated protection is weak at best. Furthermore, the increased incidence of recurrent infections described previously in patients with T cell defects (IL-12/IFN-γ deficiency) would also suggest T cells are involved in secondary immunity.

The respective contribution of these immune components to protective immunity has been further delineated in mouse models. Immunization of susceptible mice with live, attenuated strains of S. typhimurium confers near complete protection, as almost 100% of these mice survive a secondary challenge with an otherwise lethal dose of wild-type S. typhimurium [303-305]. Importantly, the use of susceptible mice and wild-type S. typhimurium to assess protection represents a robust model to determine the mediators of secondary immunity as complete protection is required for survival of these mice. In other words, understanding the immune components required for survival of susceptible mice against wild-type S. typhimurium infection may translate into immunological targets to increase vaccine efficacy in humans.

As such, in this mouse model, as opposed to human vaccine trials, the correlate of protection is the development of a T cell response [212,303]. Moreover, similar to primary infection, CD4 T cells appear to be the predominant mediators of protection, while other T cell subsets contribute a minor role but are required for maximum

65 secondary protection. For example, in susceptible strains of mice that have resolved a primary infection with attenuated S. typhimurium, depletion of CD4 T cells prior to rechallenge results in a more significant loss of protection than compared to CD8 T cell depletion [261,304]. However, β2 microglobulin deficient mice that were similarly immunized with an attenuated strain of S. typhimurium demonstrate a significant decrease in survival following wild-type S. typhimurium challenge compared to control immunized mice (40% vs. 100% survival after day 10 rechallenge) [266]. Interestingly, though, the requirement for additional T cell subsets was only observed at higher challenge inoculums (>104 CFUs i.v.) where more than 60% of the β2 microglobulin deficient mice had died by 10 days post-challenge [266]. In contrast, all β2 microglobulin deficient mice were able to survive a challenge inoculum of 102 CFUs [266]. Thus, while CD4 T cells appear to be indispensable for complete protection of susceptible mice to wild-type S. typhimurium challenge, the importance of other T cell populations is directly correlated with challenge dose.

Despite the clear requirement for T cells to secondary immunity in these experiments, the adoptive transfer of T cells from an immunized donor is insufficient to confer protection to a susceptible naïve recipient [305,306]. Conversely, transfer of immune serum only was also insufficient to confer protection suggesting antibody alone cannot provide adequate secondary immunity [305,306]. However, like additional non-

CD4 T cell subsets, B cells are also required for maximum secondary protection as immunized B cell deficient mice are unable to survive a challenge with wild-type S. typhimurium [278-280]. Interestingly, also like other non-CD4 T cell subsets, the role of

B cells becomes less important at lower challenge inoculums [280]. Consistent with the requirement of both B and T cell components to adoptively transfer protection against secondary infection with virulent S. typhimurium infection in susceptible strains of mice

[305], complete protection against secondary infection in resistant strains of mice can only be demonstrated after the adoptive transfer of both immune sera and T cells into naïve recipients [306].

In summary, secondary immunity against recurrent Salmonella enterica infection is determined by both host factors and pathogen burden. In other words, while B and T cell components alone may be able to provide some protection against low dose challenges during secondary infections, complete protection particularly against higher inocula requires both immune components. Thus, the partial protection seen in human vaccine trials may be the result of suboptimal protective B and/or T cell responses primed by currently available vaccines. Moreover, as CD4 T cells are important for activating phagocytic cells to clear intracellular pathogens as well as being required for effective antibody and memory CD8 T cell responses, the precise role of CD4 T cells in secondary immunity needs to be further elucidated.

VIII. THESIS STATEMENT

The goal of this thesis work is to determine the contribution of effector and regulatory CD4 T cell subsets to pathogenesis and protection of persistent Salmonella enterica infection using a murine model of natural host-pathogen interaction that

67 recapitulates human disease in immunocompetent individuals. In chapter 2, we determine the impact of changes in TCR signaling on the ability to prime an effective protective

Th1 CD4 T cell response against secondary infection. In chapter 3, the contribution of effector and regulatory CD4 T cells in governing the kinetics and outcome of persistent S. typhimurium infection is established. Chapter 4 identifies host and pathogen associated factors that impact the survival of antigen-specific memory CD4 T cells during primary

S. typhimurium infection. Finally, in chapter 5, we determine the efficacy and mediators of secondary immunity generated following natural S. typhimurium infection. Together, this work will provide greater insight into contribution and generation of protective Th1 and Foxp3-expressing Tregs in primary and secondary immunity against S. typhimurium infection.

Chapter 2

Naturally-occurring altered peptide ligands control Salmonella-specific

CD4+ T cell proliferation, IFN-γ production, and protective potency*

* Johanns TM, Ertelt JM, Lai JC, Rowe JH, Avant RA, Way SS (2010). J Immunol

184(2): 869.

ABSTRACT

T cell-activation required for host defense against infection is an intricately regulated and precisely controlled process. Although in vitro studies indicate three distinct stimulatory signals are required for T cell-activation, the precise contribution of each signal in regulating T cell proliferation and differentiation after in vivo infection is unknown. In this study, altered peptide ligands (APLs) derived from the protective

Salmonella-specific FliC antigen and CD4+ T cells specific for the immune-dominant

FliC431-439 peptide within this antigen were used to determine how changes in TCR stimulation impact CD4+ T cell proliferation, differentiation, and protective potency. To explore the prevalence and potential use of altered TCR stimulation by commensal or other bacterial pathogens, naturally-occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC431-439 peptide were identified and used for stimulation under both non-infection and infection conditions.

Based on this analysis, naturally-occurring APLs that prime proliferation of FliC-specific

+ CD4 T cells either more potently or less potently compared with the wild-type FliC431-439 peptide were identified. Remarkably, despite these differences in proliferation, all APLs

+ primed reduced IFN-γ production by FliC431-439-specific CD4 T cells associated with reduced T-bet expression. Moreover, after expression of the parental FliC431-439 peptide or each APL in recombinant Listeria monocytogenes, only CD4+ T cells stimulated with the wild-type FliC431-439 peptide conferred significant protection against challenge with virulent Salmonella. These results reveal important and unanticipated roles for TCR

70 stimulation in controlling pathogen-specific CD4+ T cell proliferation, differentiation, and protective potency.

INTRODUCTION

Three distinct, yet inter-related, stimulatory signals are required for the activation and differentiation of naïve T cells into cytokine-producing effector T cells [307,308].

Although naïve T cells likely receive these stimulation signals concurrently during contact with antigen-presenting cells in a coordinated fashion, each signal has also been shown to individually control unique facets in T cell activation, proliferation, and differentiation. For example, antigen specificity controlled by T cell receptor (TCR) signaling dictates which subset of T cells becomes initially activated [309,310], while costimulation signals primarily mediated by CD28 signaling prevent these newly activated T cells from becoming anergic [311,312]. In this regard, a growing list of specific cytokines that includes IL-6, IL-12, IL-21, type I IFNs, and TGF-β have each been demonstrated to provide additional stimulation signals that controls the expansion, survival, and differentiation program of newly activated CD4+ and CD8+ T cells [22,313-

320]. Therefore, coordinated stimulation through the TCR, costimulation receptors, and specific cytokine receptors each has the potential to play unique and defined roles required for synchronized T cell activation, proliferation, and differentiation.

Although there is ample evidence supporting the ability of specific cytokines to control CD4+ T cell differentiation into each distinct T-helper lineage, other T cell- stimulation signals have also been implicated to play important roles in this process.

Altered peptide ligands (APLs) containing amino acid substitutions in TCR contact residues from defined MHC class II peptide antigens have been used to characterize how

TCR stimulation can also control the CD4+ T cell differentiation regardless and independent of exogenous cytokines. For example, stimulation of CD4+ T cells from

TCR transgenic mice specific for a I-Ab restricted peptide within the Mycobacterium tuberculosis-antigen 85B (Ag85B) with wild-type Ag85B244-252 peptide primes robust

IFN-γ with minimal IL-4 production, while stimulation with a peptide variant containing a single glycine to alanine substitution within the TCR contact residues at position 248 abolishes IFN-γ production and is replaced by reciprocal IL-4 production [132]. In agreement with these results where T cells are stimulated in vitro, stimulation with APLs can also have profound effects on CD4+ T cell differentiation in vivo. For example, CD4+

T cell IFN-γ production that normally occurs after “immunization” with the human collagen IV protein is abolished and replaced by IL-4 production when a peptide variant containing a single amino acid substitution within a defined TCR contact residue is used instead [133]. Recently, the impact of TCR stimulation has also been extended to play important roles in controlling the antigen-specific CD8+ T cell response after in vivo infection. Using recombinant Listeria monocytogenes (Lm) that express either the

b parental H-2K OVA257-264 peptide or defined APLs derived from this peptide, TCR stimulation was found to dictate the kinetics of CD8+ T cell contraction and migration within lymphoid organs [321]. Interestingly, despite these differences, OVA257-264- specific CD8+ T cells were activated and formed functional memory cells similarly regardless of differences in TCR stimulation [321]. Taken together, these results indicate differences in TCR stimulation may control critical and unanticipated features in T cell differentiation and the antigen-specific T cell response during infection.

In this study, we sought to explore how differences in TCR stimulation may impact proliferation, differentiation, and protective potency for pathogen-specific CD4+ T cells. Given the importance and protective effects of CD4+ T cells in host defense against

Salmonella typhimurium infection, a defined MHC class II peptide that spans amino acids

431-439 within the protective FliC antigen of Salmonella was utilized in this study

+ [249,260,261,322,323,324]. This FliC431-439 peptide is presented to CD4 T cells by the murine MHC class II molecule, I-Ab, since CD4+ T cells from FliC-specific TCR transgenic mice derived from C57BL/6 mice expand in an antigen-specific manner after adoptive transfer into syngeneic recipient mice [273,323]. Furthermore, given the highly conserved nature of FliC and other flagellin components among diverse bacterial species, we examined the prevalence and explored potential use of altered TCR stimulation for

CD4+ T cells specific to this antigen by other bacteria. This lead to the identification of four naturally-occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC431-439 peptide. When compared with the parental FliC431-

+ 439 peptide, naturally-occurring APLs that prime proliferation of FliC-specific CD4 T cells either more or less potently were identified. Remarkably, despite these differences in proliferation, each APL compared with the parental FliC peptide primed reduced IFN-γ production and conferred diminished protection against subsequent challenge with virulent Salmonella.

RESULTS

Identification of naturally-occurring FliC431-439-derived APLs

The flagellin structural protein, FliC, is an immune-dominant antigen that confers protection against Salmonella typhimurium infection [323]. The peptide that spans amino acids 431-439 within FliC are presented by MHC class II I-Ab as CD4+ T cells with specificity for this peptide are readily identified in naïve C57BL/6 mice and cell lines derived from these mice after stimulation with heat-killed Salmonella [273,325].

Alignment with other well-characterized I-Ab peptides reveals the putative MHC class II anchor and TCR contact residues within the FliC431-439 peptide (Fig. 1) [326-329]. An essential role for amino acid positions P1, P4, P6 and P9 in direct contact and anchoring the peptide to I-Ab MHC has been demonstrated through mutational analysis and biochemical-binding/affinity assays [326,327], and these results have been confirmed by the resolved crystal structure of I-Ab-restricted peptides bound to this MHC molecule

[328,329]. These studies reveal amino acids containing large, hydrophobic aromatic side chains are predominantly found at position P1, while amino acids with small, uncharged side chains at positions P4, P6 and P9. Accordingly, the phenylalanine corresponding to residue 431 within FliC corresponds to position P1, and alanine434, threonine436, and glycine439 correspond to positions P4, P6, and P9, respectively (Fig. 1). In turn, the importance of positions P2, P3, P5, P7, and P8 for direct contact with the TCR has also been confirmed by both biochemical and crystallographic experimental approaches [326-

329]. In turn, residues asparagine432, serine433, isoleucine435, asparagine437, and leucine438

75 within FliC431-439 correspond to the TCR contact sites at positions P2, P3, P5, P7, and P8, respectively (Fig. 1). Therefore to identify potential naturally-occurring APLs within the

FliC431-439 peptide antigen, a directed BLAST search of the National Center for

Biotechnology Information (NCBI) peptide database was performed for known proteins containing substitutions in these TCR contact sites. The parental FliC431-439 peptide sequence containing substitutions incorporating each of the other 19 possible amino acids at each TCR contact residue were used for this database search. This analysis revealed four naturally-occurring FliC431-439-derived APLs that were each identified within the flagellar FliC protein homologue of various Salmonella serovars or other Gram-negative bacteria known to colonize or invade through the gastrointestinal tract (Fig. 1).

Interestingly, all of these FliC431-439-derived APLs were identified from human clinical isolates of virulent bacteria [330]. Among these FliC431-439-derived APLs, one APL contains a conserved leucine (L)  isoleucine (I) substitution at residue 438 (FliCL438I), which retains an uncharged, hydrophobic residue at the P8 position, while the other three

APLs each contain non-conservative substitutions at either P2 (FliCN432D, FliCN432E) or

P3 (FliCS433F). In the case of APLs FliCN432D and FliCN432E, an uncharged asparagine (N) residue is replaced by a negatively charged aspartate (D) or glutamate (E) residue, respectively. For APL FliCS433F, the small, hydrophilic serine (S) residue is replaced by a large hydrophobic phenylalanine (F) residue. The presence of these APLs within the immune-dominant epitope of FliC isolated from clinical samples of Salmonella and other

Gram-negative bacterial pathogens suggests altered TCR stimulation may play an important role in controlling the CD4+ T cell response to this antigen.

Characterization of FliC431-439-derived APLs in vitro

+ Salmonella FliC431-439-specific CD4 T cells derived from SM1 TCR transgenic mice were used to characterize potential differences in how each naturally-occurring APL

+ compared with the parental wild-type FliC431-439 peptide (WT FliC) primes CD4 T cell activation, proliferation, and differentiation [273]. As expected, WT FliC readily primes the proliferation of CD4+ T cells from SM1 TCR transgenic mice – more than 95% of these CD4+ T cells were CFSE dilute by day 5 after stimulation (Fig 2A). In contrast,

+ CD4 T cells stimulated with APLs FliCN432D, FliCN432E, or FliCS433F each remained

CFSEhi and had levels of CFSE comparable to cells stimulated with an irrelevant peptide control. Interestingly, the degree and kinetics of CFSE dilution for cells stimulated with

APL FliCL438I were comparable to cells stimulated with WT FliC at 10 µM (Fig. 2A). To further define potential differences in potency between APL FliCL438I and WT FliC for triggering CD4+ T cell proliferation, serial 10-fold dilutions of each peptide were used for stimulation. This analysis revealed that although CD4+ T cell proliferation by WT FliC peptide was predominantly extinguished when the concentration was reduced to 0.1 µM,

+ APL FliCL438I maintained robust CD4 T cell proliferation at this concentration and did not become extinguished until the concentration was reduced to 0.01 µM indicating this peptide is ~10-fold more potent for priming FliC-specific CD4+ T cell proliferation.

Collectively, these results indicate naturally-occurring FliC431-439-derived APLs either

+ stimulate CD4 T cell proliferation more potently (APL FliCL438I) or significantly less

77 potently (APLs FliCN432D, FliCN432E, and FliCS433F) compared with the parental WT

FliC431-439 peptide.

Based on these differences in proliferation, the impact of stimulation with each

APL compared to the parental WT FliC peptide on T cell-activation and the production of lineage-defining cytokines by FliC-specific CD4+ T cells were quantified. Consistent with their ability to readily prime proliferation, CD4+ T cells stimulated with WT FliC and APL FliCL438I both dramatically up-regulated CD25 and CD44 expression, while

CD62L expression was down-regulated (Fig. 2B). Conversely and consistent with the lack of proliferation, stimulation with APLs FliCN432E or FliCS433F did not cause appreciable changes in CD25, CD44 or CD62L expression and the expression level for each was essentially identical to cells stimulated with an irrelevant control peptide.

+ Interestingly, although APL FliCN432D did not prime detectable CD4 T cell proliferation, this APL did stimulate increased CD25 and CD44 expression albeit to a significantly lower extent than cells stimulated with either WT FliC or APL FliCL438I peptides (Fig.

2B). Therefore, the expression of cell surface markers associated with T cell-activation

(CD25 and CD44 up-regulation, CD62L down-regulation) directly correlates with the robust proliferation of FliC-specific CD4+ T cells after stimulation with WT FliC and

APL FliCL438I, while reduced or minimal changes in the expression of each marker is associated with only limited proliferation after stimulation with APLs FliCN432D,

FliCN432E or FliCS433F.

These differences in proliferation and expression of T cell activation markers were extended to explore potential differences in CD4+ T cell differentiation by

78 measuring the production of T-helper lineage-defining cytokines such as IFN-γ, IL-4, and

IL-17 representative of Th1, Th2, and Th17 lineages, respectively. Stimulation with WT

FliC peptide consistently primed a small, but defined population of IFN-γ producing

+ lo CD4 T cells that were predominantly CFSE (Fig. 3). Interestingly, while APL FliCL438I primed proliferation to a similar or greater extent compared with WT FliC, IFN-γ production was markedly reduced (Fig. 3). Moreover, the level of IFN-γ by cells stimulated with APLs FliCN432D, FliCN432E, and FliCS433F was essentially at background levels and comparable to cells stimulated with an irrelevant peptide control. Importantly, the reduced IFN-γ production by cells stimulated with each FliC431-439-derived APL compared with WT FliC peptide was not associated with a reciprocal increase in the production of either IL-4 or IL-17, as the levels for both these cytokines remained at the limits of detection for all stimulated cells quantified using both intracellular cytokine staining and ELISA.

Given the importance of the T-box transcription factor T-bet in CD4+ T cell IFN-γ production and Th1 differentiation, additional experiments quantified relative T-bet expression in FliC-specific CD4+ T cells stimulated with WT FliC or each APL peptide

[95,96]. Levels of T-bet expression were found to be directly correlated with the production of IFN-γ – the mean fluorescent intensity for T-bet expression in cells stimulated with WT FliC peptide was dramatically increased (~35-fold) compared with cells stimulated with an irrelevant control peptide, while the level of T-bet expression was reduced 70% in cells stimulated with APL FliCL438I compared with the WT FliC peptide (Fig. 3). As expected, T-bet expression for FliC-specific CD4+ T cells stimulated

79 with APLs FliCN432D, FliCN432E, or FliCS433F were each only at background levels consistent with the absence of IFN-γ production by these cells. Taken together, these results demonstrate that although APL FliCL438I and WT FliC both efficiently prime

CD4+ T cell proliferation, increased CD25 and CD44 expression, and CD62L downregulation, stimulation with WT FliC compared to APL FliCL438I primes significantly more IFN-γ which is associated with increased T-bet expression. Conversely, the other

APLs (FliCN432D, FliCN432E, or FliCS433F) each did not prime proliferation, IFN-γ production, or increased T-bet expression.

Characterization of FliC431-439-derived APLs in vivo

Based on the stark contrast in the degree of T cell-activation, proliferation, and differentiation between each naturally-occurring FliC431-439-derived APL and the WT

FliC peptide after stimulation in vitro, additional experiments sought to characterize

+ potential differences in the expansion and differentiation of FliC431-439-specific CD4 T cells after in vivo stimulation. For these experiments, purified WT FliC peptide, APL

FliCL438I, APL FliCN432D (representative of results with APLN432E and APLS433F), and an irrelevant control peptide were each intravenously inoculated into mice adoptively transferred with congenically-marked (CD45.1+) FliC-specific CD4+ T cells from SM1

TCR transgenic mice one day prior. Both purified WT FliC peptide and APL FliCL438I primed robust expansion of CD45.1+ FliC-specific CD4+ T cells – more than 100-fold increase in percentage and total numbers of CD45.1+CD4+ T cells were present in mice inoculated with WT FliC or APL FliCL438I compared with mice inoculated with irrelevant

80 control peptide (Fig. 4). By contrast but in complete agreement with the lack of proliferation after stimulation in vitro, intravenous inoculation with APL FliCN432D did not prime significant CD4+ T cell expansion as the percent and total numbers of

CD45.1+CD4+ T cells recovered from these mice were not significantly different from mice inoculated with irrelevant control peptide (Fig. 4). When cytokine production by

FliC-specific CD4+ T cells stimulated in vivo was quantified, the overall number of IFN-γ producing CD4+ T cells was markedly reduced for FliC-specific CD45.1+ cells primed with APL FliCL438I compared with WT FliC peptide (Fig. 4B). Furthermore, FliC-specific

CD45.1+CD4+ T cells produced no detectable IFN-γ after in vivo stimulation with APLs

FliCN432D or the irrelevant control peptide (Fig. 4B). Therefore despite either enhanced or reduced levels of proliferation, the markedly reduced IFN-γ production after stimulation with each APL compared with the WT FliC peptide observed in vitro is maintained after stimulation with each peptide under non-infection conditions in vivo.

Stimulation with FliC431-439-derived APLs expressed in Listeria monocytogenes

Additional experiments sought to characterize how stimulation with WT FliC peptide and each APL would impact CD4+ T cell priming during experimental infection in vivo because infection triggers complex cascades of immune cytokines and signaling molecules not reproduced by stimulation with purified peptide alone. Therefore, attenuated ΔActA Lm were engineered to express either WT FliC or each FliC431-439- derived APL behind the Lm-specific hly promoter with an N-terminus listeriolysin O- specific signal sequence that allows each recombinant protein to be secreted by the

81 bacterium (Fig. 5A). The uniform expression and secretion of the recombinant proteins that contain either the WT FliC peptide, each APL, or control peptide antigen was verified by protein immune blotting using antibody against the HA-tag (Fig. 5A).

Although protective immunity to Lm infection is predominantly mediated by CD8+ T cells, we and others have shown infection with either virulent or ΔActA Lm also primes the robust expansion of CD4+ T cells with specificity to both endogenous Lm or recombinant antigens secreted by Lm [319,331,332]. Similar to results after stimulation with purified peptide, mice infected with Lm expressing either WT FliC (rLM-WT) or

+ + APL FliCL438I (rLM-L438I) each contained robust CD45.1 CD4 T cell expansion, while mice infected with Lm expressing APL FliCN432D (rLM-N432D) contained only background levels of CD45.1+CD4+ T cells comparable to levels found in mice infected with Lm expressing an irrelevant control antigen (rLM-Control) (Fig. 5B).

Related experiments quantified IFN-γ production by FliC-specific CD45.1+CD4+

T cells after stimulation with WT FliC or each APL administered in the context of recombinant Lm infection. Since ΔActA Lm at an inocula of 106 CFUs triggers the production of cytokines such as IL-12 and type I IFN that together synergistically primes

IFN-γ production by CD4+ T cells [333], we hypothesized that the observed differences in IFN-γ production relative to the degree of proliferation for CD4+ T cells primed with

WT FliC compared with APL FliCL438I would be normalized in this highly polarizing Th1 cytokine environment. Alternatively, persistent reductions in IFN-γ production relative to

+ the degree of proliferation for CD4 T cells primed with APL FliCL438I compared with

WT FliC peptide would demonstrate a critical role for TCR stimulation in controlling

CD4+ T cell IFN-γ production even in the cytokine milieu triggered by ΔActA Lm infection. Remarkably and in complete agreement with stimulation studies using purified peptide, IFN-γ producing CD45.1+ FliC-specific CD4+ T cells were drastically reduced in mice primed with rLM-L438I compared with rLM-WT (~ 60% reduction, p <0.05) (Fig.

5B). As expected, the few cells that did not appreciably expand after infection with rLM-

N432D also did not produce detectable amounts of IFN-γ relative to mice inoculated with recombinant Lm expressing an irrelevant antigen (rLM-Control) (Fig. 5B). Additional experiments extended the time course for these experiments to characterize the survival of CD4+ T cells at later time points after infection with each recombinant Lm. By day 30 post-infection, the numbers of CD45.1+CD4+ T cells for mice infected with either rLM-

WT and rLM-L438I had contracted ~20-fold compared with day 5 levels (Fig. 5C).

Interestingly, despite this large degree of contraction, the ~60% reduction in number of

IFN-γ producing CD45.1+CD4+ T cells was maintained for rLM-L438I infected compared with rLM-WT infected mice through this later time point.

IFN-γ production dictates CD4+ T cell protective potency

Given the overall importance of CD4+ T cells and IFN-γ in host defense against intracellular bacterial pathogens like Salmonella, additional experiments sought to characterize the role of IFN-γ production by Salmonella-specific CD4+ T cells in immunity against this infection [249,260,261,322-324]. Mice primed initially with recombinant Lm expressing WT FliC or each APL were challenged with a lethal dose of virulent Salmonella typhimurium (ST) 30 days after Lm infection, and the degree of

83 protection was quantified by enumerating the number of recoverable ST CFUs from each group of mice. Compared with mice transferred with SM1 CD4+ T cells alone, significant differences in ST CFUs were found only for mice primed with rLM-WT where ~10-fold reductions (p < 0.05) in ST bacterial burden were found day 5 after challenge (Fig. 6). By contrast, mice primed with rLM-L438I, rLM-N432D, or rLM-Control each had significantly increased ST CFUs compared to mice primed with rLm-WT, at levels comparable to control mice that only received SM1 CD4+ T cells without Lm infection

(Fig. 6). This observed reduction in ST CFUs conferred by infection with rLM-WT cannot be attributed to non-specific immune activation due to ΔActA Lm infection alone because mice primed with rLM-L438I, rLM-N432D, and rLM-Control were each infected with the same overall inocula of ΔActA Lm. Furthermore, since similar numbers of FliC-specific CD4+ T cells are present both during expansion and after T cell contraction (Fig. 5) for mice primed with WT FliC and APL FliCL438I, differences in absolute number of FliC-specific CD4+ T cells alone also cannot account for reductions in ST CFUs conferred by rLM-WT compared to rLM-L438I. Taken together, these results indicate a critical role for IFN-γ production by pathogen-specific CD4+ T cells in host defense against Salmonella infection, and demonstrate how alterations in TCR stimulation can have dramatic impacts on CD4+ T cell differentiation and protective potency.

DISCUSSION

CD4+ T cell activation and differentiation requires at least three distinct, yet interrelated stimulatory signals mediated through the T cell receptor, co-stimulation receptors, and receptors for specific “inflammatory” cytokines [22,313-320]. Although these signals together uniformly stimulate T cell activation, proliferation, and differentiation, each signal has been proposed to play specific and defined roles in this process. For example, the antigen specificity for the T cell response is controlled by stimulation through the T cell receptor, while lineage differentiation is believed to be largely dictated by the presence or absence of specific cytokines. Unfortunately, since these roles have been primarily characterized following T cell stimulation in vitro or during non-infection conditions in vivo, the precise role for each stimulation signal in controlling the specific incremental steps required for T cell-activation during infection when the expression levels for all T cell stimulatory signals are drastically altered remains largely undefined.

Moreover, the impact of changes in each stimulation signal on CD4+ T cell-mediated protection to infection is unknown. Therefore, the importance of TCR stimulation in

CD4+ T cell activation were experimentally examined by measuring potential differences in proliferation, expansion, and differentiation after stimulation under non-infection and infection conditions with the Salmonella-FliC431-439 MHC class II peptide or APLs containing substitutions in TCR contact residues derived from this peptide. In these experiments, naturally-occurring altered peptide ligands containing amino acid substitutions in TCR contact residues within the FliC431-439 peptide were identified and

85 used for stimulation to explore how natural ligands from other bacterial pathogens may control the CD4+ T cell response to this antigen. Among these naturally-occurring APLs,

+ one primed FliC431-439-specific CD4 T cell proliferation more potently while others primed CD4+ T cell proliferation less potently compared with the parental wild-type FliC peptide following stimulation in vitro. Interestingly, despite differences in proliferation, all APLs primed dramatically reduced IFN-γ production that was directly associated with diminished up-regulation of T-bet expression. Importantly, the protective potency of these CD4+ T cells after challenge with virulent Salmonella was directly correlated with

IFN-γ production regardless of expansion magnitude. Collectively these results demonstrate a critical and previously unanticipated role for TCR stimulation in controlling CD4+ T cell differentiation into protective, IFN-γ producing effector T cells.

A two-step model for the initial up-regulation and eventual stabilization of T-bet expression in naïve CD4+ T cells required for IFN-γ production and Th1 differentiation has been proposed [94,334]. In this model, the initial expression of T-bet is dependent on

TCR stimulation and IFN-γ production, while the latter phase of T-bet stabilization is dependent on IL-12-receptor stimulation. Interestingly, termination of TCR stimulation permitted up-regulation of IL-12 receptor expression required for maintaining T-bet expression [94]. Our demonstration that APL FliCL438I is a more potent inducer of FliC- specific CD4+ T cell proliferation compared with WT FliC yet only weakly up-regulates

T-bet expression and IFN-γ production is consistent with this hypothesis, and suggests that the discordant expression of T-bet with proliferation potency after FliCL438I stimulation may be due to stronger TCR stimulation that inhibits the latter phase of T-bet

86 expression required for the stabilization of IFN-γ production by FliC-specific CD4+ T cells. Additional experiments that more specifically characterize the kinetics of T-bet,

IFN-γ, and IL-12 receptor expression are needed to elucidate the mechanism underlying this observed discordance between CD4+ T cell proliferation and IFN-γ production.

The identification of naturally-occurring APLs within protective immune- dominant antigens suggests that alterations in TCR stimulation may be used by pathogenic bacteria to modulate or avoid the pathogen-specific T cell response. This notion is bolstered by the identification of naturally-occurring APLs derived from

Salmonella-FliC431-439 among human clinical isolates of various invasive bacterial pathogens that we describe in this study (Fig 1). For example, APL FliCN432D has been described in over 100 unique Salmonella clinical isolates derived from serovars that have the potential to cause typhoidal or non-typhoidal disease in humans [330,335]. Moreover, this APL has also been identified in numerous clinical isolates of other invasive Gram- negative human pathogens such as Shigella sonnei, Yersinia intermedia , and both toxigenic and hemorrhagic forms of Escherichia coli [336-342]. Collectively, the identification of APLs within FliC431-439 from this diverse range of bacterial pathogens that cause clinical disease indicates pathogens containing these mutations not only retain virulence, but may be more capable of enhanced pathogenesis associated with immune evasion of protective T cells. Although our experiments examined the response for CD4+

T cells with unique specificity to one Salmonella-specific immune-dominant peptide antigen, these results nevertheless provide experimental evidence implicating alterations in CD4+ T cell TCR stimulation as a potential immune evasion strategy utilized by

87 bacterial pathogens. Therefore, further investigation characterizing the differences in virulence properties and immune response to pathogens that contain APLs in immune- dominant T cell antigens is needed, and will likely reveal novel and important information on the pathogenesis and immune response to these infections.

MATERIALS AND METHODS

Mice. C57BL/6 (I-Ab) mice were purchased from The National Cancer Institute and used

+ between 6-8 weeks of age. FliC431-439-specific (SM1) CD4 TCR transgenic mice were intercrossed with CD45.1+ mice and maintained on a Rag-1-deficient background as described [273]. All mice were housed within University of Minnesota specific pathogen- free facilities and experiments were conducted under institutional IACUC approved protocols.

b Peptides. The parental I-A -restricted wild-type FliC431-439 peptide, and each APL derived from this peptide were purchased from United Biochemical Research (≥ 90% purity;

Seattle, WA): RFNSAITNLGN (WT FliC), RFNSAITNIGN (FliCL438I),

RFDSAITNLGN (FliCN432D), RFESAITNLGN (FliCN432E), and RFNFAITNLGN

b (FliCS433F). The I-A -restricted Ag85B243-253 peptide (AYNAAGGHNAV) was used as an irrelevant stimulation control. All peptides were dissolved in DMSO (100mM), and further diluted in sterile saline to the indicated concentration used for in vitro stimulation.

For in vivo stimulation, 50 µg each peptide was diluted with saline (200 µL) and intravenously injected into mice.

T cell stimulation. For in vitro stimulation, splenocytes from SM1 TCR transgenic mice were cultured in 96-well round-bottom plates (1 X 106 cells/mL) containing the indicated concentration of each peptide dissolved in DMEM media supplemented with 10% FBS,

10 mM Hepes, 1 mM Sodium pyruvate, 2 mM L-glutamine, 50 µM 2-mercaptoethanol,

1% nonessential amino acids, and penicillin (100 U/mL) + streptomycin (100 U/mL). For some experiments, CD4+ T cells from SM1 TCR transgenic mice were labeled with

CFSE (Invitrogen Corporation, Carlsbad, CA) prior to stimulation using standard labeling conditions (5 µM for 10 minutes at room temperature). For adoptive transfer, 2 X 104

CD4+ T cells from SM1 TCR transgenic mice were intravenously inoculated into recipient mice one day prior to peptide inoculation or recombinant Lm infection.

Antibodies and other reagents for cell surface, intracellular, or intranuclear staining were purchased from BD Biosciences (San Jose, CA) or eBioscience (San Diego, CA). For measuring cytokine production by cells stimulated in vitro, brefeldin A was added to cultures for the final 5 hours prior to intracellular cytokine staining. For measuring

+ cytokine production by CD4 T cells ex vivo, splenocytes were stimulated with FliC431-439 peptide (10 µM) in the presence of brefeldin A for 5 hours prior to intracellular cytokine staining.

Bacteria. Lm ΔActA strain DPL-1942 along with methods for Lm transformation has been described previously [332,343,344]. Lm ΔActA cannot spread from infected cells into adjacent non-infected cells, is rapidly cleared after infection, and therefore allows the use of higher recombinant Lm inocula to optimally prime the expansion of pathogen- specific CD4+ T cells [331]. Briefly, recombinant Lm expressing either the parental wild- type FliC431-439 peptide or each APL derived from this peptide were generated by cloning the coding sequence for each into the “open” pAM401-based expression construct that

90 allows transcription behind the Lm-specific hly promoter and secretion based on the

LLO-specific signal sequence[331,344]. Specifically, the coding and non-coding sequences that correspond to each peptide (Table 1) were annealed together and ligated into the PstI and StuI sites of this “open” vector. Relevant portions of this construct were verified by DNA sequencing. Lm protein preparation, SDS gel electrophoresis, and blotting using anti-HA antibody (clone HA-11, Covance) were performed as described

[331,344]. For infection, Lm was grown to log phase in brain heart infusion media (BD

Biosciences, San Jose, CA) containing chloramphenicol (20 µg/mL) at 37oC, washed and diluted with saline to a final concentration of 1 X 106 CFUs per 200 µL and injected intravenously into mice as described [331,344]. The virulent Salmonella typhimurium

(ST) strain SL1344 has been described [273,323], and was similarly propagated as Lm in brain heart infusion broth at 37oC. For infections, 1 X 102 ST CFUs were washed and diluted in saline (200 µL) and injected intravenously into mice.

Statistics. The differences in mean numbers of recoverable bacterial CFUs between groups of mice were evaluated using the Student’s t test with p <0.05 as statistically significant (GraphPad Prism Software).

b Figure 1. Alignment of FliC431-439 with other I-A peptides. Putative TCR contact or

MHC anchor residues within each peptide are indicated by black and gray font, respectively. Numbers in parentheses indicate the amino acid residues that correspond to each position for the FliC431-439 peptide compared with the indicated residues from antigen-85B (Ag85B) or pigeon cytochrome C (PCC) (top). Alignment of each naturally-

93 occurring FliC431-439 –derived APL compared with the parental FliC431-439 peptide

(bottom).

Figure 2. FliC-specific CD4+ T cell proliferation and activation after in vitro stimulation.

A. CFSE dilution in SM1 CD4+ T cells after stimulation with WT FliC peptide or each

APL (line histogram) at the indicated concentration, or after stimulation with Control peptide (shaded histogram, 10 µM) for 5 days. Numbers in each plot indicate the percent

CFSElo cells. B. CD25, CD44 and CD62L expression by FliC-specific CD4+ T cells after stimulation with 10 µM WT FliC or each APL (line histogram) or Control peptide 94

(shaded histogram) for 5 days. These results are representative of three independent experiments each with similar results.

Figure 3. Reduced IFN-γ and T-bet expression after stimulation with each APL compared with WT FliC peptide. Percent IFN-γ-producing CD4+ T cells after stimulation with each indicated peptide (10 µM) (top). T-bet expression after stimulation with WT

FliC or each APL (line histogram) or Control peptide (shaded histogram) (bottom).

Numbers in each plot indicate mean fluorescent intensity of T-bet staining. These results are representative of at least two independent experiments each with similar results.

Figure 4. Pathogen-specific CD4+ T cell expansion and cytokine production after in vivo stimulation. A. Percent FliC-specific CD45.1+ T cells among CD4+ splenocytes day 5 after intravenous injection of each peptide (50 µg). B. Total number FliC-specific

CD45.1+ T cells among splenocytes for mice described in A (left), and total number IFN-

γ-producing CD45.1+CD4+ splenocytes after peptide stimulation (shaded bar) or no stimulation control (open bar) (right) for mice described in A. These results are representative of two independent experiments each with similar results containing six mice per group. Bar, standard error.

Figure 5. FliC-specific CD4+ T cell expansion and cytokine production after recombinant

Lm infection. A. Construct map indicating placement of coding sequences for WT FliC and each APL within the pAM401-based expression vector (Phly, Lm hly promoter; SS, signal sequence of hly; HA, hemagglutinin tag; cat, chloramphenicol acetlytransferase)

(left). Western blot of supernatant protein from rLM-WT (lane 1), rLM-L438I (lane 2), rLM-N432D (lane 3), and rLM-CONTROL (lane 4) (right). B,C. Total number of FliC- specific CD45.1+CD4+ cells (left) and IFN-γ-producing CD45.1+CD4+ cells (right) after peptide stimulation (shaded bar) or no stimulation control (open bar) among splenocytes day 3 (B) and day 30 (C) after infection with each recombinant Lm. These results are combined from at least two independent experiments each with similar results containing four to six mice per group. Bar, standard error. 97

Figure 6. WT FliC peptide expressed in recombinant Lm confers protection to

Salmonella. Number of recoverable Salmonella CFUs in the spleen of mice day 5 after challenge with virulent Salmonella typhimurium. Each group of mice were adoptively transferred with FliC-specific CD4+ T cells and initially infected with the indicated recombinant Lm or no Lm infection 30 days prior to Salmonella challenge. These data represent eight to fourteen mice per group combined from four independent experiments each with similar results. Bar, standard error. *, p < 0.05.

Chapter 3

Regulatory T Cell Suppressive Potency Dictates the Balance between

Bacterial Proliferation and Clearance during Persistent Salmonella

Infection*

* Johanns TM, Ertelt JM, Rowe JH, Way SS (2010). PLoS Pathog 6(8): e1001043.

ABSTRACT

The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3-4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden was associated with robust effector T cell activation. Using Foxp3GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3+

Treg suppressive potency. In complementary experiments using Foxp3DTR mice that allow the targeted ablation of regulatory T cells, the significance of these changes in Treg suppressive potency on infection outcome was verified by enumerating the changes in bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

100

INTRODUCTION

Typhoid fever is a systemic, persistent infection caused by highly adapted host- specific strains of Salmonella [202,203,345]. Human typhoid is caused predominantly by

S. enterica serotype Typhi [205], while mice develop a typhoid-like disease following S. enterica serotype Typhimurium infection. Interestingly, the early stages of this infection, in both mice and humans, are usually asymptomatic or associated with only mild, non- specific “flu-like” symptoms [205,346]. This represents a stark contrast to other Gram- negative bacterial pathogens (e.g. Escherichia coli, Neisseria meningitidis, Haemophilus influenza) that primarily cause acute infection and immediately trigger robust systemic symptoms after tissue invasion. Thus, the inflammatory response is blunted early after infection with Salmonella strains that cause persistent infection, and this feature likely facilitates long-term pathogen survival [345]. On the other hand, the blunted inflammatory response to systemic Salmonella infection also minimizes immune- mediated damage to host tissues that may outweigh the immediate risk posed by the pathogen itself [249]. Thus, dampening the immune response provides potential advantages to pathogen and host during persistent Salmonella infection.

Regulatory T cells (Tregs) were initially identified as a CD25-expressing subset of CD4+ T cells required for maintaining peripheral immune tolerance to self-antigen.

However more recent studies clearly demonstrate their importance extends to controlling the immune response during infection [163,347-349]. In this regard, the functional importance of Tregs has been best characterized for pathogens that cause persistent

101 infection. For example, depletion of CD25+CD4+ Tregs is associated with enhanced effector T cell activation and reduced pathogen burden during Leishmania major infection [350]. Similarly, reconstituting T cell-deficient mice with CD25+CD4+ Tregs abrogates enhanced pathogen clearance that occurs after reconstitution with CD25- depleted CD4+ T cells [151,350]. These complementary experimental approaches initially used to identify the role of CD25+ Tregs in host defense during L. major infection have since been reproduced after infection with numerous other bacterial, viral, and parasitic pathogens [149,152,154,158,163,351,352]. Interestingly, Treg-mediated immune suppression can also play “protective” roles for infections where host injury caused by the immune response outweighs the damage caused by the pathogen itself, [149,152], or when pathogen persistence is required for maintaining protection against secondary infection [350,353]. Together, these findings suggest Treg-mediated immune suppression can provide both detrimental and protective roles in host defense against infection.

Despite these observations, identifying the functional importance of Tregs during in vivo infection has been limited, in part, by the lack of unique markers that allow their discrimination from other CD4+ T cell subsets. In this regard, the majority of infection studies have experimentally manipulated Tregs based on surrogate markers such as CD25 expression on CD4+ T cells. However, since CD25 expression is also a marker for activated T cells with no suppressive function, identifying Tregs based on CD25 expression does not allow discrimination between these functionally distinct T cell subsets. These limitations have been recently overcome by the identification of Foxp3 as the master regulator for Treg differentiation, and the generation of transgenic mice that

102 allow precise identification or targeted manipulation of Tregs based on Foxp3 expression

[354-356]. These include Foxp3GFP reporter mice that allow ex vivo Foxp3+ Treg isolation by sorting for GFP-expressing cells, and Foxp3DTR transgenic mice that co-express a high affinity diphtheria toxin receptor (DTR) with Foxp3 [135,150]. Intriguingly, the first infection study using Foxp3DTR mice for Treg ablation revealed somewhat paradoxical roles for Foxp3+ Tregs in host defense. Within the first fours days after intravaginal herpes simplex virus 2 (HSV-2) infection, reduced inflammatory cell infiltrate and increased viral burden were found at the site of infection in Treg-ablated compared with

Treg-sufficient mice [161]. These effects were not limited to HSV-2, nor were they restricted to the mucosal route of infection as increased pathogen burden associated with

Foxp3+ Treg ablation also occurred after parenteral infection with lymphocytic choriomeningitis virus (LCMV) and West Nile virus [160,161]. Whether these Treg- mediated reductions in pathogen burden are limited to these specific viral pathogens, or represent re-defined roles for Tregs based on their manipulation using Foxp3-specific reagents are currently undefined. Therefore, additional studies using representative mouse models of other human infections and Foxp3-specific reagents for Treg manipulation are required. In this study, the role of Foxp3+ Tregs in controlling immune cell activation and the balance between pathogen proliferation and clearance during the natural progression of persistent bacterial infection was examined after infection with virulent Salmonella.

103

RESULTS

Persistent Salmonella infection in F1 129Sv X C57BL/6 mice

Commonly used inbred mouse strains have discordant levels of innate resistance to virulent S. enterica serotype Typhimurium based primarily on whether a functional allele of Nramp1 is expressed [255,257]. For example, C57BL/6 mice express a functionally defective, naturally occurring variant of Nramp1 and thus, are inherently susceptible to infection with virulent Salmonella dying within the first few days from uncontrolled bacterial replication. By contrast, 129Sv mice, which express wild-type

Nramp1 (Nramp1-sufficient), are inherently more resistant developing a persistent infection instead [251,274]. Since transgenic mouse tools for Treg manipulation based on

Foxp3-expression are available primarily on the susceptible, Nramp1-defective C57BL/6 background, we sought to exploit the autosomal dominant resistance to Salmonella conferred by wild-type Nramp1, and the X-linked inheritance of Foxp3 transgenic mice by examining infection in resistant F1 129Sv X C57BL/6 mice [274]. Similar to results after infection with virulent Salmonella in 129Sv mice, progressively increasing bacterial burdens are found throughout the first 3-4 weeks after infection in F1 129Sv X C57BL/6 mice (Fig. 7A). By contrast, Nramp1-defective C57BL/6 mice died within the first week after infection from overwhelming bacterial replication despite a 100-fold reduction in

Salmonella inocula (Fig. 7A). The progressively increasing bacterial burden within the first 3-4 weeks after Salmonella infection in F1 129Sv X C57BL/6 mice parallels dramatic changes in both spleen size and absolute number of splenocytes (Fig. 7B, 7C).

104

Each of these parameters increased within the first three weeks after infection and declined subsequently at later time points that directly coincide with changes in

Salmonella bacterial burden (Fig. 7A-C). These findings demonstrate an interesting dichotomy in infection tempo between early (first 3-4 weeks) and later time points during persistent Salmonella infection in resistant F1 129Sv X C57BL/6 mice.

Delayed T cell activation early after Salmonella infection

Given the importance of T cells in host defense against Salmonella [261,324,357], the expansion and activation kinetics for CD4+ and CD8+ T cells during this persistent infection were each enumerated. Although the absolute numbers of both cell types increased in parallel with the absolute numbers of splenocytes, a progressive and steady increase in percent CD4+ T cells became readily apparent beginning week three post- infection (Fig. 7D). By contrast, the percent CD8+ T cells remained essentially unchanged throughout these same time points. Additional phenotypic characterization revealed that the percent activated (CD44hiCD62Llo) CD4+ and CD8+ T cells both increased sharply beginning week 3, and were sustained at high levels through week 7 after infection (Fig. 8A). Furthermore, the kinetics of T cell activation based on CD44 and CD62L expression directly paralleled the kinetics whereby CD4+ and CD8+ T cells each became primed for IFN-γ production (Fig. 8B). Thus, the kinetics of CD44 and

CD62L expression and IFN-γ production each reveal delayed T cell activation early after infection, not peaking until weeks 3 to 4, that is followed by more sustained T cell activation thereafter.

105

Given the durability whereby T cells maintain changes in CD44 and CD62L expression, and IFN-γ production after activation, the expression of more transient T cell activation markers such as CD25 and CD69 were also quantified throughout persistent

Salmonella infection. CD25 and CD69 expression on CD4+ and CD8+ T cells each peaked between weeks 3 and 4 post-infection (Fig. 8C). However consistent with the transient nature of their expression, CD25 and CD69 expression each declined to baseline levels over the next 2 to 3 weeks. Thus, the sharp increase in T cell activation that occurs between weeks 3 and 4 after Salmonella infection is confirmed using both transient

(CD25, CD69) and more stable (CD44, CD62L, IFN-γ) markers of T cell activation.

Interestingly, the sharp increase in T cell activation beginning week 3 after infection directly parallels when reductions in bacterial burden begins to occur, and suggests dampened T cell activation early after infection allows progressively increasing bacterial burden, while enhanced T cell activation later facilitates bacterial clearance.

CD4+ T cell-mediated Salmonella clearance during persistent infection

To determine the overall importance and individual contribution provided by each

T cell subset in bacterial clearance during the natural course of persistent Salmonella infection, the impacts of CD4+ and/or CD8+ T cell depletion were determined. Anti- mouse CD4 and anti-mouse CD8 depleting antibodies were administered beginning day

31 post-infection. In initial studies, we found that 750 µg of each could deplete the respective T cell subset with ≥ 99% efficiency even in Salmonella-infected mice that contain expanded T cell numbers (Fig. 9A). With sustained CD4+ T cell depletion,

106 significantly increased numbers of recoverable Salmonella CFUs were found at day 6

(day 31+6) after the administration of anti-mouse CD4 compared with isotype control antibody (Fig. 9B). Moreover, the magnitude of this difference became even more pronounced by day 14 (day 31+14) after antibody treatment. By contrast, CD8+ T cell depletion alone or together with CD4+ T cell depletion did not cause significant changes in Salmonella bacterial burden except in the spleen day 14 after antibody treatment where combined depletion of both CD4+ and CD8+ T cells resulted in increased numbers of recoverable Salmonella CFUs compared to CD4+ T cell depletion alone (Fig. 9B).

Together, these results demonstrate an essential role for CD4+ T cells in the clearance of persistent Salmonella infection, and these findings are consistent with the previously reported requirement for this T cell subset in controlling the replication of attenuated

Salmonella in susceptible Nramp1-defective mice [261]. Moreover, an essential role for

CD4+ T cells in host defense during persistent infection in resistant mice is further supported by the sharp increase in overall percentage and activation of these cells which coincides with reductions in Salmonella bacterial burden beginning week 3 post-infection

(Fig. 7, 8).

Parallel expansion of Foxp3+ Tregs and non-Treg CD4+ cells during persistent infection

The requirement for CD4+ T cells in bacterial clearance during persistent

Salmonella infection may reflect contributions from either Foxp3-negative effector or

Foxp3+ regulatory T cells (Tregs). To characterize the relative contributions of each

107

CD4+ T cell subset during persistent infection, our initial studies enumerated the percent

Foxp3+ cells among CD4+ T cells and the expansion kinetics of Foxp3+ and Foxp3- negative CD4+ T cells during persistent infection. Interestingly despite dramatic shifts in the percent and absolute number of CD4+ T cells among splenocytes, the percent Foxp3+

Tregs among CD4+ T cells remains remarkably stable and essentially unchanged at approximately 10% throughout the infection (Fig. 10A, 10B). By extension, the absolute numbers of Foxp3+ Tregs and Foxp3-negative effector CD4+ T cells were also found to expand in parallel (Fig. 10C). These findings suggest shifts in the ratio of Foxp3+ Tregs among non-Treg effector CD4+ T cells alone does not account for the shift in relative T cell activation and change in infection tempo at early compared to late time points during persistent Salmonella infection.

Dynamic shifts in Foxp3+ Treg suppressive potency

Since defined inflammatory cytokines and pathogen associated molecular patterns have each been shown to control Treg suppressive potency after stimulation in vitro [358-

363], we explored the possibility that intact pathogens and the ensuing immune response would also dictate shifts in Treg suppressive potency after infection in vivo. By extension, these shifts in relative Treg suppressive potency may also impact the activation of non-Treg effector cells and overall tempo of persistent infection.

Accordingly, we compared the suppressive potency for Foxp3+ Tregs isolated at early

(day 5) and late (day 37) time points during persistent Salmonella infection. These specific time points where chosen because they reflect highly pronounced contrasts in T

108 cell activation and directional changes in Salmonella bacterial burden, yet have comparable bacterial burdens (Fig. 7, 8). Nramp1-sufficient F1 Foxp3GFP reporter hemizygous male mice derived by intercrossing 129Sv males with Foxp3GFP/GFP females

(on the C57BL/6 background) that simultaneously allow persistent Salmonella infection and for all Tregs to be isolated based on cell sorting for GFP+Foxp3+ cells were used in these experiments [135] (Fig. 11A). By first enriching for CD4+ cells using negative selection, GFP+Foxp3+ Tregs could be routinely isolated from naïve and Salmonella- infected F1 Foxp3GFP reporter mice each with ≥ 99% purity (Fig. 11B). Potential differences in suppressive potency for GFP+Foxp3+ Tregs isolated at each time point after infection were quantified by measuring their ability to inhibit the proliferation of responder CD4+ T cells isolated from naïve CD45.1 congenic mice after non-specific stimulation in vitro using previously defined methods [364-366].

Compared with Tregs isolated from F1 Foxp3GFP reporter mice prior to infection, the suppressive potency of Tregs isolated from mice day 5 after Salmonella infection was enhanced (Fig. 11C). At the same Treg to responder T cell ratio, Foxp3+ Tregs from mice day 5 after infection consistently inhibited responder CD45.1+ T cell proliferation (CFSE dilution) more efficiently. These differences in suppression were eliminated when a 2- fold reduction in Treg to responder cell ratio from mice day 5 post-infection compared with undiluted Tregs from naïve mice were co-cultured with a fixed number of naïve responder cells (Fig. 11C). In sharp contrast to increased suppression that occurs at this early post-infection time point, the suppressive potency for Tregs isolated from mice day

37 after infection was significantly reduced. Compared with Tregs isolated from mice 5

109 days after infection, the efficiency whereby Tregs isolated day 37 post-infection inhibited the proliferation of responder CD45.1+ T cells was reduced approximately 4-fold; and compared with Tregs isolated from naïve mice, their suppressive potency was reduced approximately 2-fold (Fig. 11C). In other words, a 50% reduction in Treg to responder cell ratio for Tregs isolated from naïve mice, and a 75% reduction in ratio for Tregs from mice day 5 after infection each suppressed responder cell proliferation to the same extent as undiluted Foxp3+GFP+ Tregs isolated from mice day 37 after infection. These results demonstrate that although the ratio of Foxp3+ Tregs and non-Treg effector CD4+ T cells remains unchanged, shifts in Treg suppressive potency that directly parallel the kinetics of T cell activation and infection tempo occur during the progression of persistent

Salmonella infection.

In complementary experiments, the relative degree of Treg-mediated suppression during Salmonella infection was further characterized using newly developed in vivo approaches. Specifically the expansion of adoptively transferred antigen-specific T cells after stimulation with cognate peptide at defined time points during persistent infection was enumerated. This approach exploits the use of F1 129Sv X C57BL/6 mice as recipients for adoptively transferred T cells from TCR transgenic mice on the C57BL/6 background [367]. As a control to identify the overall contribution of Tregs in suppressing the expansion of adoptively transferred T cells in vivo, F1 Foxp3DTR hemizygous male mice derived from intercrossing 129Sv males with Foxp3DTR/DTR female mice on the C57BL/6 background, which allows targeted ablation of Foxp3+

Tregs by administering low-dose diphtheria toxin (DT) were used initially [150]. We

110 found 1.0 µg (50 µg/kg) DT given on two consecutive days was sufficient for ≥ 99% ablation of Foxp3+ Tregs, and continued DT dosing (0.2 µg every other day) was able to maintain this level of Treg ablation in Foxp3DTR mice on the F1 background (Fig. 12A).

These results are consistent with the reported efficiency whereby Foxp3+ Tregs are selectively ablated in Foxp3DTR mice on the C57BL/6 background [150]. Although in vivo injection of cognate OVA257-264 peptide could stimulate only modest levels of expansion for adoptively transferred T cells from OT-1 TCR transgenic mice in Treg- sufficient mice, the expansion magnitude was increased > 50-fold in Treg-ablated F1

Foxp3DTR mice (Fig. 12B). Importantly, the expansion of these adoptively transferred T cells was antigen-dependent because very few cells could be recovered from either Treg- ablated or Treg-sufficient recipient mice without peptide stimulation. Thus, Tregs actively suppress the expansion of peptide stimulated antigen-specific T cells in vivo, and the relative expansion of these exogenous cells is a reflection of Treg suppressive potency.

Using this approach, the relative expansion of exogenous T cells from OT-1 TCR transgenic mice after adoptive transfer into Salmonella infected F1 129Sv X C57BL/6 and stimulation with cognate OVA257-264 peptide was enumerated. The percent and total numbers of OT-1 T cells was increased 4-fold and 5-fold, respectively, after adoptive transfer into mice at late (day 37) compared with early (day 5) time points during persistent infection (Fig. 12C). Thus, the in vivo environment at later compared with early time points during persistent Salmonella infection is significantly more permissive for peptide-stimulated T cell expansion. These results, together with the reductions in

111 suppressive potency for Foxp3+GFP+ cells isolated ex vivo from mice at early compared with late time points (Fig. 12C), and the critical role for Foxp3+ Tregs in controlling exogenous T cell expansion in response to cognate peptide (Fig. 12A, 12B) clearly illustrate reductions in Treg suppressive potency occur from early to late points during persistent Salmonella infection. Furthermore, given the sharp dichotomy in infection tempo at these specific time points, these results suggest enhanced Treg suppression early after infection restrains effector T cell activation that allows progressively increasing

Salmonella bacterial burden, while diminished Treg suppression at later time points allows enhanced T cell activation that more efficiently controls the infection.

Dynamic regulation of Treg-associated molecules that control suppression

Multiple Treg-associated cell surface and secreted molecules have been implicated to mediate immune suppression by these cells. For example, increased expression of CTLA-4, IL-10, Tgf-β, Granzyme B, ICOS, PD-1, and CD39 each have been shown independently to coincide with enhanced Treg suppressive potency

[166,174,182,184,187,191,368-371], while expression of other Treg cell-intrinsic molecules (e.g. GITR, OX40) each parallel reductions in suppressive potency [372-374].

Although the relative importance of each defined molecule varies significantly depending upon the experimental model used, the relative expression of Treg cell-intrinsic signals that either stimulate or inhibit suppression likely dictates the overall suppressive potency of Tregs. Therefore, we quantified the relative expression of each molecule on Foxp3+

Tregs to explore how the observed shifts in suppression potency from early to late time

112 points during persistent Salmonella infection correlate with changes in their expression

(Fig. 13A and Fig. 14). Consistent with the drastic reduction in suppressive potency, significant shifts in expression for some Treg-associated molecules between day 5 and day 37 post-infection were identified. For example, molecules that have independently been associated with diminished Treg suppression potency such as reduced CTLA-4 and increased GITR expression were found for Foxp3+ Tregs from mice day 5 compared with day 37 after infection [174,372,373] (Fig. 13A). By contrast, more modest or minimal changes were found for other Treg-associated molecules implicated to mediate suppression (e.g. CD39, IL-10, Granzyme B, PD-1, and Tgf-β) [182,184,187,191,368-

370,374] (Fig. 14). Thus, reduction in Treg suppressive potency during the progression of persistent Salmonella infection directly parallels reduced CTLA-4 and increased GITR expression that each independently correlates with this shift in suppression.

Salmonella FliC-specific CD4+ T cells expand during persistent infection

Given the importance of pathogen-specific Tregs in controlling pathogen-specific effector cells in other models of persistent infection [153,375,376], the expansion kinetics and relative expression of Treg-associated effector molecules were also characterized for

Salmonella-specific Tregs. The best characterized Salmonella-specific, I-Ab-restricted

MHC class II antigen is the flagellin FliC431-439 peptide [323]. Using tetramers with specificity for this antigen and magnetic bead enrichment, naïve C57BL/6 mice have

+ been estimated to contain ~20 FliC431-439-specific CD4 T cells [325]. Using these same

+ techniques, we find similar numbers of FliC431-439-specific CD4 T cells in naïve F1 mice

113 prior to Salmonella infection (Fig. 13B). As predicted after Salmonella infection, the

+ numbers of these FliC431-439-specific CD4 T cells expand reaching ~10-fold and 20-fold increased cell numbers day 5 and 37 post-infection, respectively (Fig. 13B). Interestingly,

+ + for FliC431-439-specific CD4 cells identified in this manner, ~10% were Foxp3 in F1 mice prior to and at each time point after infection (Fig. 13B). Thus, FliC431-439-specific

Tregs and effector T cells expand in parallel during this persistent infection, and these results are consistent with the stable percentage of Foxp3+ Tregs among bulk CD4+ T cells (Fig. 10). Although the relatively small number (~ 1-2 cells per mouse) of FliC431-

+ 439-specific Foxp3 Tregs in naïve mice precluded further analysis beyond these absolute

+ cell numbers, the expansion of FliC431-439-specific Tregs and non-Treg effector CD4 T cells at early and late time points after infection allowed the relative expression of likely determinants of Treg suppression to be characterized. FliC431-439-specific Tregs were found to down-regulate CTLA-4 and up-regulate GITR expression, as infection progressed from early to late time points to a similar extent in FliC431-439-specific compared with bulk Tregs at these same time points after infection (Fig. 13A, 13C).

Thus, the relative expression of Treg-intrinsic molecules known to stimulate or impede immune suppression occurs for both pathogen-specific and bulk Foxp3+ Treg cells, and these changes directly coincide with reductions in their suppressive potency that occurs from early to late time points during persistent infection.

Reduced impact of Treg-ablation from early to late time points during persistent infection

114

To more definitively identify the relative importance of Treg-mediated immune suppression on the progression of persistent Salmonella infection, the impacts of Treg ablation on infection tempo and T cell activation were enumerated at early and late time points after infection. Given the striking contrasts in suppressive potency for Foxp3+

Tregs, directional changes in bacterial burden, and effector T cell activation between mice day 5 versus day 37 post-infection, the relative impact caused by Treg ablation using F1 Foxp3DTR mice (Fig. 12A) on infection tempo beginning at these time points were enumerated. In agreement with their essential role in maintaining and sustaining peripheral tolerance [150], Treg-ablated mice began to appear lethargic and dehydrated beginning day 8 after the initiation of DT treatment in Salmonella-infected mice. Thus, the impacts of Treg ablation on infection tempo and T cell activation were limited to discrete 7 day windows during persistent Salmonella infection. For mice that received DT beginning day 5 post-infection, significantly reduced numbers of recoverable Salmonella

CFUs were found for Treg-ablated F1 Foxp3DTR compared with Treg-sufficient F1

Foxp3WT control mice 6 days after the initiation of DT treatment (day 5+6) (Fig. 15A).

These reductions in bacterial burden after Treg ablation early after infection were paralleled by significantly increased T cell activation (percent CD44hiCD62lo T cells)

(Fig. 15B). Importantly, the reductions in Salmonella bacterial burden in Treg-ablated mice cannot be attributed to non-specific effects related to DT treatment because both

Treg-ablated F1 Foxp3DTR and Treg-sufficient F1 Foxp3WT control mice each received identical doses of this reagent, nor could they be attributed to cell death-induced inflammation triggered by dying Tregs because no significant reductions in recoverable

115

CFUs were found for F1 Foxp3DTR/WT mice where ~50% Tregs express the high affinity

DT receptor and are eliminated following DT treatment (Fig. 16). By contrast, Treg ablation beginning later after infection (day 37) when T cells are already highly activated caused no significant change in Salmonella bacterial burden and only a modest incremental increase in T cell activation between Treg-ablated F1 Foxp3DTR compared with Treg-sufficient F1 Foxp3WT control mice (Fig. 15A, 15B). Thus, the relative impact of Treg ablation at early and late time points on infection outcome directly parallel the differences in their suppressive potency. Together, these results demonstrate enhanced

Treg suppressive potency at early time points after infection restrains effector T cell activation and allows progressively increasing bacterial burden. By extension, Treg ablation at these early time points markedly increases T cell activation and significantly reduces the bacterial burden (Fig. 15A, 15B). Reciprocally, at later time points after infection when Treg suppressive potency is diminished, the relative contribution of

Foxp3+ Tregs on T cell activation and bacterial clearance is reduced (Fig. 15A, 15B).

Thus, dynamic regulation of Treg suppression dictates the balance between pathogen proliferation and clearance during the course of persistent Salmonella infection.

Given the drastic shifts in Treg-associated expression of CTLA-4 and GITR that each correlates with the reduced suppressive potency of these cells from early to late time points during persistent Salmonella infection, additional experiments sought to identify the relative importance of these molecules in dictating infection tempo using well characterized CTLA-4 blocking (clone UC10-4F10) or GITR-stimulating (clone DTA-1) monoclonal antibodies [179,373,377,378]. Consistent with the essential role for CTLA-4

116 in Treg suppression during non-infection conditions in vivo [174], significant reductions in Salmonella recoverable CFUs and accelerated T cell activation were found with

CTLA-4 blockade initiated beginning day 5 after Salmonella infection, and the magnitude of these changes paralleled those following DT-induced Treg-ablation in

Salmonella-infected F1 Foxp3DTR mice (Fig. 15C, 15D). Since Foxp3-negative cells also express CTLA-4, albeit at significantly reduced levels compared with Foxp3+ Tregs (Fig.

13), we further explored the relative contribution of CTLA-4 blockade in the absence of

Foxp3+ Tregs. Consistent with the reduced levels of CTLA-4 expression on Foxp3- negative CD4+ T cells, the effects of CTLA-4 blockade were eliminated with Foxp3+

Treg ablation (Fig. 17). By extension, at later time points after infection (day 37) when

CTLA-4 expression is down regulated on Foxp3+ Tregs, no significant change in

Salmonella bacterial burden or T cell activation occurred with CTLA-4 blockade (Fig.

15C, 15D). By contrast to these results with CTLA-4 blockade that directly recapitulates the effects of Treg ablation at early and late time points during persistent infection, treatment with a monoclonal antibody that stimulates cells through GITR caused no significant changes in Salmonella bacterial burden or T cell activation when initiated at either early or late time points during persistent infection (Fig. 15E, 15F). Together, these results suggest the dynamic regulation of Treg suppressive potency during Salmonella infection is predominantly mediated by shifts in CTLA-4 expression, and reduced CTLA-

4 expression by Tregs during the progression of this persistent infection dictates reduced suppression with enhanced effector T cell activation and bacterial clearance.

117

DISCUSSION

The balance between immune activation required for host defense, and immune suppression that limits immune-mediated host injury is stringently regulated during persistent infection [249,347]. Although Tregs have been widely implicated to control the activation of immune host defense components during infection, their role in dictating the natural progression of persistent infection remains undefined. In this study, we report two distinct phases of effector T cell activation with opposing directional changes in pathogen burden in a mouse model of persistent Salmonella infection. Delayed T cell activation associated with increasing bacterial burden occurs early, while enhanced T cell activation that parallels reductions in pathogen burden occurs later during infection. Remarkably, significant reductions in Treg suppressive potency between early and late infection time points directly coincide with these differences in infection tempo. In complementary experiments, the significance of these shifts in Treg suppressive potency were verified by directly enumerating the relative impact of Treg ablation on infection tempo at early and late infection time points. Together, these results demonstrate dynamic changes in

Foxp3+ Treg suppressive potency dictate the natural course and progression of this persistent infection.

Along with two recent studies characterizing infection outcome with Foxp3+ Treg ablation after mucosal HSV-2, systemic LCMV, and footpad West Nile virus infection

[160,161], these are the first studies to characterize the importance of Tregs during infection using Foxp3DTR transgenic mice. These results comparing infection outcome after Treg manipulation based on their lineage-defining marker, Foxp3, allow the

118 importance of Tregs to be more precisely characterized compared with other methods that identify and manipulate Tregs using surrogate markers (e.g. CD25 expression) that are not expressed exclusively by these cells. Interestingly, while Treg ablation caused increased pathogen burden, delayed arrival of acute inflammatory cells, and accelerated mortality after HSV-2, LCMV, and West Nile virus infection [160,161], we find contrasting reductions in pathogen burden and increased T cell activation with Treg ablation at early, but not late time points during persistent Salmonella infection.

However, the reductions in Salmonella pathogen burden with early Treg ablation are consistent with reduced Mycobacterium tuberculosis pathogen burden after partial Treg depletion using bone marrow chimera mice reconstituted with mixed cells containing congenically-marked Foxp3+ Tregs and Foxp3-deficient cells [155]. Together, these studies comparing infection outcome after Treg ablation using Foxp3-specific reagents highlight interesting and divergent functional roles for Foxp3+ Tregs during specific infections. The reasons that account for these differences – whether they are related to differences between bacterial versus viral pathogens or between pathogens that primarily cause acute versus persistent infection, are important areas for additional investigation, and require the characterization of infection outcomes after Treg manipulation using

Foxp3-specific reagents with other pathogens.

The dynamic regulation of Treg suppressive potency during Salmonella infection we demonstrate here is consistent with the ability of inflammatory cytokines and purified

Toll-like receptor (TLR) ligands to each control Treg suppression after stimulation in vitro [358-363]. However, since these stimulation signals in isolation trigger opposing

119 directional changes in suppressive potency, the specific contribution for each on changes in Treg suppression during infection is unclear. Therefore, the cumulative impact of multiple TLR ligands expressed by intact pathogens and the ensuing immune response on changes in Treg suppression is best characterized for Tregs isolated directly ex vivo after infection. The increased suppressive potency for Foxp3+ Treg at early time points after

Salmonella infection we demonstrate here is consistent with the increased suppressive potency for CD25+CD4+ cells isolated day 5 after Plasmodium yoelii and day 10 after

HSV-1 infection, as well as CD25+CD4+ cells isolated in the acute (day 12) and chronic phase (day 28) after Heligmosomoides polygyrus infection [148,352,379]. However, our results build upon and extend the significance of these findings in three important respects. First, by isolating Tregs based on Foxp3 rather than CD25 expression, the limitations imposed by contaminating non-Treg CD25+ effector T cells in subsequent ex vivo functional analysis is bypassed.

Secondly, although an increase in CD25+CD4+ T cell suppression early after infection when pathogen proliferation occurs, potential shifts in Treg suppression at later time points during the natural progression of persistent infections has not been previously demonstrated. In this regard, the relatively short time interval that separates pathogen proliferation and clearance during persistent Salmonella infection is ideally suited for comparing differences in relative importance and suppressive potency for Tregs during these contrasting stages of infection. Using this model, we demonstrate significant reductions in Treg suppressive potency between early and late time points after infection that enables robust immune cell activation required for pathogen clearance. Despite these

120 changes in suppressive potency, the percent Treg among bulk and Salmonella FliC- specific CD4+ T cells each remained relatively constant throughout infection. These findings are consistent with the stable ratio of Tregs to effector CD4+ T cells during other models of persistent infection, and represent a striking contrast to the selective priming and expansion of pathogen-specific Foxp3-negative CD4+ T cells that occurs after acute

Listeria monocytogenes infection [153,155,331,380]. Thus, the priming and expansion of pathogen-specific Tregs may be an important distinguishing feature between pathogens that cause acute rather than persistent infection.

The development and refinement of methods for MHC class II tetramer staining and magnetic bead enrichment has allowed the precise identification of very small numbers of T cells with defined specificity from naïve mice [325]. Using these techniques, we find in this and a recent study [331] that Foxp3+ Tregs comprise

+ approximately 10% of CD4 T cells with specificity to both the FliC431-439 and 2W1S52-68 peptide antigens, respectively. Together, these results suggest previously under- appreciated overlap in the repertoire of antigens recognized by Foxp3+ Tregs compared with non-Treg CD4+ effectors in naïve mice [381-383]. However, more considerable overlap in the specificity of these two cell types is consistent with the TCR repertoires of human peripheral Tregs and non-Tregs based on genomic analysis of TCR sequences

[384,385]. Thus, additional studies that examine the percent Tregs among CD4+ T cells with other defined antigen-specificities using recently developed tetramer based enrichment techniques are warranted.

121

Lastly, by enumerating the relative expression of defined Treg-associated molecules that have been implicated to directly mediate or inhibit suppression, the complexity whereby Tregs maintain the balance between immune activation and suppression becomes more clearly defined. For example, shifts in suppressive potency for

Tregs isolated from early compared to late time points during persistent infection are paralleled by significant changes in the expression of numerous Treg cell-intrinsic molecules that have been demonstrated in other experimental models to control and/or mediate suppression [166,371] (Fig. 13 and Fig. 14). In particular, the drastic reductions in suppressive potency that occurs for Tregs isolated from mice day 5 compared with day

37 after infection is associated with significant reductions in CTLA-4 expression and

+ + increased expression of GITR on both bulk Foxp3 CD4 Tregs and Salmonella FliC431-

439-specific Tregs (Fig. 13). Based on these results, the relative contributions of CTLA-4 and GITR in controlling suppression by Foxp3+ Tregs during persistent infection were investigated using antibody reagents that block CTLA-4 or stimulate cells through GITR.

We find that CTLA-4 blockade alone is sufficient to recapitulate the effects of Treg ablation on Salmonella infection tempo, while GITR stimulation had no significant effect

(Fig. 15). These results are consistent with the recent demonstration that CTLA-4 expression on Foxp3+ Tregs is essential for maintaining peripheral tolerance [165,174].

Our results expand upon these findings by demonstrating the importance of dynamic

CTLA-4 expression on Tregs during persistent infection that controls the kinetics of effector T cell activation and overall infection tempo.

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The increase in Treg suppressive potency at early time points after Salmonella infection is consistent and may provide the mechanistic basis that explains the relative immune suppression previously observed during this infection [386-390]. Interestingly, increased Treg suppressive potency early after infection has also been described after viral and parasitic pathogens [148,352,379]. Is enhanced Treg suppression early after infection advantageous for the host, the pathogen, or both? Our ongoing studies are aimed at identifying the signals activated during Salmonella infection that trigger these changes, and Treg-intrinsic molecules that sense and dictate this augmentation in suppressive potency. Perhaps more intriguing are the molecular signals during natural infection that trigger reductions in Treg suppression that transform blunted immune effectors early after infection into more potent mediators of pathogen clearance. Given the multiple known pathogen-associated molecular patterns expressed by Salmonella

(e.g. LPS, flagellin, porins, and CpG DNA) that each stimulate immune cells through defined Toll-like and other pattern recognition receptors [391-396], together with the enormous potential for cell intrinsic TLR-stimulation on Tregs to alter their suppressive potency [358-360,363,397], it is tempting to hypothesize that shifts in the expression of individual, multiple, or cumulative TLR ligands during persistent infection controls the relative expression of Treg-associated molecules that mediate suppression. In this regard, our ongoing studies are also aimed at identifying the Salmonella-specific ligands, and their corresponding host receptors that dictate these reductions in Treg suppression during the progression of this persistent infection. We believe these represent important prerequisites for developing new therapeutic intervention strategies aimed at accelerating

123 the transition to pathogen clearance and further unraveling the pathogenesis of typhoid fever caused by Salmonella infection.

124

MATERIALS AND METHODS

Ethics Statement. These experiments were conducted under University of Minnesota

IACUC approved protocols (0705A08702 and 1004A80134) entitled "Regulatory T cells dictate immunity during persistent Salmonella infection". The guidelines followed for use of vertebrate animals were also created by the University of Minnesota IACUC.

Mice. 129Sv males and C57BL/6 females were purchased from the National Cancer

Institute. F1 mice were generated by intercrossing 129Sv males with C57BL/6 females.

F1 Foxp3GFP/- and F1 Foxp3DTR/- hemizygous males and F1 Foxp3DTR/WT heterozygous females were derived by intercrossing 129Sv males with Foxp3GFP/GFP or Foxp3DTR/DTR females, respectively [135,150]. Both Foxp3GFP/GFP and Foxp3DTR/DTR females have been backcrossed to C57BL/6 mice for over 15 generation. OT-1 TCR transgenic mice that contain T cells specific for the OVA257-264 peptide were maintained on a RAG-deficient

CD90.1+ background. All mice were used between 6-8 weeks of age and maintained within specific pathogen-free facilities.

Bacteria. The virulent Salmonella enterica serotype Typhimurium strain SL1344 has been described [251]. For infections, SL1344 was grown to log phase in brain heart infusion media at 37ºC, washed and diluted with saline to a final concentration of 1 X 104 colony forming units (for infection in F1 mice) or 1 X 102 colony forming units (for infection in C57BL/6 mice) per 200 µL, and injected intravenously through the lateral tail

125 vein. At the indicated time points after infection, mice were euthanized and the number of recoverable Salmonella CFUs enumerated by plating serial dilutions of the spleen and liver organ homogenate onto agar plates.

Reagents. Antibodies and other reagents for cell surface, intracellular, or intranuclear staining were purchased from BD Biosciences (San Jose, CA) or eBioscience (San Diego,

CA), and used according to the manufacturers’ recommendations. For measuring cytokine production by T cells, splenocytes were stimulated ex vivo with anti-mouse CD3 and anti-mouse CD28 (each at 5 µg/mL) in the presence of brefeldin A for 5 hours prior to intracellular cytokine staining. Antibodies used for depletion, blocking or stimulation experiments were purchased from BioXcell (West Lebanon, NH). For T cell depletions, purified anti-mouse CD4 (clone GK1.5) and anti-mouse CD8 (clone 2.43) antibodies were diluted to a final concentration of 750 µg per 1 mL in sterile saline and injected intraperitoneally on days 31 and 34 post-infection. Additional injections were given on days 38 and 41 post-infection in experiments where depletion was maintained up to 14 days. For CTLA-4 blockade and GITR stimulation, anti-mouse CTLA4 (clone UC10-

4F10), anti-mouse GITR (clone DTA-1), or isotype control antibodies (hamster IgG or rat

IgG, respectively) were diluted to a final concentration of 500 µg per 1 mL in sterile saline and injected intraperitoneally beginning either day 5 or day 37 post-infection followed by an additional injection of 250 µg of the same antibody three days later

[179,373,377,378]. For Foxp3+ Treg ablation, purified diphtheria toxin (DT; Sigma-

Aldrich, St. Louis, MO) was dissolved in saline and administered intraperitoneally to F1

126

Foxp3WT control, F1 Foxp3DTR/-, or F1 Foxp3DTR/WT mice at 50 µg/kg body weight for two consecutive days beginning at indicated time point after infection, and then maintained on a reduced dose of DT thereafter (10 µg/kg body weight every other day).

In vitro and in vivo suppression assays. For enumerating relative Treg suppression in vitro, Foxp3+GFP+ Tregs were isolated from F1 Foxp3GFP/- mice by enriching CD4+ splenocytes first with negative selection using magnetic bead cell isolation kits (Miltenyi

Biotec, Auburn, CA). Foxp3+GFP+ Tregs were further purified by staining and sorting for

CD4+GFP+ cells using a FACSAria cell sorter. The purity of CD4+Foxp3+ cells post-sort was verified to be >99%. Responder CD4+ T cells were isolated from naïve CD45.1+ mice, CFSE-labeled under standard conditions (5 µM for 10 min), and co-cultured in 96- well round bottom plates (2 X 104 cells/100 µL) at the indicated ratio of purified

Foxp3+GFP+ Tregs and responder CD45.1+CD4+ T cells. The relative suppressive potency for Tregs was enumerated by comparing the proliferation (CFSE dilution) in responder cells after co-culture and stimulation with anti-mouse CD3 and anti-mouse

CD28 (1 µg/mL) for 4 days. For enumerating relative Treg suppression in vivo, 2 X 104 T cells from OT-1 TCR transgenic mice on a RAG CD90.1+ background were diluted in

200 µL sterile saline and injected intravenously at the indicated time points relative to

Treg ablation or Salmonella infection followed by intravenous injection of purified

OVA257-264 peptide (400 µg) the following day. For each experiment, the degree of OT-1

T cell expansion was enumerated five days later.

127

MHC tetramer staining and enrichment. MHC class II tetramer staining and enrichment were performed as described [325,331]. Briefly, splenocytes were harvested at the indicated time points after infection and incubated with 5-25 nM PE or APC-conjugated

FliC431-439-specific tetramer in Fc block for 1 hour at room temperature. These cells were then incubated with anti-PE or anti-APC magnetic beads (Miltenyi Biotec, Auburn ,CA) for 30 minutes on ice and column purified according to the manufacturer’s instructions.

The bound and unbound fractions were stained with fluorochome-labeled antibodies for

b cell surface and intracellular staining. The absence of I-A FliC431-439 tetramer staining on

CD8+ T cells, and among CD4+ T cells in the unbound fraction of cells after bead enrichment were used as independent markers to verify the specificity of tetramer staining using methods described (data not shown) [325].

Statistics. The differences in number of recoverable bacterial CFUs, and the number and percent T cells among from different groups of mice were evaluated using the Student’s t test (GraphPad, Prism Software) with p < 0.05 taken as statistical significance.

128

Figure 7. Tempo of persistent Salmonella infection in F1 129Sv X C57BL/6 mice. A.

Recoverable CFUs at the indicated time points after infection from the spleen (top) and liver (bottom) after infection with 104 S. enterica serotype Typhimurium (strain SL1344) in F1 129Sv X C57BL/6 (left) or 102 in C57BL/6 (right) mice. †, all mice died or were moribund. B. Spleen size in F1 129Sv X C57BL/6 mice at the indicated time points after infection. Absolute number of splenocyte cells (C) and percent CD4+ and CD8+ cells (D) in F1 129Sv X C57BL/6 mice at the indicated time points after infection. These data reflect eight to ten mice per time point representative of three independent experiments.

Bar, standard error.

129

Figure 10. T cell activation kinetics during persistent Salmonella infection. A. Percent

CD44hiCD62Llo cells among CD4+ and CD8+ T cells at the indicated time points after infection with 104 S. enterica serotype Typhimurium in F1 129Sv X C57BL/6 mice. B.

Percent IFN-γ producing CD4+ and CD8+ T cells after Salmonella infection and ex vivo stimulation with anti-CD3/CD28 antibody (black histogram) or no stimulation control

(shaded histogram) at the indicated time points after infection. C. Expression levels of

CD25 (top) and CD69 (bottom) by CD4+ and CD8+ T cells at the indicated time points post-infection (black histograms) compared to naïve F1 control mice (shaded histograms). These data reflect eight to ten mice per time point representative of three independent experiments. 130

Figure 9. CD4+ T cells are required for reductions in Salmonella pathogen burden during persistent infection. A. Percent CD4+ and CD8+ T cells 14 days after treatment with each indicated antibody in mice beginning day 31 after Salmonella infection. B. Recoverable

Salmonella CFUs in the spleen (left) and liver (right) for mice treated with each antibody for six days (31 + 6) or 14 days (31 + 14). These data reflect six to twelve mice per time point representative of three independent experiments each with similar results. Bar, standard error. *, p <0.05; **, p <0.001.

131

Figure 10. Parallel expansion of Foxp3+ and Foxp3-negative CD4+ T cells during persistent Salmonella infection. Representative FACS plots (A) and composite data (B) indicating percent Foxp3+ cells among CD4+ T cells at the indicated time points after infection with 104 Salmonella in F1 129Sv X C57BL/6 mice. C. Total numbers of

Foxp3+CD4+ Tregs and Foxp3-negative non-Treg CD4+ T cells among splenocytes during persistent infection. These data reflect six to eight mice per time point representative of three independent experiments each with similar results. Bar, standard error.

132

Figure 11. Dynamic regulation of Treg suppressive potency during persistent Salmonella infection. A. Percent Foxp3+ (left) and GFP+ (right) cells among CD4+ T cells from F1 and F1 Foxp3GFP/- mice. B. Expression of Foxp3+ after cell sorting for GFP+CD4+ cells from F1 Foxp3GFP/- mice at the indicated time points after infection. C. (Top) Percent

CFSElo cells among CD45.1+CD4+ responder T cells (Tresp) after co-culture with the indicated ratio of Foxp3+GFP+CD4+ Tregs isolated from mice at indicated time point after infection and stimulation with anti-CD3/CD28 (line histogram) or no stimulation

(shaded histogram). (Bottom) Relative suppression of CFSE dilution in CD45.1+CD4+ responder T cells by Foxp3+GFP+CD4+ Tregs isolated at indicated time point after infection normalized to the suppression conferred by Foxp3+GFP+ Tregs from naïve mice co-cultured with responder T cells at a 1:1 ratio (dotted line). These data are representative of three independent experiments each with similar results.

133

Figure 12. Shifts in Treg-mediated in vivo suppression during persistent Salmonella infection. A. Representative FACS plots demonstrating the efficiency whereby Foxp3+

Tregs are ablated with DT treatment in F1 Foxp3DTR compared with F1 Foxp3WT (F1) control mice. The numbers indicate the percent Foxp3-expressing among CD4+ T cells after DT treatment. B. Representative FACS plots demonstrating percent (top) CD90.1+

OT-1 T cells among CD8+ splenocytes and total number (bottom) of CD90.1+CD8+ splenocytes in Treg-sufficient (F1) or Treg-ablated (F1 Foxp3DTR) mice day 5 after injection of OVA257-264 peptide or no peptide controls. C. Representative FACS plots

(top) demonstrating percent CD90.1+ OT-I T cells among CD8+ splenocytes and composite data depicting percent and total number CD90.1+CD8+ splenocytes after adoptive transfer into mice at the indicated time points after Salmonella infection and peptide stimulation. These data represent six to ten mice per group combined from three independent experiments each with similar results. Bar, standard error. **, p <0.01. 134

Figure 13. Expression of Treg-associated effector molecules during persistent

Salmonella infection. A. The relative expression of CTLA-4 and GITR on Foxp3+ Tregs or Foxp3-negative CD4+ T cells at the indicated time points during persistent infection.

+ B. Expansion of Salmonella FliC431-439-specific CD4 T cells, and Foxp3-expression among these cells after staining with FliC:I-Ab tetramer and magnetic bead enrichment.

The numbers in each plot represent the average cell number and percent Foxp3+ cells from 12 mice per time point combined from four independent experiments. C. Expression

+ + of CTLA-4 or GITR on FliC431-439-specific Foxp3 Tregs and Foxp3-negative CD4 T cells at early (day 5) and late (day 37) time points during persistent Salmonella infection.

These data reflect six mice per time point representative of two independent experiments each with similar results. Bar, standard error.

135

Figure 14. Expression of additional Treg-associated effector molecules during persistent

Salmonella infection. The relative expression of defined Treg cell-intrinsic molecules known to either enhance (CD39, Granzyme B, ICOS, IL-10, PD-1, Tgf-β) or impede

(OX40) suppression on Foxp3+ Tregs or Foxp3-negative CD4+ T cells at the indicated time points during persistent infection. These data reflect six mice per time point representative of two independent experiments each with similar results. Bar, standard error.

136

137

Figure 15. Relative impacts after Treg ablation on the tempo of persistent Salmonella infection. A. Number of recoverable Salmonella CFUs from spleen (top) and liver

(bottom) in Treg-ablated F1 Foxp3DTR compared with Treg-sufficient F1 control mice when DT treatment was initiated at either early (day 5) or late (day 37) time points during persistent infection. B. Percent CD44hiCD62Llo among CD4+ T cells in Treg-ablated F1

Foxp3DTR compared with Treg-sufficient F1 control mice when DT treatment was initiated on either day 5 or day 37 during persistent infection. C. Number of recoverable

Salmonella CFUs from spleen (top) and liver (bottom) following CTLA-4 blockade beginning at either early (day 5) or late (day 37) time points post-infection in F1 mice. D.

Percent CD44hiCD62Llo among CD4+ T cells when CTLA-4 blockade was initiated at either early (day 5) or late (day 37) time points post-infection. These data reflect six to seven mice per group combined from two independent experiments each with similar results. Bar, standard error. *, p < 0.05. E. Number of recoverable Salmonella CFUs from spleen (top) and liver (bottom) following treatment with GITR-stimulating antibody beginning at either early (day 5) or late (day 37) time points post-infection in F1 mice. F.

Percent CD44hiCD62Llo among CD4+ T cells when GITR stimulation was initiated at either early (day 5) or late (day 37) time points post-infection. These data reflect six to ten mice per group combined from two to three independent experiments each with similar results. Bar, standard error. *, p < 0.05.

138

Figure 16. Relative impact of Treg ablation on recoverable Salmonella CFUs in F1 control, Foxp3DTR/- and Foxp3DTR/WT mice. Number of recoverable Salmonella CFUs from the spleen (top) and liver (bottom) in Treg-ablated F1 Foxp3DTR/- and partially Treg- ablated F1 Foxp3DTR/WT mice compared with Treg-sufficient F1 control mice six days after the initiation of DT treatment beginning day 5 post-infection. These data reflect eight to twelve mice per group combined from three independent experiments each with similar results. Bar, standard error. *, p < 0.05.

139

Figure 17. Foxp3+ Treg ablation eliminates the effects of CTLA-4 blockade early after

Salmonella infection. Number of recoverable Salmonella CFUs (left) from spleen and liver following CTLA4 blocking (α-CTLA4) or isotype control (hamster IgG) antibody, and DT treatment in F1 Foxp3DTR mice each beginning day 5 post-infection, and harvested six days thereafter (day 11 post-infection). Percent CD44hiCD62Llo among

CD4+ T cells (right) for each group of Salmonella infected mice. Bar, standard error.

140

Chapter 4

Activated monoclonal and endogenous antigen-specific CD4 T cells

demonstrate differences in survival during Salmonella infection

141

ABSTRACT

Infection with host-adapted strains of Salmonella results in a systemic, persistent disease. While CD4 T cells are required for control of this pathogen, the appearance of a protective CD4 T cell response is generally delayed until several weeks after infection. A number of mechanisms have been proposed that contribute to this delayed CD4 T cell response, however most studies have largely focused on the bulk CD4 T cell response.

Thus, the kinetics of the antigen-specific CD4 T cell response following Salmonella infection remains relatively unclear. We report herein that a monoclonal population of antigen-specific TCR transgenic CD4 T cells rapidly expand early but fail to persist into memory during Salmonella infection. Moreover, the impaired survival of this monoclonal population of antigen-specific CD4 T cells was associated with expression of virulence genes encoded by the SPI-2 locus of Salmonella. Interestingly, the Salmonella-induced loss of activated CD4 T cells was not complete as tetramer staining revealed a population of antigen-specific memory cells within the endogenous CD4 T cell pool despite a concurrent Salmonella infection. Thus, these results suggest that Salmonella infection induces the selective loss of a subpopulation of antigen-specific CD4 T cells, yet within a given population of antigen-specific CD4 T cells, there exists a subset of cells that can escape this mechanism and persist indefinitely.

142

INTRODUCTION

Host-adapted strains of Salmonella enterica are able to cause a persistent, systemic infection in their respective hosts. In humans, Salmonella enterica serotype

Typhi (S. typhi) can cause a systemic infection that results in a prolonged disease referred to as typhoid fever [222]. Similarly, a comparable disease state occurs in mice following

Salmonella enterica serotype Typhimurium (S. typhimurium) infection [249,398,399].

Thus, specific strains of Salmonella enterica appear to have acquired the ability to efficiently invade and survive indefinitely within a respective host.

Cell-mediated immunity is critical for protection against Salmonella enterica infection in both mice and humans [34,259,324]. It has further been demonstrated in mouse models that the generation of an IFN-γ producing Th1 CD4 T cell response is the primary mediator of pathogen control and clearance [251,260,270,400]. Likewise, patients with defects in either the IFN-γ or IL-12 pathways, which are both essential for the differentiation and effector function of Th1 CD4 T cells, are highly susceptible to recurrent, systemic Salmonellosis [268,269]. Unfortunatley, the appearance of a protective Th1 CD4 T cell response is surprisingly delayed not peaking until the third to fourth week post-infection [274,400]. Hence, the ability to impede or prevent the development of a protective CD4 T cell response represents a potential means by which

Salmonella enterica is able to evade the host immune response to establish infection.

Indeed, several mechanisms associated with Salmonella enterica infection have been shown to suppress the CD4 T cell response [387,389,390,401]. However, all of these

143 proposed mechanisms of Salmonella-associated immune suppression have focused on the bulk CD4 T cell population. Therefore, the kinetics of the antigen-specific CD4 T cell response during Salmonella enterica infection remains poorly defined.

In an attempt to address this question, a recent study demonstrated the early expansion but rapid culling of antigen-specific CD4 T cells following S. typhimurium infection [402]. However, the use of a highly susceptible strain of mice, which possess a functionally defective NRAMP1 protein, necessitated the use of an attenuated strain of S. typhimurium, and therefore limits these findings as it applies to the development of an antigen-specific CD4 T cell response in fully immunocompetent mice following wild- type S. typhimurium infection. Furthermore, the antigen-specific CD4 T cell response was restricted to a monoclonal TCR transgenic cell population. Thus, whether this phenomenon is more broadly applicable to the polyclonal endogenous population of CD4

T cells following S. typhimurium has yet to be determined.

In this study, we adopted the B6.129F1 mouse model, which possess a functional

NRAMP1 protein and therefore develop a persistent disease following infection with wild-type S. typhimurium, to examine the antigen-specific CD4 T cell response in the context of a more natural host-pathogen relationship. Furthermore, as B6.129F1 mice are derived by intercrossing C57BL/6 and 129/Sv mice, which share the I-Ab haplotype, cells from TCR transgenic mice on the C57BL/6 background and I-Ab restricted MHC class II tetramers can simultaneously be utilized to track monoclonal and endogenous antigen- specific CD4 T cell responses, respectively.

144

We demonstrate herein using a monoclonal population of TCR transgenic T cells that antigen-specific CD4 T cells rapidly expand early after wild-type S. typhimurium infection but display impaired survival becoming virtually undetectable by later time points. Furthermore, the loss of this population of cells was not due to suboptimal priming conditions but dependent upon expression of virulence factors encoded by the

SPI-2 gene locus of S. typhimurium. Interestingly, within the polyclonal endogenous population of T cells, antigen-specific CD4 T cells primed during S. typhimurium infection could be readily observed up to three weeks later using tetramer staining. Thus, these results suggest that during wild-type S. typhimurium infection there is a selective loss of a subset of antigen-specific CD4 T cells that is dependent upon SPI-2 expression by S. typhimurium, however a subpopulation of antigen-specific CD4 T cells exists within the endogenous population that is able to escape and persist to later time points of infection.

145

RESULTS

Expansion and loss of monoclonal transgenic CD4 T cells during wild-type S. typhimurium infection

Delayed kinetics in the expansion of and production of IFN-γ by the bulk CD4 T cell response has been reported following wild-type S. typhimurium infection of resistant mice (i.e. functional NRAMP1 expression) with the peak of the response occurring approximately 4 weeks post-infection and gradually contracting toward baseline over the subsequent 6-8 weeks [274]. In contrast, FliC-specific TCR transgenic CD4 T cells (SM1

T cells), which recognize an immunodominant epitope within the flagellin protein of S. typhimurium, adoptively transferred into susceptible C57BL/6 mice (i.e. functionally defective NRAMP1 expression) have been shown to expand early after infection with an attenuated strain of S. typhimurium peaking at day 3 and rapidly contracting to undetectable levels by day 10 [270,402]. The incongruent expansion kinetics described in these previous studies may be due to a disconnect between the antigen-specific compared to the bulk CD4 T cell response, or reflect inherent differences in the priming of CD4 T cells following attenuated versus wild-type S. typhimurium infection. To address this question, we sought to track the antigen-specific CD4 T cell response following wild- type S. typhimurium infection using congenically marked (CD45.1+) FliC-specific SM1

CD4 T cells adoptively transferred into resistant B6.129F1 mice.

Wild-type S. typhimurium (ST) efficiently primes the expansion of adoptively transferred FliC-specific SM1 CD4 T cells as there was nearly a 100-fold expansion in

146 total number of CD45.1+ CD4 T cells by day 5 post-infection compared to uninfected control mice (Fig. 18). However, despite the significant expansion of CD45.1+ CD4 T cells, these cells contracted by more than 99.8% between day 5 and day 21 (used herein as a “memory” time point) to a level that was below the limit of detection (Fig. 18B).

Importantly, no appreciable expansion of CD45.1+ CD4 T cells was observed following infection with the aflagellated (FliC-deficient), virulent S. typhimurium strain, BC490

(STΔFliC) compared to the uninfected control group demonstrating that expansion of

SM1 CD4 T cells was in fact antigen-dependent and not due to non-specific, bystander activation (Fig. 18A). These results demonstrate that antigen-specific CD4 T cells do, in fact, expand early after infection with wild-type S. typhimurium, however, these cells do not persist, which is consistent with the kinetics observed in C57BL/6 mice following attenuated S. typhimurium infection [402]. Thus, the rate of expansion of antigen-specific

CD4 T cells following S. typhimurium infection does not seem to be influenced by pathogen virulence, and the activation and expansion kinetics of this T cell population is not accurately represented by the bulk CD4 T cell population.

The failure to observe SM1 CD4 T cells at later time points following S. typhimurium infection may be due to the massive splenomegaly that follows S. typhimurium infection [400,403] precluding the detection of a relatively small, persistent

SM1 T cell population post-contraction, or alternatively, may be due to suboptimal priming conditions during S. typhimurium infection required for SM1 CD4 T cell memory generation. To bypass these limitations, we employed a recombinant strain of attenuated Listeria monocytogenes engineered to express the FliC protein (LM-FliC),

147 which has been shown previously to prime the generation of a robust memory population of SM1 CD4 T cells in C57BL/6 mice (~5,000 cells/spleen) [404] allowing the examination of persistence at later time points. Consistent with the results in C57BL/6 mice, CD45.1+ SM1 CD4 T cells underwent vigorous expansion (~1000-fold) by day 5, which was approximately 10-fold greater than observed following ST infection (Fig. 18).

In contrast to ST infection, however, the expansion following LM-FliC was followed by a subsequent contraction phase that resulted in a readily detectable population of cells

(~50,000 cells/spleen) at day 21 that was approximately 5% of the initial peak response

(Fig. 18). Surprisingly, though, co-infection with LM-FliC and ST resulted in CD45.1+

SM1 CD4 T cells contracting by more than 99.9% between day 5 and day 21 post- infection which completely abrogated the development of the memory SM1 T cell population observed following LM-FliC priming alone (Fig. 18). Importantly, the peak expansion of CD45.1+ SM1 CD4 T cells following LM-FliC and ST infection was comparable to expansion observed following LM-FliC alone suggesting that the presence of ST did not alter the initial priming of these cells and the loss of SM1 CD4 T cells following S. typhimurium infection occurs after the initial priming stage (Fig. 18B).

Moreover, a similar absence of memory SM1 CD4 T cells was observed following co- infection with LM-FliC and the aflagellated strain of ST (LM-FliC+STΔFliC; Fig. 18) implicating a more general effect of S. typhimurium infection on the persistence of SM1

CD4 T cells that is not dependent upon direct expression of the antigen by the pathogen itself. Together, these results suggest that S. typhimurium induces the loss of a

148 monoclonal population of antigen-specific CD4 T cells subsequent to the initial priming of these cells preventing there survival into later time points of infection.

Loss of monoclonal antigen-specific CD4 T cells is dependent upon Salmonella expression of SPI-2 associated virulence genes

Salmonella enterica possesses several virulence factors that are believed to play an essential role in the establishment of persistent infection within susceptible hosts

[249,398]. Salmonella pathogenicity island 2 (SPI-2) is a plasmid-associated locus of virulence genes that encodes for 16-18 effector proteins, which have been associated with the intracellular survival of Salmonella enterica [405]. In C57BL/6 mice, a 6-fold greater percentage of adoptively transferred SM1 CD4 T cells were present at day 6 post- infection following infection with a SPI-2 deficient strain of S. typhimurium (STΔSPI-2) compared to an isogenic, wild-type strain (ST) suggesting that expression of SPI-2 encoded virulence genes is potentially involved in the loss of antigen-specific CD4 T cells in S. typhimurium infection [402]. However, since STΔSPI-2 remains highly virulent to the susceptible C57BL/6 mice used in this study, the effect of SPI-2 on the survival of antigen-specific CD4 T cells beyond 6 days post-infection could not be examined [402]. Thus, the effect of SPI-2 encoded virulence genes on the persistence of antigen-specific CD4 T cells during S. typhimurium infection remains unclear.

To address this question, the development of a memory population of SM1 CD4 T cells was assessed following co-infection with LM-FliC and STΔSPI-2. In contrast to the lack of a CD45.1+ SM1 CD4 T cell population at day 21 in mice infected with LM-FliC

149 and ST, there was a clear memory population of CD45.1+ SM1 CD4 T cells in mice infected with LM-FliC and STΔSPI-2 that was comparable to control mice infected with

LM-FliC alone (Fig. 19). Furthermore, the absence of SPI-2 associated gene expression had no appreciable impact on the overall expansion of SM1 CD4 T cells as the number of

CD45.1+ CD4 T cells was comparable following infection with LM-FliC alone or co- infected with either ST or STΔSPI-2 at day 5 (Fig. 19B). However, less than 0.01% of the total number of CD45.1+ CD4 T cells present at day 5 remained at day 21 in mice co- infected with LM-FliC and ST compared to approximately 7% remaining in mice infected with LM-FliC alone or LM-FliC plus STΔSPI-2 (Fig. 19B). Together, these results demonstrate a role for SPI-2 encoded virulence genes in preventing the development of a memory SM1 CD4 T cell population during S. typhimurium infection that preferentially impacts the survival rather than priming of these cells.

Endogenous antigen-specific CD4 T cells are primed and persist following

Salmonella infection

The results observed thus far using an adoptively transferred population of monoclonal SM1 CD4 T cells may not be representative of the entire antigen-specific endogenous CD4 T cell response that consists of a broad range of TCR avidities for any given antigen. Indeed, such a discrepancy has been reported previously between adoptively transferred monoclonal TCR transgenic CD4 T cells and the endogenous CD4

T cell population in which transferred SMARTA transgenic CD4 T cells, specific to the

LCMV derived epitope GP61-80, failed to persist following infection with a virulent

150 recombinant L. monocytogenes strain expressing the cognate antigen whereas GP61-80- specific memory CD4 T cells were readily detected by tetramer staining within the endogenous T cell population [406]. Therefore, to address if the loss of antigen-specific

CD4 T cells following S. typhimurium infection applies to all antigen-specific CD4 T cells or limited to a subset of the population, the expansion and persistence of endogenous CD4 T cells was assessed using MHC class II tetramers. For these experiments, mice were infected with an attenuated recombinant L. monocytogenes strain engineered to express the 2W1S epitope, an immunodominant peptide variant derived from the murine MHC class II I-Eα protein (LM-2W1S) [331], and the endogenous antigen-specific CD4 T cell response was tracked using MHC class II I-Ab:2W1S tetramer staining [325]. In C57BL/6 mice, the 2W1S-specific CD4 T cell response has a high naïve precursor frequency (~200 cells/mouse) [325] and generates a robust memory population following LM-2W1S infection that is roughly 10% of the day 7 response or

~10,000 cells per spleen [331]. This large antigen-specific memory CD4 T cell population allows for the evaluation of a persistent endogenous 2W1S tetramer positive

CD4 T cell population above background levels at later time points during S. typhimurium infection.

Consistent with the findings in C57BL/6 mice infected with LM-2W1S [331], the

I-Ab:2W1S tetramer positive CD4 T cell population in B6.129F1 mice underwent rapid expansion following LM-2W1S infection by day 7 post-infection (Fig. 20A).

Subsequently, the 2W1S-specific CD4 T cell population contracted to about 20% of the peak response and a clear population of 2W1S-specific memory CD4 T cells (~20,000

151 per spleen) was readily detectable at day 21 (Fig. 20). Interestingly, co-infection of ST along with LM-2W1S did not alter the peak expansion or persistence of this endogenous antigen-specific CD4 T cell population at early or late time points, respectively (Fig. 20).

Thus, these results demonstrate that the loss of antigen-specific CD4 T cells during S. typhimurium infection is limited to a subset of antigen-specific cells represented by the monoclonal SM1 CD4 T cells yet within the endogenous CD4 T cell population there exists a subset of antigen-specific cells that are capable of escaping this S. typhimurium- related cell death. Moreover, this data is consistent with the observation in C57BL/6 mice and the LCMV GP61-80 response where a disconnect between tracking the monoclonal transgenic and endogenous tetramer positive populations has been described [406].

To explore the possibility that the differences in persistence between monoclonal

TCR transgenic and endogenous tetramer-positive CD4 T cells during S. typhimurium is due to a selective effect of S. typhimurium infection on the survival of adoptively transferred cells, we tracked the 2W1S-specific CD4 T cell response within a population of transferred splenocytes from a congenically marked (CD45.1+) B6.129F1 donor following LM-2W1S and ST co-infection (Fig. 21A). Thus, if there is a preferential loss of antigen-specific CD4 T cells following S. typhimurium infection due to adoptive transfer, then the donor-derived (CD45.1+) 2W1S-specific CD4 T cell population would be undetectable at later time points following co-infection with LM-2W1S and ST whereas the endogenous (CD45.1-) 2W1S-specific CD4 T cell population would remain uneffected. As such, there was a comparable population of CD45.1+ 2W1S tetramer positive CD4 T cells after LM-2W1S plus ST infection as after LM-2W1S infection

152 alone (~1000 cells/spleen) (Fig. 21B, 21C) suggesting there was not a preferential loss of adoptively transferred cells following S. typhimurium infection. Furthermore, the percentage of 2W1S tetramer positive CD4 T cells among the CD45.1+ CD4 T cell population at day 7 (0.89%) and day 21 (0.17%) was equivalent to the percentage of

2W1S tetramer positive CD4 T cells within the endogenous (CD45.1-) CD4 T cell population at the same time points (0.67% and 0.1%, respectively) following LM-2W1S and ST co-infection (Fig. 21B, 21C) demonstrating that the expansion and survival of adoptively transferred antigen-specific CD4 T cells is similar to endogenous cells.

Persistence of an endogenous FliC-specific CD4 T cell population primed during

Salmonella infection

To address whether the loss of monoclonal FliC-specific SM1 CD4 T cells and survival of an endogenous 2W1S-specific CD4 T cells during S. typhimurium infection is related to the respective antigens rather than a difference between monoclonal and endogenous cell populations, the endogenous FliC-specific CD4 T cell response was quantified at later time points following LM-FliC and STΔFliC co-infection. Importantly,

STΔFliC was used to avoid the possibility of activating newly-recruited recent thymic emigrants due to persistently expressed antigen by ST at later time points of infection which would confound the interpretation of survival of cells activated early after infection. At 21 days post-infection, a population of FliC-specific tetramer positive CD4

T cells was readily detectable following LM-FliC and STΔFliC co-infection (Fig. 22A) that was comparable in total number (19950 cells/spleen) to what was measured in mice

153 infected with LM-FliC alone (13680 cells/spleen) and about 10-fold higher than what was observed in control mice infected with STΔFliC alone (2260 cells/spleen; Fig. 22B).

While the overall magnitude of the FliC tetramer positive population at this time point was reduced compared to the 2W1S tetramer positive population (Fig. 20B), this is consistent with what would be expected based on differences in initial antigen-specific

CD4 T cell precursor frequency between these respective antigens [325]. Therefore, the impaired survival of antigen-specific CD4 T cells during S. typhimurium infection is not dependent upon the antigen-specificity of the T cell response (i.e. FliC vs. 2W1S) but rather represents a selective effect on a subpopulation of cells within the larger endogenous population for a given antigen (i.e. monoclonal FliC-specific SM1 T cells vs. endogenous FliC tetramer positive CD4 T cells).

154

DISCUSSION

Pathogens that cause persistent infections have evolved multiple strategies which permit them to avoid immunological detection and survive indefinitely within a compatible host [249,398,399]. For many persistent intracellular pathogens including

Salmonella enterica, host defense against this class of pathogens is dependent upon the generation of a protective CD4 T cell response [34,324]. Thus, evasion or inhibition of

CD4 T cell immunity represents a potentially advantageous evasion mechanism for establishing a persistent infection. Indeed, a number of in vitro and in vivo studies have proposed multiple means by which S. typhimurium is able to impede the development of a CD4 T cell response upon infection [390,400-402,407-410]. However, these immune evasion strategies have largely been studied in models of susceptible mouse strains infected with attenuated S. typhimurium. Moreover, the effects of these various immunosuppressive mechanisms on the development and kinetics of an antigen-specific

CD4 T cell response has not been fully evaluated. Therefore, using a resistant B6.129F1 strain of mice, we sought to address the kinetics and survival of antigen-specific CD4 T cells following wild-type S. typhimurium infection.

Adoptively transferred monoclonal FliC-specific TCR transgenic SM1 CD4 T cells were initially used to track the antigen-specific CD4 T cell response during wild- type S. typhimurium. These results demonstrated that while antigen-specific CD4 T cells expand early after infection, they fail to persist throughout S. typhimurium infection (Fig.

18). Furthermore, using a recombinant strain of L. monocytogenes that expresses the FliC

155 antigen, a persistent population of SM1 CD4 T cells could be primed, however, co- infection of S. typhimurium completely abrogates the survival of this population of cells

(Fig. 18). Interestingly, since the level of expansion remained equivalent while the extent of contraction became augmented following co-infection with S. typhimurium, we conclude that S. typhimurium infection is preferentially targeting the survival of these cells demonstrated by their lack of persistence rather than the initial activation or priming as implied by comparable expansion magnitude (Fig. 18). Moreover, the impaired survival of SM1 CD4 T cells was further shown to be dependent upon the presence of

SPI-2 encoded virulence genes by S. typhimurium as co-infection with a SPI-2 deficient strain of S. typhimurium did not quantitatively impact the memory population of SM1

CD4 T cells generated by L. monocytogenes expressing FliC (Fig. 19). Interestingly, though, this loss of antigen-specific CD4 T cells does not appear to universally effect all antigen-specific CD4 T cells as a population of antigen-specific CD4 T cells was present within the endogenous CD4 T cell population that persisted despite co-infection of wild- type S. typhimurium with recombinant L. monocytogenes expressing either the FliC or

2W1S antigen (Fig. 20, 22). Together, these data support a model where antigen-specific

CD4 T cells expand early after wild-type S. typhimurium infection, however the subsequent survival of these cells is differentially effected by S. typhimurium in a SPI-2 dependent manner.

While our results are consistent with several previously published observations, they expand upon these earlier findings in three important ways. First, the loss of SM1

CD4 T cells following S. typhimurium infection has been observed in C57BL/6 mice

156

[270,402], however the increased susceptibility of these mice (due to a functionally defective NRAMP1 protein) necessitates the use of attenuated strains of S. typhimurium to generate a persistent infection. As the increased host susceptibility and decreased pathogen virulence of this model may not fully represent the host-pathogen interaction that occurs during natural persistent Salmonella enterica infection, it is important to evaluate the antigen-specific CD4 T cell response using wild-type S. typhimurium. Thus, our results support the early expansion and loss of SM1 CD4 T cells after S. typhimurium infection and that this impaired survival is not related to host susceptibility or pathogen virulence. Moreover, the co-infection of mice with a recombinant L. monocytogenes vector, a potent inducer of memory T cell responses, further suggests that the lack of a memory response is not due to suboptimal priming of SM1 CD4 T cells during

Salmonella infection but a subsequent and complete loss of this monoclonal population of antigen-specific T cells.

Secondly, our results demonstrate that SPI-2 gene expression is involved in the impaired memory generation of SM1 CD4 T cells following S. typhimurium infection.

While a role for SPI-2 encoded virulence genes has been implicated in the loss of SM1

CD4 T cells in C57BL/6 mice, S. typhimurium deficient in the SPI-2 gene locus remain highly virulent to this susceptible strain of mice, thus precluding examination of the effect of SPI-2 expression on the survival of SM1 CD4 T cells beyond 6 days after infection [402]. Therefore, these data expand upon this initial observation by examining the kinetics of these cells at later time points of infection and further extend these

157 findings to demonstrate that the impact of SPI-2 expression is preferentially on the survival of SM1 CD4 T cells not on the early priming or expansion of these cells.

Lastly, the persistence of antigen-specific cells within the endogenous CD4 T cell population despite the complete loss of the monoclonal SM1 CD4 T cell population following S. typhimurium infection shown here is consistent with previous data tracking the LCMV derived, GP61-80 specific CD4 T cell response following recombinant virulent

L. monocytogenes infection where a similar preferential loss of a monoclonal population of antigen-specific CD4 T cells was observed while a memory response was generated within the endogenous population [406]. The fact that a similar phenomenon is described for multiple antigens using different infection models in different strains of mice suggests that this disconnect between monoclonal TCR transgenic and polyclonal endogenous

CD4 T cell responses is a more global phenomenon and has important ramifications for the interpretation and extrapolation of data obtained using either strategy alone to track the antigen-specific CD4 T cell response as neither may wholly represent the response of the entire antigen-specific CD4 T cell population.

Interestingly, our results demonstrating early expansion of antigen-specific CD4 T cells that peak around 5-7 days post-infection are inconsistent with the expansion kinetics of the bulk CD4 T cell population which reportedly peaks around 4 weeks after wild-type

S. typhimurium infection in resistant B6.129F1 mice [274,400]. Based on our findings that survival of CD4 T cells within a given population of antigen-specific cells is variable following S. typhimurium infection, one potential explanation is that there is preferential activation of antigen-specific CD4 T cells that are prone to SPI-2 mediated cell death

158 early after infection whereas the subset of antigen-specific CD4 T cells that have increased survival potential are not activated until later time points. Alternatively, SPI-2 expression may be downregulated as the course of the infection progresses permitting antigen-specific CD4 T cells that would otherwise not persist to survive following activation resulting in the eventual accumulation of these cells at later time points. A third possibility is that the expression of pro-survival signals delivered by costimulatory molecules [411,412] or cytokines [413-415] may become preferentially upregulated at later time points of infection which can then sustain the survival of susceptible populations of antigen-specific CD4 T cells that are preferentially lost early after S. typhimurium infection. Future studies are needed to more fully understand the factors that determine if there is indeed a difference in which stage of infection different antigen- specific CD4 T cells become activated and what characteristics of the activated CD4 T cell response dictates which cells will persist versus be eliminated during Salmonella infection. In addition, further experiments will be required to more specifically elucidate the mechanism by which SPI-2 encoded virulence genes are able to induce to loss of certain antigen-specific CD4 T cells.

Overall, as the generation of a protective CD4 T cell response is critical for the control of S. typhimurium infection, understanding the principles underlying the generation of a sustained population of antigen-specific CD4 T cells has important implications for both defining the pathogenesis of persistent Salmonella enterica infection as well as designing more effective preventative and therapeutic treatment strategies.

159

MATERIALS AND METHODS

Mice. All strains of C57BL/6 and 129Sv mice were purchased from the National Cancer

Institute, and B6.129F1 mice were generated by intercrossing C57BL/6 female with 129Sv male mice as described [274,367]. CD45.1+ B6.129F1 mice were generated by intercrossing

129Sv males with C57BL/6 females homozygous for the CD45.1 congenic marker. FliC431-

+ + 439-specific (SM1) CD4 TCR transgenic mice were intercrossed with CD45.1 C57BL/6 mice and maintained on a Rag-1-deficient C57BL/6 background as described previously

[273]. In adoptive transfer experiments, donor splenocytes were isolated and homogenized into single cell suspensions then resuspended to a concentration of either 5 X 104 SM1 CD4

T cells or 5-10 X 107 CD45.1+ B6.129F1 splenocytes per 200 µL PBS and injected intravenously one day prior to infection. All mice were generated and maintained in specific pathogen-free facilities and used between 6-8 weeks of age. Experiments were conducted under University of Minnesota IACUC approved protocols.

Bacteria and infections. The S. enterica serotype Typhimurium (S. typhimurium) strain,

SL1344, and the aflagellated isogenic strain, BC490, have been described previously

[273,323]. The SPI-2 deficient HH109 and wild-type isogenic SL12023 strains of S. typhimurium were generously donated by Dr. Stephen McSorley (Univ. of Minnesota) and have been described elsewhere [402,416]. For infections with S. typhimurium, the indicated strain was grown to log phase in brain heart infusion (BHI) media (BD Biosciences, San

Jose, CA) at 37ºC, washed and diluted to 1 X 104 CFUs/ 200µL with PBS and injected

160 intravenously through the lateral tail vein. At the indicated time points after infection, the number of recoverable Salmonella CFUs was quantified by plating serial dilutions of organ homogenates onto BHI agar plates. The recombinant Listeria monocytogenes ΔActA strain

(DPL-1942) expressing the FliC and 2W1S peptides along with methods for LM transformation have been described previously [331,332,343,344,404]. For infections with L. monocytogenes (LM), bacteria were grown to log phase in brain heart infusion media containing chloramphenicol (20 µg/mL) at 37oC, washed and diluted with PBS to a final concentration of 5 X 106 CFUs per 200 µL and injected intravenously.

Reagents for flow cytometry and MHC tetramer staining. Antibodies and other reagents for flow cytometry were purchased from BD Biosciences (San Jose, CA) or eBioscience

(San Diego, CA). For MHC class II tetramer staining, CD4 T cells were isolated from the splenocyte population by negative selection using magnetic beads and columns (Miltenyi

Biotec, Auburn, CA). The purified CD4 T cell population was then stained with 5-25 nM of the indicated PE-conjugated tetramer as previously described [325,331]. The MHC class II tetramers, I-Ab:FliC and I-Ab:2W1S, used in this study were generously provided by Drs. Stephen McSorley and Marc Jenkins, respectively.

161

Figure 18. Expansion and loss of a monoclonal population of FliC-specific SM1 CD4 T cells during Salmonella infection. FliC-specific SM1 CD4 T cells (CD45.1+) were adoptively transferred into naive B6.129F1 mice and infected the following day with wild-type S. typhimurium (strain SL1344, ST), isogenic, aflagellated S. typhimurium

(STΔFliC), recombinant L. monocytogenes expressing FliC (LM-FliC), both LM-FliC and ST, or both LM-FliC and STΔFliC. At the indicated days post-infection, the percent

CD45.1+ cell among total CD4+ splenocytes (A) and the total numbers of CD45.1+CD4+ splenocytes (B) were enumerated and the averages are presented. Numbers in (B) represent percent CD45.1+CD4+ splenocytes remaining at day 21 compared to day 5 and was calculated from averages of total number CD45.1+CD4+ splenocytes at day 5 and day

21 (B). These results represent data from three independent experiments with 6-10 mice per group.

162

Figure 19. Loss of FliC-specific SM1 CD4 T cells during Salmonella infection is SPI-2 dependent. One day after adoptive transfer of FliC-specific SM1 CD4 T cells, mice were infected with LM-FliC, LM-FliC plus wild-type S. typhimurium (strain SL12023, ST), or

LM-FliC plus isogenic SPI-2 deficient S. typhimurium (STΔSPI-2). On the indicated days post-infection, the percent CD45.1+ cell among total CD4+ splenocytes (A) and total numbers of CD45.1+CD4+ splenocytes (B) were enumerated and the averages are presented. Numbers in (B) represent percent CD45.1+CD4+ splenocytes remaining at day

21 compared to day 5 and calculated as described in Figure 1. These results represent data from two independent experiments with 5-7 mice per group.

163

Figure 20. Persistence of an endogenous 2W1S-specific memory CD4 T cell population during Salmonella infection. B6.129F1 mice were infected with recombinant L. monocytogenes expressing 2W1S (LM-2W1S), LM-2W1S plus ST (strain SL1344), or

ST alone. On the indicated days post-infection, CD4+ splenocytes were purified and stained with I-Ab:2W1S tetramer. The percent 2W1S tetramer positive cells among enriched CD4+ splenocytes (A) and the total numbers of 2W1S tetramer positive CD4+ splenocytes (B) were enumerated and the averages are presented. Numbers in (B) represent percent CD45.1+CD4+ splenocytes remaining at day 21 compared to day 5 and calculated as described in Figure 1. These results represent data from at least two independent experiments with 6-10 mice per group.

164

165

Figure 21. Expansion and persistence of transferred 2W1S-specific memory CD4 T cells primed during Salmonella infection. CD45.1+ B6.129F1 splenocytes were adoptively transferred into age and sex-matched B6.129F1 recipient mice. Transferred

(CD45.1+CD4+) splenocytes routinely composed approximately 5% of the splenic CD4 T cell population. Numbers in (A) represent the relative frequency of I-Ab:2W1S positive cells among endogenous (CD45.1-) and transferred (CD45.1+) CD4+ splenocytes in uninfected recipient mice. On the indicated days post-infection, CD4+ splenocytes were purified and stained with I-Ab:2W1S tetramer. The percent (B) and total number (C) of

2W1S tetramer positive cells among CD45.1+ (left) and CD45.1- (right) CD4 T cells were enumerated and the averages are presented. Numbers in bar graphs (C) represent percent

2W1S tetramer positive cells among CD45.1+ and CD45.1- CD4+ splenocytes present at day 21 compared to day 7. These results represent data from two independent experiments with six mice per group.

166

Figure 22. Persistence of an endogenous FliC-specific memory CD4 T cell population generated during Salmonella infection. B6.129F1 mice were infected with LM-FliC, LM-

FliC and STΔFliC, or STΔFliC. On day 21 post-infection, CD4+ splenocytes were purified and stained with I-Ab:FliC tetramer. The percent (A) and total number (B) of

FliC tetramer positive cells among enriched CD4+ splenocytes were enumerated and the averages are presented. These results represent data from at least three independent experiments with 6-8 mice per group.

167

Chapter 5

Early eradication of persistent Salmonella infection primes antibody-

mediated protective immunity to recurrent infection

168

ABSTRACT

Typhoid fever is a systemic, persistent infection caused by host-specific strains of

Salmonella. Although the use of antibiotics has reduced the complications associated with primary infection, recurrent infection remains an important cause of ongoing morbidity and mortality. Surprisingly, little is known regarding how antibiotic eradication of primary infection impacts the priming of protection against secondary infection. Using a murine model of persistent Salmonella infection, we demonstrate protection against recurrent infection is sustained despite early eradication of primary infection using antimicrobials. Interestingly, in this model, protection is not mediated by CD4+ or CD8+

T cells as depletion of these cells either alone or in combination prior to rechallenge does not abrogate protection. Instead, infection followed by antibiotic-mediated clearance primes robust levels of Salmonella-specific antibody that can adoptively transfer protection to naïve mice. Thus, eradication of persistent Salmonella infection primes and sustains antibody-mediated protective immunity to recurrent infection.

169

INTRODUCTION

Typhoid fever is a persistent, systemic infection caused by host-adapted strains of

Gram-negative bacteria within the Salmonella genus. The development and use of antimicrobials with bactericidal activity against Salmonella has transformed this once debilitating and often fatal infection into a readily treatable condition. Unfortunately, even with appropriate antibiotic treatment, recurrent disease occurs in 5 to 15% of individuals after the discontinuation of antimicrobial therapy [214-217]. Molecular genotyping and phenotyping of S. enterica serotype Typhi (S. typhi) isolates from individuals with primary and recurrent infection suggest recurrent typhoid may be caused by either re-activation of latent or secondary infection. However, for individuals living in endemic areas where re-exposure is essentially unavoidable, protection from recurrent infection is paramount, while the significance of molecular comparisons between

Salmonella isolates for distinguishing re-activation and secondary infection is less relevant.

Regardless of the specific etiology, clinical evidence demonstrates recurrent infection is less severe and of shorter duration than primary infection [205]. Moreover, after challenge with virulent Salmonella, significantly reduced rates of typhoid fever and infection relapse occur for human volunteers previously recovered from typhoid compared with naïve individuals [293]. Similarly, reduced attack rates of Salmonella infection have been reported for individuals with prior Salmonella infection during a natural outbreak among military personnel exposed to infected food handlers [299].

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These epidemiological features of human typhoid suggest naturally-acquired primary

Salmonella infection does confer some protection against secondary infection.

Protection from recurrent disease triggered by primary infection is also reproduced in animal models of Salmonella infection. For example, natural recovery from experimental typhoid fever protected chimpanzees from fever, bacteremia, and systemic inflammation after secondary challenge with virulent Salmonella [417].

Similarly, in the murine model of S. enterica serotype Typhimurium (S. typhimurium) infection, which recapitulates many clinical features of human typhoid, primary infection with live attenuated Salmonella mutants confers a high level of protection against secondary challenge with virulent Salmonella [323,418]. Thus, animal models of typhoid infection allow the potential impact of antibiotic treatment in priming protective immunity to be more precisely characterized. In this regard, a recent study using a mouse infection model reported significantly reduced protection after early Salmonella eradication compared with more sustained primary infection [419]. These findings suggest antimicrobial therapy, while beneficial for curtailing the more severe symptoms and sequelae of primary infection, may also blunt the priming of protective immunity conferred by natural infection. However, the high susceptibility of Nramp1-deficient

C57BL/6 mice to virulent S. typhimurium required the eradication of primary infection within two days, and therefore the effects of antibiotic-mediated clearance of primary infection during the later and persistent phase of this infection remains undefined. To bypass this limitation, the potential impact of infection eradication with antibiotics on protection against secondary Salmonella infection in Nramp1-sufficient mice that are

171 naturally resistant and develop persistent infection was investigated in this study.

Together, these results demonstrate highly significant levels of protection mediated by

Salmonella-specific antibody despite antibiotic-mediated early eradication of persistent infection.

172

RESULTS

Protective immunity against recurrent Salmonella infection despite early infection eradication

The reduced severity, shorter duration, and lowered attack rates of recurrent compared with primary Salmonella infection in humans suggest natural infection primes some protective effects against secondary infection [293,299]. Moreover, these protective features of primary typhoid against recurrent infection are readily reproduced in non- human primate and rodent models of infection [151,157,323,353,417,418]. The notion that prolonged or persistent Salmonella antigen sustains protective T cell-mediated immunity against recurrent infection is consistent with results from other infection models where pathogen-specific CD4+ T cells have been implicated to mediate protection

[151,157,353]. Importantly, however, since persistent Salmonella infection is increasingly being treated with antibiotics, the potential impacts of pathogen eradication on priming protection against the recurrence of this infection needs to be better defined.

In this regard, Nramp1-sufficient 129Sv or B6.129F1 mice that each develop persistent infection with virulent S. typhimurium [251,274] represent ideal models for experimentally addressing the role of antigen persistence in host defense against recurrent infection. In initial experiments, we evaluated the susceptibility to recurrent infection for

S. typhimurium infected mice treated with enrofloxacin beginning day 20 after primary infection. Enrofloxacin is a fluoroquinolone derivative used in veterinary medicine, and this dose has been shown to eradicate systemic Salmonella infection within 3 days

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[419,420]. Significant protection against recurrent infection was found for mice with eradicated primary infection because these mice contained ≥100-fold reductions in recoverable CFUs by day 5 post-challenge in both the spleen and liver (Fig. 23A).

Importantly, this reduction in number of recoverable Salmonella CFUs was not due to residual enrofloxacin as both infected and naive mice were treated and withdrawn from this antibiotic in parallel. Together, these results indicate protection against recurrent infection is primed despite eradication of primary Salmonella infection.

To explore if earlier eradication of primary Salmonella infection would impact protection against recurrent infection, the protection primed by eradication of primary infection day 5 compared with day 20 was investigated. Interestingly, both groups of mice were protected against recurrent infection to a similar extent despite the earlier eradication of Salmonella infection (Fig 23B). Importantly, these protective effects could not be attributed to reduced efficiency of antibiotic eradication of virulent S. typhimurium that causes persistent infection in Nramp1-sufficient mice because enrofloxacin readily and efficiently eradicated the infection within the first 5-7 days after treatment in all mice

(Fig 24).

Additional experiments sought to identify if these reductions in pathogen burden for mice treated with antibiotics during primary Salmonella infection is associated with protection following challenge with a higher dose of S. typhimurium (106 CFUs) that normally causes lethal infection even in Nramp1-sufficient mice. Remarkably, we found that eradication of primary infection with antibiotics beginning day 5 completely rescued these mice from lethal Salmonella infection, while naïve control mice treated with

174 antibiotics in parallel all succumbed within the first 5-7 days after this high dose infection

(Fig. 25). Together, these results demonstrate protection against recurrent infection is both primed and sustained despite early eradication of primary Salmonella infection.

Neither CD4+ nor CD8+ T cells directly mediate protection against recurrent

Salmonella infection

T cells are important mediators of host defense against Salmonella infection because mice with targeted defects in CD4+ and CD8+ T cells are defective in controlling both primary and secondary defects even with attenuated Salmonella mutant strains [260-

263,266,421]. However, given the intricate interplay between T cells and other immune cell subsets, the use of mice with targeted and complete defects in these specific cell types cannot discriminate between whether these cells are required for priming protection by other immune mediators, or if T cells are the actual cellular mediators of protective immunity. To overcome these limitations, and to determine the specific roles of CD4+ and CD8+ T cells in mediating protection against recurrent Salmonella infection after early infection eradication, we enumerated the susceptibility to recurrent Salmonella infection following CD4+ and CD8+ T cell depletion either alone or in combination.

Surprisingly, CD4+ and CD8+ T cell depletion either alone or in combination (Fig. 26A) had no significant effect on the level of protection primed by primary Salmonella infection with antibiotic treatment. Importantly, these results could not be explained by inefficient T cell depletion because each subset was found to be depleted ≥ 99% (Fig.

26B). Taken together, these results demonstrate CD4+ and CD8+ T cells are non-essential

175 mediators of protection against recurrent infection primed by early eradication of primary

Salmonella infection.

Salmonella-specific antibodies are primed and confer protection after early infection eradication

Given the sustained protection against recurrent infection even after CD4+ and

CD8+ T cell depletion, the serological response in “immune” mice primed by early antibiotic eradication of primary infection was enumerated. Although Salmonella-specific antibody titers were drastically increased in Salmonella-infected compared with naïve control mice as excepted, the titer of Salmonella-specific IgG and IgA were surprisingly unchanged between antibiotic treated and persistently infected mice (Fig. 27). To determine if Salmonella-specific antibody primed by primary Salmonella infection eradicated with early antibiotic treatment can mediate protection against recurrent infection, the protective effects conferred by adoptively transferred serum from these mice was enumerated. Compared with control mice, serum from antibiotic-treated mice conferred significant ≥10-fold reductions in recoverable Salmonella CFUs in the spleen and liver 5 days post-challenge (Fig. 28A). Importantly, these protective effects cannot be explained by potential non-specific effects of serum transfer alone because serum from naïve mice failed to confer any change in susceptibility to Salmonella infection (Fig.

28B). Together, these results demonstrate Salmonella-specific antibody is primed and confers protection against recurrent infection despite early antibiotic-mediated resolution of primary S. typhimurium infection.

176

DISCUSSION

Given the widespread use of antimicrobials to eradicate and reduce the long-term complications associated with human typhoid, identifying how this therapy impacts the priming of protective immunity to recurrent infection is an important area for investigation. Although human epidemiological data demonstrating reduced attack rates of recurrent infection in individuals with primary typhoid treated with antibiotics

[293,299] suggest protective immunity is generated, these studies are limited by relatively small sample size, large heterogeneity in immune response between individuals, and wide fluctuations in Salmonella inocula during natural human infection even during outbreak-type infections. Therefore, to more definitively address this question, the impact of antibiotic-mediated clearance of primary Salmonella infection on protective immunity to recurrent infection was investigated using a murine model of persistent Salmonella infection where each of these parameters could be more precisely controlled. We demonstrate that protection is primed against recurrent Salmonella infection and sustained equally whether antibiotics are administered during the early or later phases of primary persistent Salmonella infection. These results have important implications for treating and preventing recurrent Salmonella infection especially in typhoid endemic areas where re-infection is essentially unavoidable.

Furthermore, using immunological tools and experimental techniques that are more readily performed in rodent models of infection, the mediators of protective immunity primed by antibiotic-mediated eradication of primary Salmonella infection

177 were also identified. We demonstrate that protection against recurrent infection is largely mediated by Salmonella-specific antibody because not only are high titers of Salmonella- specific antibody primed, but increased resistance against infection could be transferred with sera from “immune”, but not naïve mice. These findings are consistent with the protection against human typhoid conferred by the Vi polysaccharide vaccine that primes a T cell-independent, Salmonella-specific serological response [220,244,301,422,423], and the significantly reduced levels of protection against secondary Salmonella infection in B cell (antibody)-deficient mice [278-280]. Importantly, since B cells are also potent antigen presenting cells, the lack of protection against recurrent infection in B cell- deficient mice does not discriminate between the antibody producing and antigen presenting roles of these cells [292]. To clarify these different roles, the protective effects of serum containing high titer Salmonella antibody primed by early eradication of primary infection were clearly demonstrated in the adoptive serum transfer experiments we describe in this report (Fig. 27). Together, these results indicate an important role for

Salmonella-specific antibody in protective immunity against recurrent infection.

Surprisingly and in sharp contrast to the role for T cells in host defense against primary infection, neither CD4+ nor CD8+ T cells were essential mediators of protective immunity against recurrent infection because depletion of each cell type individually or in combination did not increase susceptibility to secondary Salmonella infection in antibiotic treated mice. Despite these observations, T cells clearly contribute and play an important role in host defense against Salmonella infection. Mice with targeted defects in

CD4+ T cells are highly susceptible to and do not eradicate even attenuated strains of

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Salmonella, and similar defects in host defense against primary Salmonella infection are found with T cell depletion [260-263,266,419,421]. These experimental results in mouse typhoid are consistent with the clinical observation that recovery from human typhoid and reductions in typhoid-associated complications such as gastrointestinal bleeding or perforation each correlates with the development of cell-mediated immunity

[211,265,424]. Nevertheless, since T cells also play important and critical roles in the maturation and activation of antibody producing cells, these loss-of-function experimental approaches cannot exclude that T cells are mediating protection through enhanced and sustained help to Salmonella antibody producing cells. Accordingly, this loss-of-function approach using mice that completely lack each immune cell subset does not allow the precise roles of antibody or T cells in mediating protection against recurrent infection to be identified. Our experiments comparing differences in susceptibility to recurrent infection with T cell ablation immediately prior to secondary infection bypasses these important limitations, and allows the requirement for T cells to be more definitively identified. These experiments demonstrate neither CD4+ nor CD8+ T cells are direct mediators of immunity against recurrent Salmonella infection in this infection model.

Our results demonstrating both significant reductions in Salmonella CFUs and increased mouse survival despite early antibiotic eradication of primary infection are also consistent with the increased survival of antibiotic treated mice after S. typhimurium infection in Nramp1-deficient mice [419]. While antibiotic eradication of primary infection conferred significantly prolonged and increased survival for C57BL/6 mice that are uniformly susceptible to even low inocula of virulent Salmonella in this recent study,

179 we demonstrate that early antibiotic eradication of primary infection confers 100% survival even after infection with increased doses of virulent Salmonella in Nramp1- sufficient B6.129F1 mice. In this regard, infection in Nramp1-sufficient mice that develop persistent instead of acute infection with S. typhimurium more directly recapitulates the blunted inflammatory response immediately after S. typhi infection in humans. Nevertheless the results presented here clearly demonstrate that early eradication of primary Salmonella infection primes sustained protection against secondary infection.

Moreover, a protective role for Salmonella-specific antibody that is generated despite early infection eradication is demonstrated. These findings suggest vaccination strategies that prime a more robust and sustained serological response compared with primary infection will be more efficacious for preventing Salmonella typhoid infection.

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MATERIALS AND METHODS

Mice. C57BL/6 and 129Sv mice were purchased from the National Cancer Institute.

B6.129F1 mice were generated by intercrossing C57BL/6 female with 129Sv male mice as described [274,367]. All mice were generated and maintained in specific pathogen-free facilities and used between 6-8 weeks of age. All experiments were conducted under

University of Minnesota IACUC approved protocols.

Bacteria, infections, and antibiotic treatment. The virulent S. enterica serotype

Typhimurium (S. typhimurium) strain, SL1344, has been described [273,323]. For infections,

S. typhimurium was grown to log phase in brain heart infusion (BHI) media at 37ºC, washed and diluted with saline and injected intravenously through the lateral tail vein. At the indicated time points after infection, the number of recoverable Salmonella CFUs was quantified by plating serial dilutions of organ homogenates onto BHI agar plates. Where indicated, enrofloxacin (Baytril) was added to the drinking water (2 mg/mL) of infected mice beginning either five or twenty days post-infection. Mice were allowed unrestricted access to antibiotic-treated water through day 40 post-infection. In some experiments, antibiotic treated mice were re-challenged with 1 X 104 or 1 X 106 CFUs of SL1344 five days after antibiotic withdrawal.

Reagents for cell staining, antibody ELISA, and cell depletion. Antibodies and other reagents for flow cytometry and ELISA were purchased from BD Biosciences (San Jose,

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CA) or eBioscience (San Diego, CA). For ELISA, flat bottom 96-well plates were coated

7 with 1.25 X 10 heat-killed SL1344 diluted in 0.1 M NaHCO3 and incubated overnight at

4ºC. Wells were then blocked with 1% albumin, assayed with serial dilutions of serum from either naïve or Salmonella-infected mice followed by biotinylated anti-mouse isotype specific antibodies, and developed with streptavidin conjugated to peroxidase and O- phenylenediamene substrate. For CD4+ and CD8+ T cell depletion, 500 µg of purified anti- mouse CD4 (clone GK1.5) and/or anti-mouse CD8 (clone 2.43) antibody (BioXCell) were intraperitoneally inoculated one day prior to Salmonella infection. For transfer, serum was collected from antibiotic treated or control mice, and transferred intravenously into naïve recipient mice (350 to 400 µL/mouse) one day prior to Salmonella infection.

Statistics. The difference in number of recoverable bacterial CFUs were evaluated using the Student’s t test (GraphPad, Prism Software) with p < 0.05 taken as statistically significant.

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Figure 23. Protection to secondary Salmonella infection despite early eradication of primary infection. A. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice with eradication of primary Salmonella beginning day 20 after infection, or without primary Salmonella infection treated with antibiotics in a similar fashion (naïve). B. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice with eradication of primary Salmonella beginning day

5 or 20 after infection, or without primary Salmonella infection treated with antibiotics in a similar fashion (naïve). These results are representative of two independent experiments each containing 3-5 mice per group. Bar, one standard error. *, p < 0.05.

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Figure 24. Enrofloxacin supplementation efficiently eradicates persistent S. typhimurium infection in B6.129F1 mice. Number of recoverable Salmonella CFUs in the spleen and liver at the indicated time points after infection for mice without antibiotic treatment

(open squares), or for mice treated with enrofloxacin (2mg/ml) in the drinking water beginning day 5 post-infection (solid squares). These results are representative of two independent experiments each containing 3-5 mice per group per time point.

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Figure 25. Protection from lethal rechallenge with virulent S. typhimurium. Percent survival following re-challenge with a normally lethal dose of virulent Salmonella (106

CFUs) in mice treated with antibiotics beginning 5 days post-primary infection (open squares) or naïve mice given antibiotics in parallel (solid squares). The number of mice in each group is as indicated and combined from two independent experiments.

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Figure 26. T cell depletion does not impact protection from secondary infection conferred by antibiotic eradicated primary Salmonella infection. A. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice with

CD4+ and CD8+ T cells depleted beginning one day prior to secondary Salmonella infection. B. Representative FACS plots demonstrating the efficiency of CD4+ and CD8+

T cell in vivo depletions. The numbers in each plot indicate the percent cells in each gate.

These results are combined from two independent experiments containing 7-10 mice per experimental group.

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Figure 27. Salmonella-specific antibody production despite early eradication of primary

Salmonella infection. Antibody titers of Salmonella-specific IgM, IgG, and IgA in the serum of naïve mice (open circles), mice 40 days post-infection treated with enrofloxacin beginning day 5 (filled squares) or no antibiotic treatment (open squares). These results represent two independent experiments containing 4-6 mice per group.

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Figure 28. Adoptively transferred serum from “immune” mice confers protection to naïve recipients. A. Number of recoverable Salmonella CFUs in the spleen and liver day

5 post-challenge for mice receiving serum from mice eradicated of primary Salmonella or no transfer control mice. B. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice receiving serum from antibiotic treated mice without

Salmonella or no transfer control mice. These results are representative of two independent experiments containing 6-8 mice per group. Bar, one standard error. *, p <

0.05.

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Chapter 6

Concluding Statement

The aim of this thesis work was to identify the factors that drive the generation of a protective CD4 T cell response and further define the contribution of CD4 T cell subsets in primary and secondary immunity against persistent Salmonella enterica infection using a murine model that recapitulates the natural host-pathogen relationship.

In Chapter 2, naturally-occurring APLs were identified within an immune- dominant CD4 T cell epitope of Salmonella enterica. Using a recombinant Listeria monocytogenes delivery system to prime memory Th1 CD4 T cells, these APLs were able to differentially prime the expansion and activation of antigen-specific CD4 T cells compared to the parental peptide but no APL stimulated the generation of a protective monoclonal pathogen-specific Th1 CD4 T cell memory population as seen following parental peptide stimulation. The lack of functional capacity of these cells to protect inherently-susceptible mice against wild-type S. typhimurium challenge was associated with impaired expression of T-bet induced by priming with the APLs compared to the parental peptide resulting in diminished production of IFN-γ following restimulation. In

Chapter 3, using a natural persistent S. typhimurium infection mouse model, the delayed generation of a protective Th1 CD4 T cell response was demonstrated. Additionally, employing an experimental system that allowed the select tracking and targeted ablation of Foxp3+ regulatory CD4 T cells (Tregs) throughout persistent S. typhimurium infection, 189 enhanced Foxp3+ Treg suppressive function was observed during the early phase of infection that was associated with impaired effector Th1 development and disease progression. Conversely, decreased Foxp3+ Treg function at later stages associated with downregulation of CTLA-4 expression permitted protective Th1 CD4 T cell development to progress leading to subsequent reductions in pathogen burden. In Chapter 4, using

TCR transgenic T cells and tetramer reagents to track monoclonal and endogenous antigen-specific CD4 T cell responses, respectively, during persistent S. typhimurium infection, the selective loss of a monoclonal antigen-specific CD4 T cell population but preservation of an endogenous antigen-specific memory CD4 T cell population primed during the early phase of S. typhimurium infection was demonstrated. Furthermore, the loss of monoclonal antigen-specific CD4 T cells was due to impaired survival following activation due to Salmonella-associated virulence factors encoded by the SPI-2 gene locus. Finally, in Chapter 5, secondary immunity against reinfection with wild-type S. typhimurium was shown to be antibody-mediated and CD4 T cell independent.

Furthermore, while this protective antibody response was able to protect mice against an otherwise lethal secondary challenge, it did not provide sterilizing immunity.

The generation of a protective IFN-γ producing Th1 CD4 T cell response is required for protection against primary infection with S. typhimurium infection

[260,261,270,305]. Furthermore, protection by Th1 CD4 T cell appears to be delayed until the later phases of infection (i.e. after 3-4 weeks post-infection), which corresponds to the delayed developmental kinetics of IFN-γ producing T cells during persistent S. typhimurium infection [260,261,274,323]. However, these studies were done under

190 conditions where the natural host-pathogen interaction was manipulated by the use of susceptible strains of mice that necessitated infection with attenuated strains of S. typhimurium in order to establish a persistent infection. Moreover, the relative contribution of CD4 and CD8 T cells, the predominant IFN-γ producing populations at later phases of persistent S. typhimurium infection ([274] and Fig. 8), have not been addressed. Our data, using a model of natural host-pathogen interactions, demonstrates that select depletion of CD4 T cells results in a gradual loss of protection during the later phase of primary persistent S. typhimurium infection that is more pronounced with the additional depletion of CD8 T cells (Fig. 9). As CD8 T cell depletion alone had no significant effect on protection, we conclude that, consistent with previous data in susceptible strains of mice, CD4 T cells are the predominant T cell population in protection and required for pathogen control whereas CD8 T cells can play a compensatory role in the absence of CD4 T cells. Furthermore, as Tregs have been implicated to be important for the proper coordination of an effector T cell response during persistent infection [161,162], it is possible that the loss of protection following

CD4 depletion is due to a preferential loss of regulatory rather than effector CD4 T cell populations. However, the select ablation of Foxp3-expressing Tregs at this later stage of infection did not significantly impact the level of protection (Fig. 15). Thus, we propose that the effector CD4 T cell population as opposed to the regulatory subset is critical for maintaining the balance in the host-pathogen interaction during later stages of persistent

S. typhimurium infection.

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The importance of CD4 T cells in protection during primary S. typhimurium infection combined with the delayed kinetics in the development of a protective CD4 T cell response suggests a theory that host-adapted strains of Salmonella enterica have co- evolved mechanisms that impede the generation of protective CD4 T cell responses in their respective hosts to favor pathogen persistence. Consistent with this model, we describe three such mechanisms that support this idea. Firstly, the identification of naturally-occurring altered peptide ligands within an immunodominant CD4 T cell epitope of S. typhimurium that suboptimally stimulates the expansion or upregulation of effector functions in an antigen-specific CD4 T cell population compared to the parental peptide (Fig. 1-5) implies that there may be a selection for immunodominant CD4 T cell epitopes that result in defective CD4 T cell activation. Additionally, the increased suppressive function of Tregs that impedes the early activation of effector CD4 T cells during persistent S. typhimurium infection (Fig. 11 and 15) would implicate mechanisms possessed by this pathogen that preferential activate regulatory compared to effector CD4

T cell subsets during this early stage of infection creating a suppressive environment (Fig.

12) that favors growth of the pathogen during this time frame. Lastly, the presence of virulence factors encoded by the SPI-2 gene locus of S. typhimurium that induce the death of antigen-specific CD4 T cells early after infection (Fig. 19) further supports the establishment of a suppressive environment that is non-conducive to the generation of a protective host CD4 T cell response to favor pathogen persistence. Importantly, however, our results demonstrating the existence of a population of antigen-specific CD4 T cells within the endogenous pool that can evade these various evasion mechanisms reveals that

192 these evasion strategies are incomplete and future experiments determining the factors that prime and/or permit the survival of this population of effector cells has important implications for the design of therapeutic interventions that can skew the host-pathogen interaction to favor the host during persistent Salmonella enterica infection.

Regarding secondary immunity against recurrent Salmonella enterica infection, the mediators of protection are currently incompletely understood. Our results demonstrate that vaccination of mice with a L. monocytogenes vector primed a population of antigen-specific memory CD4 T cells that can provide a slight but significant reduction in bacterial burden against S. typhimurium infection (Fig. 6).

Conversely, however, CD4 T cells are dispensable for protection against secondary infection generated following clearance of primary infection (Fig. 26). These apparently discordant results may reflect differences in the role of CD4 T cells in protection of susceptible compared to resistant mice where CD4 T cells are important for protection in susceptible mice but not required in resistant mice. This theory is in accordance with previous data demonstrating that serum from immunized mice is sufficient to protect resistant mice against secondary S. typhimurium infection whereas both serum and T cells are required to confer protection to susceptible mice [304-306]. Alternatively, while the observation that antibody alone can confer some protection is consistent with the field trial data examining the protective efficacy of Vi polysaccharide [244,245], however our data demonstrates that this protection is incomplete as it was unable to provide sterilizing immunity (Fig. 28), thus protection following Vi polysaccharide vaccination may similarly provide incomplete protection and therefore higher infection inoculums may be

193 able to overwhelm this protection resulting in clinical disease. Thus it is possible that the role of CD4 T cells in secondary immunity is to act in an additive effect to antibody- mediated protection that would augment protection provided by antibody alone.

So how can these results be used to design more effective vaccine regimens?

Based on the observations that host-adapted strains of Salmonella enterica have evolved mechanism that impair the generation of a protective Th1 CD4 T cell response by the host, overcoming these pathogen-associated evasion strategies represents a critical step in the future design of more effective vaccination strategies. For example, if strains of Salmonella enterica have undergone a selection process during co-evolution with its respective natural hosts resulting in CD4 T cell epitopes that generate a suboptimal effector response, then its possible that using natural Salmonella-derived antigens to prime a protective CD4 T cell response during immunization would similarly lead to the generation of a suboptimal secondary immune response. As such, both the Ty21a vaccination and natural primary infection prime a secondary immune response that is generated using such naturally-derived Salmonella antigens, and it would stand to reason that the poor protective efficacy afforded after each of these immunization strategies is that the endogenous Salmonella antigens present in both of these situations prime suboptimal memory Th1 CD4 T cell responses. Thus, a search for novel superagonistic

APLs derived from endogenous immune-dominant CD4 T cell epitopes on Salmonella enterica that are capable of priming a more robust protective Th1 CD4 T cell memory response may represent a more promising approach to designing future vaccine

194 candidates. Proof-of-concept for this approach has been demonstrated in a mouse model of chronic hepatitis virus infection [425]. The authors identified a superagonist APL against a natural sub-dominant viral epitope that primed a cytolytic CD8 T cell response with significantly higher functional avidity toward the parental epitope and protected against the selection of escape mutants resulting in abrogated disease progression in infected mice [425]. Therefore, future experiments aimed at identifying similar superagonist CD4 T cell epitopes specific to Salmonella enterica and elucidating their impact on protection against secondary infection are important areas of future study.

Additionally, the enhanced Treg suppressive function limiting the development of a protective Th1 CD4 T cell response during the early phase of disease progression represents another potential therapeutic target to enhance vaccine efficacy. An intriguing approach to overcome this mechanism of suppression is to simultaneously inhibit Tregs while priming an effector CD4 T cell response during vaccination. A similar approach is currently being used in the cancer field where antagonistic CTLA-4 antibody is being used in conjunction with other vaccination or induction therapies for the treatment of metastatic melanoma [426,427]. The proposed mechanism of action is to temporarily break Treg-induced tolerance in order to permit the more effective generation of an anti- tumor CD4 and CD8 T cell response [426,427]. Thus far, this strategy has met with considerable success and is currently in Phase III clinical trials [426,427]. Based on the observation that downregulation or inhibition of CTLA-4 on Tregs during S. typhimurium infection results in a robust T cell response associated with increased pathogen clearance, it is intriguing to speculate a similar strategy may be effective in vaccine development

195 against S. typhimurium as well. Therefore, anti-CTLA-4 blockade combined with the live, attenuated Ty21a vaccine strain of S. typhi represents a potential approach to generate a more robust secondary immune response by current vaccines to improve their efficacy.

Lastly, while it is possible that current vaccines and natural infection prime suboptimal Th1 CD4 T cell responses which may explain the lack of a contribution of

CD4 T cells to protection against secondary infection over antibody alone, an alternative explanation is that Ty21a vaccination and natural infection prime CD4 T cell responses that provide inadequate B cell help resulting in an antibody response that is comparable in protective efficacy to the T cell independent Vi polysaccharide response. In other words, if the critical role of CD4 T cells in secondary immunity is to facilitate a more efficacious pathogen-specific B cell antibody response, then strategies to augment CD4 T cell help to B cells would improve vaccine efficacy. This idea is supported by the preliminary success of a Vi polysaccharide vaccine conjugated to an irrelevant, recombinant Pseudomonas aeruginosa exotoxin protein to generate a T cell dependent Vi polysaccharide antibody response. In a recent field trial, this Vi-conjugated vaccine was able to achieve a three-year efficacy of over 90% in children between the ages of 3-5 in

Vietnam [428], which is far superior to what has been observed to date with other licensed vaccines and is associated with significantly elevated titers of IgG Vi-specific antibodies compared to levels seen in with unconjugated Vi polysaccharide and Ty21a vaccines [428]. Thus, determining the characteristics that comprise a highly-protective antibody response against Salmonella enterica infection as well as identifying the factors

196 that drive the development of a CD4 T cell response that provides the appropriate help to generate the optimal pathogen-specific antibody response may improve upon the initial success of the Vi conjugated vaccine.

Overall, the findings presented within this thesis have provided important insight into the role of effector and regulatory CD4 T cells as it pertains to their effects on influencing the pathogenesis and protection in a natural host-pathogen relationship during persistent S. typhimurium infection as well as to the contribution of CD4 T cells to primary and secondary immunity. This work has important implications in the design of more effective therapeutic and prevention strategies that can potentially reduce the global disease burden associated with this and related persistent infections.

197

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