Probiotics are live nonpathogenic microorganisms capable of positively impacting the host by improving the natural gut microbial balance and promoting animal health. In ovo technology represents one means to administer probiotics and promote early colonization of beneficial bacteria for stimulating intestinal and immune system development, and warding off enteric threats. Three studies were conducted to evaluate the effects of in ovo administration of Primalac in broilers. The objective of the first study was to determine the effects of administering probiotics in ovo on hatchability, post-hatch performance and expression of immune-related genes in the ileum and cecal tonsils. On embryonic day 18, 360 eggs were injected with water, 1x105, 1x106, or 1x107 probiotic bacteria. Another 90 eggs remained uninjected as a negative control group. Measurements and tissue samples were taken on day of hatch (DOH) and days (d) 4, 6, 8, 15, and 22. A subsequent study was conducted to evaluate the effects of administering Primalac in ovo and in the diet on broiler chick hatchability, post-hatch performance, immune organ weights and ileal immune-related gene expression. At embryonic day 18, 1977 eggs were either not injected (negative control), dry-punched, or injected with 1x106 or 1x107 probiotic bacteria. Performance parameters were measured on DOH and d4, 6, 8, 14, 19, and 42, while immune organ weights and tissue samples were taken on DOH and d4, 6, 8, 14, and 20. A third study investigated the effects of in ovo administration of Primalac on hatchability, performance, immune organ weights, and lesion scores in broiler chicks exposed to Eimeria sp. At embryonic day 18, 210 eggs were injected with either sterile water or 1x106 probiotic bacteria. On d3 post-hatch, half of the chicks from each treatment group were challenged with a mixed inoculum of Eimeria acervulina, E. maxima, and E. tenella. Measurements and tissue samples were taken on DOH and d3, 9 and 15. The results of these studies suggest that in ovo Primalac supplementation does not negatively impact hatchability, enhances performance, modulates intestinal gene expression, and provides protection against a mixed Eimeria infection. / Ph. D.

For nearly forty years diethylstilbestrol (DES) was administered to pregnant women to maintain healthy pregnancies. During this time, it is estimated that several million men and women have been exposed to DES during sometime of their life. The most common period of exposure was during fetal development. Although rarely used for the maintenance of pregnancy now, its current medical use is restricted to certain clinical situations such as breast and prostate cancer therapies in adults. Thus, DES exposure spans the entire lifetime, from prenatal to geriatric age. Since the early 1950s, health risks were beginning to be associated with prenatal DES treatment. So far only reproductive problems such as infertility, neoplastic diseases of the cervix and vagina and testicular cancers have been well-documented in DES cases. Immunological abnormalities associated with DES are only now beginning to be recognized. Self-reported cases and questionnaire-based studies have revealed increased incidence of infections and autoimmune diseases in DES exposed people. Animal studies that have examined the immunological effects of DES treatment are largely restricted to one gender, or to one dose of DES or to the developmental period. This is an important issue since human exposure to DES occurred in both men and women, at all ages and, at a wide-range of doses. The purpose of these studies was to investigate the immunological consequences resulting from the exposure to DES. Since sensitivity can vary between genders, dose and at the time of exposure, it is critical to investigate the DES-induced immunological changes during all stages of life in both genders. To address these critical gaps in the literature, we examined the immunomodulatory effects of adult and prenatal exposure to DES in males and females. Our findings show that DES effects were evident in both the thymus and spleen. DES markedly affected the apoptosis of thymocytes and the ability of splenic lymphocytes to proliferate in response to stimulants and secrete vital cytokines such as interferon-gamma. Our notable findings were that in-utero exposure to DES resulted in profound alterations in lymphocyte functionality, which were noticed as late as one-year of age. This suggests that alterations to the in utero environment can have deleterious consequences that may be long lasting. These studies have profound implications to the humans and animals exposed to DES, and indirectly to a whole range of other estrogenic compounds. / Master of Science

Feed costs are approximately 70% of total production cost for poultry producers. Poultry diets in the United States generally consist of 2 grains: corn and soybean meal. In recent years, the cost of these grains has dramatically increased. Due to these price increases, producers seek alternative feeds that provide adequate nutrition, and are also more affordable than "traditional" grains. Cottonseed meal is one alternative that is both affordable and an excellent source of crude protein. However, cottonseed meal contains gossypol, a pigment toxic to chickens. This study had two main objectives. The first objective was to determine if silymarin, an extract from milk thistle, could offset or prevent gossypol toxicosis. The second objective was to determine if divergent selection for humoral immune response would have an impact on the ability of the chicken to cope with gossypol toxicosis. Two preliminary studies were conducted. One determined basal activities of liver detoxification enzymes at various ages. The other determined concentrations of gossypol and silymarin that should be added to the diet to elicit a response. The information gathered from the second preliminary study was used to conduct the final experiment. In the final experiment, chickens from each of 2 lines selected for humoral immunity were exposed to diets containing gossypol, silymarin, gossypol and silymarin, and a control. Humoral immunity had no impact on the ability of the chicken to cope with gossypol toxicosis. Silymarin did not alleviate gossypol toxicosis. Future studies will focus on using a lower gossypol concentration in the diet. / Master of Science

An attractive strategy to improve efficacy of current Influenza A Virus (IAV) vaccines is to promote protective CD4 T cell responses. Antiviral CD4 T cell responses are predominantly classified as strong IFN-gamma producing T helper type 1 (Th1) cells. Yet, the role of IFN-gamma in protection against IAV is still unclear, suggesting absolute Th1 polarization may be expendable for effective IAV immunity. Here we test this hypothesis using models that restrict the deficiency of known transcriptional regulators of Th1 immunity in only CD4 T cells, avoiding indirect impact on other immune cell responses. We find the 'master regulator' of Th1 cells, the T-box transcription factor T-bet (Tbx21), to be dispensable for CD4 T cell-mediated protection from lethal IAV but important for maximizing the magnitude of effector responses by regulating cell trafficking to the lung. While donor Tbx21-/- responses gain Th17 characteristics, they still produce substantial IFN-gamma, suggesting their Th1 attributes may still be required for protection. Interestingly, Tbx21-/- cells expressed more Eomesodermin (Eomes), a paralog of T-bet, but Eomes-/- cells retain WT-like responses suggesting minor roles for Eomes in presence of T-bet. In contrast, Tbx21-/-Eomes-/- cells, completely lose their Th1 identity but remarkably exhibit stronger inflammation-induced Th17 attributes than Tbx21-/- cells. Strikingly, we find in vitro Th17-primed Tbx21-/-Eomes-/- effectors lose their plasticity to become Th1 but instead strengthen their Th17-ness in IAV infected mice but still protect. Finally, we observed that protection of previously primed Tbx21-/- and Tbx21-/-Eomes-/- mice from a lethal unrelated IAV strain required T cell mediated immunity. Our observations thus demonstrate novel roles for Eomes in broadening the scope of protective mechanisms against IAV. Furthermore, we decisively demonstrate protective roles for prototypical non-plastic Th17 responses in primary and secondary responses, thus increasing the scope of target mechanisms relevant for CD4 T-cell based vaccination strategies against viral pathogens.

Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

<p><strong>INTRODUCTION: </strong>Pre-clinical and clinical data strongly support the feasibility of employing immunotherapy as a strategy to treat cancer.</p> <p><strong>METHODS: </strong>Using the B16F10 murine melanoma model, we have been investigating mechanisms of T cell-mediated antitumor immunity following immunization with dopachrome tautomerase (DCT), a melanoma-associated antigen.</p> <p><strong>RESULTS: </strong>In <strong>Chapter 2</strong>, we uncovered an interesting dichotomy whereby DCT-specific CD4⁺ T cell-mediated tumor protection and autoimmunity are dependent on IL-4/STAT-6 and IFN-g/STAT-4, respectively. Our data also revealed that this phenomenon is extrinsic of CD4⁺ T cell polarization.</p> <p>To gain further insight into the targets recognized by CD4⁺ T cells, we conducted in <strong>Chapter 3</strong> extensive CD4⁺ T cell epitope mapping experiments using overlapping peptide libraries. Interestingly, while we were able to identify “helper” epitopes within DCT that were required for maximal CD8⁺ T cell expansion, we were unable to identify “effector” epitopes responsible for tumor rejection. Further examination of the requirements for the generation of CD4⁺ T cell effector epitopes showed that post-translational modifications of the protein were involved.</p> <p>In <strong>Chapter 4</strong>, we investigated the modest efficacy afforded by DCT immunization in the context of established B16F10 melanomas. Using intratumoral transcriptional analysis, we demonstrated that the vaccine rapidly promoted an IFN-g-dependent immunosuppressive state inside the tumor. Concurrent treatment with the immunomodulatory antibodies anti-4-1BB and anti-PD-1 effectively counteracted this tumor immunosuppression, resulting in complete regression of tumors and long-term survival in 70% of the mice.</p> <p><strong>CONCLUSIONS: </strong>The research described in this thesis sheds new light into the mechanisms by which vaccine-mediated CD4⁺ T cell responses participate to tumor rejection and autoimmunity. Moreover, our findings indicate that cancer vaccine-induced tumor immunosuppression significantly limits tumor regression, emphasizing the requirement of combinatorial approaches for successful cancer immunotherapy. Overall, our research offers new insight for future vaccine development.</p> / Doctor of Philosophy (Medical Science)

La maladie de Newcastle est une maladie virale hautement contagieuse et dévastatrice chez la volaille. Bien que la biosécurité et lhygiène puissent savérer suffisantes pour lutter contre lintroduction de la maladie, la vaccination préventive est considérée comme une précaution supplémentaire et a été rendue obligatoire dans la plupart des pays européens dès les années 90 suite aux épidémies de ND. Cependant, lobservation dépizooties sporadiques témoigne des limites des programmes de prévention actuels. Dans ce contexte, de nombreuses alternatives vaccinales sont étudiées afin de limiter le risque dune infection par le NDV et de réduire la transmission virale, tout en prévenant les signes cliniques et la mortalité. Durant ce travail, la vaccination à un jour à laide dun vaccin atténué a été privilégiée et lamélioration de son efficacité par la co-administration avec ladjuvant chitosan et une primo-vaccination in ovo avec un vaccin recombinant rHVT-ND a été investiguée. Lobjectif de cette thèse consiste à étudier de manière approfondie limmunité induite chez le poulet par la vaccination contre la ND et à établir une corrélation entre les réponses immunitaires mesurées et la protection. Ces expériences ont montré linfluence du tropisme de la souche vaccinale de NDV sur linduction de limmunité à médiation cellulaire et sur le degré de la réponse immune locale en anticorps au niveau des tractus respiratoire et digestif. Cette réponse immune cellulaire spécifique du NDV a pu être renforcée par la co-administration du chitosan avec le vaccin atténué. Linterférence des anticorps maternels sur linduction dune réponse immune spécifique lors dune vaccination à un jour a également été validée. Cet impact négatif a pu être limité par une primo-vaccination in ovo avec un vaccin recombinant rHVT-ND. Enfin, le programme de vaccination combinant une primo-vaccination in ovo avec une vaccination à un jour avec un vaccin atténué co-administré avec le chitosan a permis daméliorer la protection contre les signes cliniques de la maladie mais également de diminuer lexcrétion virale lors dune infection virulente. Cette plus forte protection peut être corrélée avec linduction dune meilleure immunité à médiation cellulaire et une réponse immune locale accrue. Newcastle disease is a highly contagious and devastating condition of poultry. Although good biosecurity and hygiene practices can be sufficient to fight against this disease, the preventive vaccination is thought to be an additional precaution and is rendered compulsory in many European Union countries since the 90 after ND epidemic. Nevertheless, enzootics and epizootics that are sometimes reported point out the limitations of current vaccination regimens. In this context, different approaches have been investigated in order to prevent infection by NDV, reduce the transmission of the virus and eliminate clinical signs and mortality. In this work, we have selected the vaccination at day-old with attenuated ND vaccine and we have attempted to increase its efficacy by the co-administration with chitosan adjuvant and by an in ovo injection of a rHVT-ND vaccine. The aim of this present work was to evaluate the immunity induced by ND vaccination and the establishment of the correlation between the recorded measurement and the protection. These experiments showed the influence of the tropism of the ND vaccine strain on the NDV-specific antibody-mediated immunity in the respiratory and digestive tracts and on the cell-mediated immunity. This NDV-specific cellular immune response was improved by the co-administration of chitosan with the ND attenuated vaccine. It was also observed that the presence of MDA interferes with the establishment of a persisting good protective immunity after a single day-old vaccination. The use of rHVT-ND vaccine applied in ovo as primo-vaccination allowed overcoming this negative interference of MDA. Finally, the innovative rHVT-ND/live ND-chitosan vaccination regimen provided the highest protection against mortality and morbidity as well as the strongest reduction of virus shedding, which could be related to a higher measured cellular immune response and local antibody-mediated immunity.

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but their tendency to display low immunogenicity in humans has hindered their usage, despite excellent tolerability and safety profiles. Various approaches have been used to improve the immunogenicity of DNA vaccines. Recent human study data re-established the value of DNA vaccines, especially in priming high-level antigen-specific antibody responses. Data suggests that innate immune responses to the DNA vaccine plasmid itself contribute to the immunogenicity of DNA vaccines, however the underlying mechanisms responsible remain unclear. In this dissertation, we investigate the role of innate immunity in shaping antigen-specific adaptive immune responses following DNA vaccination. The current belief is that the cytosolic DNA sensing pathways govern DNA vaccine immunogenicity. To date, only the type I interferon inducing STING/TBK1 regulatory pathway has been identified as required for DNA vaccine immunogenicity. Surprisingly, neither the upstream receptor nor the downstream signaling molecules in this pathway have been characterized. I therefore investigated a candidate cytosolic DNA receptor, as well as the downstream transcription factors required for generation of antigen-specific immune responses. Additionally, the effects of pro-inflammatory signaling on DNA vaccine immunogenicity have yet to be comprehensively studied. Previous studies have only provided indirect evidence for the role of inflammatory v signaling in DNA vaccination. As such, I also investigated the role of the DNA sensing AIM2 inflammasome in DNA vaccination. My data indicates that AIM2 is a key modulator in DNA vaccination via a previously unrecognized connection to type I interferon. Importantly, this marks the first time a DNA vaccine sensor has been identified. Of note, this dissertation represents a departure from many published works in the field. Whereas previous studies have mostly utilized model antigens and only focused on the adaptive immune responses generated, I analyzed the effects on innate immunity as well. Using various innate gene knockout murine models, I quantified antigen-specific humoral and T cell responses, as well as serum cytokine and chemokines following immunization with a clinically relevant DNA vaccine. Overall, this data provides a basis for understanding the mechanisms of DNA vaccination, allowing for the design of more effective vaccines.

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.

T cell development in the thymus produces multiple lineages of cells, including conventional naïve CD4+ and CD8+ T cells, regulatory T cells, and innate T cells. Innate T cells encompass γδ T cells, invariant natural killer (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and H2-M3-restricted cells (Berg, 2007). Although they are a minor subset of all thymocytes, innate T cells develop in the thymus and share characteristics of the innate and adaptive immune systems (Berg, 2007). These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation (Berg, 2007). However, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8+ T cells develop as innate cells that share characteristics with memory T cells (Atherly et al., 2006b; Broussard et al., 2006; Fukuyama et al., 2009; Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). One of these signaling proteins, inducible T cell kinase (Itk) is a nonreceptor protein tyrosine kinase that signals downstream of the T cell receptor (TCR) (Berg et al., 2005). Upon TCR activation, Itk is activated and recruited to the TCR signaling complex, where Itk interacts with Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), linker for activation of T cells (LAT), and phospholipase C γ1 (PLCγ1) (Berg et al., 2005). Thus, in Itk-deficient mice, TCR signaling is disrupted, which results in mature CD4- CD8+ (CD8SP) thymocytes that are CD44high, CD62Lhigh, CD122+, and CXCR3+ and that express high levels of the transcription factor, Eomesodermin (Eomes) (Atherly et al., 2006b; Broussard et al., 2006; Weinreich et al., 2010). Recently, it was determined that the development of these innate CD8SP thymocytes in itk-/- mice is dependent on IL-4 produced in the thymic environment by a poorly characterized subset of CD3+ thymocytes expressing the transcriptional regulator, promyelocytic leukemia zinc finger (PLZF) (Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). Here we show that a sizeable proportion of mature CD4+ CD8- (CD4SP) thymocytes in itk-/- mice also develop as Eomesodermin+ innate T cells. These Eomes+ innate CD4+ T cells are CD44high, CD62Lhigh, CD122+, and CXCR3+ (Atherly et al., 2006b; Broussard et al., 2006; Dubois et al., 2006; Weinreich et al., 2010). Surprisingly, neither CD4SP nor CD8SP innate thymocytes in itk-/- mice are dependent on γδ T cells for their development as was previously hypothesized (Alonzo and Sant'Angelo, 2011). Instead, both subsets of innate itk-/- T cells require the presence of a novel PLZF-expressing, SAP-dependent thymocyte population that is essential for the conversion of conventional CD4+ and CD8+ T cells into Eomesodermin-expressing innate T cells with a memory phenotype. This novel subset of PLZF-expressing SAP-dependent innate T cells preferentially home to the spleen and mesenteric lymph nodes and have a restricted TCR repertoire. Thus, we have christened this subset as CD4+ PLZF + MAIT-like cells. We have characterized multiple subsets of innate T cells that expand in the absence of Itk. Therefore, we were interested in how innate T cells respond to infection. Although Itk KO mice have defects in cytolytic function and cytokine production during an acute infection, these mice are able to clear viral infections (Atherly et al., 2006a; Bachmann et al., 1997). Hence, we hypothesized that Itk-deficient memory CD8+ T cells would be able to provide protection upon a challenge infection. Conversely, we found this not to be true although Itk-deficient memory CD8+ T cells were present in similar frequencies and cell numbers as WT memory CD8+ T cells at 42 days post-infection. Furthermore, Itk-deficient memory CD8+ T cells were able to produce IFNγ and exert cytolytic function upon stimulation. Although the function of Itk-deficient memory CD8+ T cells appeared to be intact, we found that these cells were unable to expand in response to a challenge infection. Remarkably, conventional memory CD8+ T cells lacking Itk were able to expand and form protective memory responses upon challenge. Thus, the inability of Eomes+ innate CD8+ T cells to form protective memory responses does not appear to be intrinsic to cells deficient in Itk. This thesis is divided into six major chapters. The first chapter will provide an introduction to T cell development and the role of Itk in T cell development. Additionally, it will introduce a variety of innate T cell subsets that will be discussed throughout this thesis and will provide an overview of CD4+ and CD8 + T cell differentiation during infection. This section will explain the role of Itk in CD4+ helper T cell differentiation and describe how Itk-deficient CD8+ T cells respond to acute infection. The introduction will also discuss the generation of conventional memory CD8+ T cells. The second chapter will provide the details of the experimental procedures used in this thesis. The third chapter will describe the characterization and development of Eomes+ innate CD4+ T cells that develop in the absence of Itk. Additionally, this chapter will address the subset of PLZF+ innate T cells that induce the expression of Eomes in innate T cells. The fourth chapter will further characterize and explore the development of itk-/- CD4+ PLZF+ MAIT-like T cells. The fifth chapter will examine the role of Eomes + innate CD8+ T cells in protective memory responses. Chapters three through five will display work that is in preparation to be submitted to a peer-reviewed journal. The sixth chapter will discuss the results of this thesis and their implications.