Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous immunity.

Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

Na Serra do Espinhaço Meridional, o predomínio de uma litologia quartzítica, associada a processos tectônicos diversos, proporcionou a formação de um complexo sistema de falhas, fraturas e dobramentos que, aparentemente, controlam a sedimentação quaternária e a rede de drenagem. Esta última, por sua vez, possui importante papel na formação de ambientes hidromórficos e na formação de turfeiras. Baseado na hipótese de que as turfeiras se formam em decorrência do controle exercido por estruturas quartzíticas sobre a rede de drenagem, gerando hidromorfismo, este trabalho objetiva estudar a relação entre morfoestratigrafia, hidrologia e os processos de formação das turfeiras a partir de uma análise espacial do meio físico, da configuração do embasamento rochoso, analisado pelo Radar de Penetração do solo (GPR), e de mudanças climáticas ocorridas no Pleistoceno e Holoceno. A análise do meio físico foi realizada a partir de uma compilação de cartas geológicas da área e do mapeamento das principais feições geomorfológicas, analisadas a partir de modelos gerados em sistemas de informação geográfica (SIG). A partir de uma análise detalhada dos produtos supracitados, foram selecionadas cinco microbacias hidrográficas com padrões geomorfológicos favoráveis a formação de turfeiras (planícies quartzíticas, vales suspensos, vales encaixados e vales alongados ladeado por vertentes suaves). Foram realizadas análises das geoformas e da drenagem por estereoscopia e transeções com o GPR. A cronologia dos eventos, bem como, a estratigrafia e a origem da MO foram analisadas por datações por C14, granulometria e fracionamento isotópico da MO, respectivamente. As turfeiras se formam, preferencialmente, em depressões formadas sobre litotipos mais susceptíveis ao intemperismo confinados entre quartzitos altamente resistentes onde a umidade tende a ser mantida. Dados do GPR mostram que as turfeiras ocorrem também associadas ao excesso de umidade mantido por soleiras quartzíticas em subsuperfície nos vales alongados adaptados a falhas e nos vales suspensos. Os soterramentos de turfeiras foram favorecidos por lineamentos tectônicos de direção E - W que controlam parcialmente a drenagem e a sedimentação na porção mediana da área. As variações nos processos sedimentares apresentam forte relação com mudanças climáticas ocorridas no Holoceno e Pleistoceno. Pelo menos seis períodos de mudanças climáticas foram observadas: entre 30.251 a 12.418 anos AP o clima favoreceu a deposição de MO; de 12.418 a 7.890 anos AP os processos erosivos foram mais intensos; de 7.890 a 3.280 anos AP o clima quente e úmido favoreceu o deposito de MO e, nos últimos 2.590 anos AP, o clima foi semelhante ao atual com 3 períodos breves favoráveis a deposição de turfa observado por camadas discretas de MO. / The morphology of the Serra do Espinhaço Meridonal is associated with various tectonic processes. In combination with the prevalence of a quartzite lithology this caused the formation of a complex system of faults, fractures and folds that control the Quaternary sedimentation and drainage network. Impeded drainage cause hydromorphic environments in which organic matter (OM) may accumulate, causing the formation of peatlands. Based on the hypothesis that the formation of peatlands in this area is controlled by impeded drainage of quartzite structures, the aims of this thesis were (i) to study the relationship between morphology, stratigraphy and hydrology, (ii) to determine the influence of this relationship on the formation of peatlands, and (iii) to interpret this in the context of climatic changes in the Pleistocene and Holocene. This will be done by a spatial analysis of the physical environment of five watersheds. The watersheds were selected based on geomorphological patterns that favor peat formation, including quartzite plains, hanging valleys and elongated valleys flanked by gentle slopes. The configuration of the rocky basement will be determined with Ground Penetrating Radar (GPR), a compilation of geological maps of the area will be made, and the main geomorphological features will be examined using Geographic Information Systems (GIS) models. The chronology, stratigraphy and origin of OM were analyzed by C14 dating, particle size and isotopic fractionation, respectively. The results indicate that the peatlands are preferentially formed on rock types other than quartzite, which are occasionally located between the highly resistant quartzite rocks. Because this material is more susceptible to weathering, depressions were formed in which water tends to accumulate, thereby providing conditions that favor peat formation. However, GPR data showed that peatlands were also formed on quartzite rocks when the subsurface showed elongated valleys adapted to crashes and hanging valleys. Burried peat was found at some places, which was related to tectonic lineament patterns with an east-west direction. These tectonic lineament patterns partially controlled the drainage and sedimentation in the central part of the area. Variation in sedimentary processes (peat formation vs. mineral influx/erosion) was found to be strongly related to climatic changes in the Holocene and Pleistocene. At least six periods of climate change were observed: between 30.251 and 12.418 yr BP climate favored the deposition of OM, between 12,418 and 7890 yr BP erosion processes were more intense; between 7.890 - 3.280 yr BP a hot and humid climate favored peat formation, and the last 2.590 years the climate was similar to the current favorable with three brief periods of peat deposition were observed.

This dissertation addresses the role for Janus Kinase 3 (Jak3) in CD4+ T cell homeostasis and function. Jak3 is a protein tyrosine kinase whose activity is essential for signals mediated by the γc dependent cytokines IL-2, -4, -7, -9, -15, and -21. Previous data have demonstrated that peripheral CD4+ T cells from Jak3-deficient mice have a memory phenotype and are functionally impaired in both proliferative and IL-2 responses in vitro. Interestingly, Jak3/γc activity has been previously shown to play a role in the prevention of T cell anergy. These studies were initiated to more precisely define the role for Jak3/γc cytokines in the prevention of T cell anergy and the maintenance of functional CD4+ T cell responses. We began to address this question by assessing global gene expression changes between wild type and Jak3-/- CD4+ T cells. These data indicate that Jak3-/- CD4+ T cells have an increase in gene expression levels of inhibitory surface receptors as well as immunosuppressive cytokines. Further analyses confirmed that Jak3-deficient T cells express high levels of PD-1, secrete a Trl-type cytokine profile following direct ex vivo activation, and suppress the proliferation of wild type T cells in vitro. These characteristics indicate that CD4+ Jak3-/- T cells share properties with regulatory T cell subsets that have an important role in peripheral tolerance and the prevention of autoimmunity. We next addressed whether these regulatory characteristics were T cell intrinsic or rather the result of expanding in a Jak3-deficient microenvironment characterized by a number of immune abnormalities and a disrupted splenic architecture. Jak3-/- CD4+ T cells proliferate in vivoin a lymphopenic environment and selectively acquire regulatory T cell characteristics in the absence of any additional activation signals. While the precise mechanism by which Jak3-deficient T cells acquire these characteristics remains unclear, our data indicate that one important component is a T cell-intrinsic requirement for Jak3 signaling. These findings indicate several interesting aspects of T cell biology. First, these studies, demonstrate that the homeostatic proliferation of CD4+ T cells is not dependent on signaling via γc-dependent cytokine receptors. And, second, that the weak activation signals normally associated with homeostatic expansion are sufficient to drive Jak3-/- T cells into a non-conventional differentiation program. Previous data indicate that, for wild type T cells, signaling through both the TCR as well as γc-dependent cytokine receptors promote the homeostatic proliferation of T cells in lymphopenic hosts. Since Jak3-/- T cells are unable to receive these cytokine signals, their proliferation is likely to be wholly dependent on TCR signaling. As a consequence of this TCR signaling, Jak3-/- T cells proliferate, but in addition, are induced to up regulate PD-1 and to selectively activate the IL-10 locus while shutting off the production of IL-2. Since this fate does not occur for wild type T cells in a comparable environment, it is likely that the unique differentiation pathway taken by Jak3-/- T cells reflects the effects of TCR signaling in the absence of γc-dependent cytokine signaling. Interestingly, wild type T cells undergoing homeostatic expansion in lymphopenic hosts show many common patterns of gene expression to freshly-purified unmanipulated Jak3-/- T cells. For instance, micro array analysis of gene expression in wild type CD4+ T cells after lymphopenia induced homeostatic expansion show a similar pattern of upregulation in surface markers (PD-1 and LAG-3), and cytokine signaling molecules (IL-10 and IFN-γ cytokine, receptors, and inducible gene targets) to that of Jak3-/- CD4+ T cells immediately ex vivo. These data suggest that the process of homeostatic proliferation normally induces immune attenuation and peripheral tolerance mechanisms, but that full differentiation into a regulatory T cell phenotype is prevented by γc-dependent cytokine signals. Taken together these data suggest that Jak3 plays an important role in tempering typical immune attenuation mechanisms employed to maintain T cell homeostasis and peripheral tolerance.

Na Serra do Espinhaço Meridional, o predomínio de uma litologia quartzítica, associada a processos tectônicos diversos, proporcionou a formação de um complexo sistema de falhas, fraturas e dobramentos que, aparentemente, controlam a sedimentação quaternária e a rede de drenagem. Esta última, por sua vez, possui importante papel na formação de ambientes hidromórficos e na formação de turfeiras. Baseado na hipótese de que as turfeiras se formam em decorrência do controle exercido por estruturas quartzíticas sobre a rede de drenagem, gerando hidromorfismo, este trabalho objetiva estudar a relação entre morfoestratigrafia, hidrologia e os processos de formação das turfeiras a partir de uma análise espacial do meio físico, da configuração do embasamento rochoso, analisado pelo Radar de Penetração do solo (GPR), e de mudanças climáticas ocorridas no Pleistoceno e Holoceno. A análise do meio físico foi realizada a partir de uma compilação de cartas geológicas da área e do mapeamento das principais feições geomorfológicas, analisadas a partir de modelos gerados em sistemas de informação geográfica (SIG). A partir de uma análise detalhada dos produtos supracitados, foram selecionadas cinco microbacias hidrográficas com padrões geomorfológicos favoráveis a formação de turfeiras (planícies quartzíticas, vales suspensos, vales encaixados e vales alongados ladeado por vertentes suaves). Foram realizadas análises das geoformas e da drenagem por estereoscopia e transeções com o GPR. A cronologia dos eventos, bem como, a estratigrafia e a origem da MO foram analisadas por datações por C14, granulometria e fracionamento isotópico da MO, respectivamente. As turfeiras se formam, preferencialmente, em depressões formadas sobre litotipos mais susceptíveis ao intemperismo confinados entre quartzitos altamente resistentes onde a umidade tende a ser mantida. Dados do GPR mostram que as turfeiras ocorrem também associadas ao excesso de umidade mantido por soleiras quartzíticas em subsuperfície nos vales alongados adaptados a falhas e nos vales suspensos. Os soterramentos de turfeiras foram favorecidos por lineamentos tectônicos de direção E - W que controlam parcialmente a drenagem e a sedimentação na porção mediana da área. As variações nos processos sedimentares apresentam forte relação com mudanças climáticas ocorridas no Holoceno e Pleistoceno. Pelo menos seis períodos de mudanças climáticas foram observadas: entre 30.251 a 12.418 anos AP o clima favoreceu a deposição de MO; de 12.418 a 7.890 anos AP os processos erosivos foram mais intensos; de 7.890 a 3.280 anos AP o clima quente e úmido favoreceu o deposito de MO e, nos últimos 2.590 anos AP, o clima foi semelhante ao atual com 3 períodos breves favoráveis a deposição de turfa observado por camadas discretas de MO. / The morphology of the Serra do Espinhaço Meridonal is associated with various tectonic processes. In combination with the prevalence of a quartzite lithology this caused the formation of a complex system of faults, fractures and folds that control the Quaternary sedimentation and drainage network. Impeded drainage cause hydromorphic environments in which organic matter (OM) may accumulate, causing the formation of peatlands. Based on the hypothesis that the formation of peatlands in this area is controlled by impeded drainage of quartzite structures, the aims of this thesis were (i) to study the relationship between morphology, stratigraphy and hydrology, (ii) to determine the influence of this relationship on the formation of peatlands, and (iii) to interpret this in the context of climatic changes in the Pleistocene and Holocene. This will be done by a spatial analysis of the physical environment of five watersheds. The watersheds were selected based on geomorphological patterns that favor peat formation, including quartzite plains, hanging valleys and elongated valleys flanked by gentle slopes. The configuration of the rocky basement will be determined with Ground Penetrating Radar (GPR), a compilation of geological maps of the area will be made, and the main geomorphological features will be examined using Geographic Information Systems (GIS) models. The chronology, stratigraphy and origin of OM were analyzed by C14 dating, particle size and isotopic fractionation, respectively. The results indicate that the peatlands are preferentially formed on rock types other than quartzite, which are occasionally located between the highly resistant quartzite rocks. Because this material is more susceptible to weathering, depressions were formed in which water tends to accumulate, thereby providing conditions that favor peat formation. However, GPR data showed that peatlands were also formed on quartzite rocks when the subsurface showed elongated valleys adapted to crashes and hanging valleys. Burried peat was found at some places, which was related to tectonic lineament patterns with an east-west direction. These tectonic lineament patterns partially controlled the drainage and sedimentation in the central part of the area. Variation in sedimentary processes (peat formation vs. mineral influx/erosion) was found to be strongly related to climatic changes in the Holocene and Pleistocene. At least six periods of climate change were observed: between 30.251 and 12.418 yr BP climate favored the deposition of OM, between 12,418 and 7890 yr BP erosion processes were more intense; between 7.890 - 3.280 yr BP a hot and humid climate favored peat formation, and the last 2.590 years the climate was similar to the current favorable with three brief periods of peat deposition were observed.

Virus-specific cellular immune responses have been shown to be important in the control of viral replication in several animal and human virus models. Cells of both the CD8+ and CD4+T cell lineages have been shown to play protective roles during viral infections by exerting effector functions that can kill infected host cells or inhibit the production and spread of infectious virions. The continued spread of HIV-1 infection throughout the world, as well as the lack of a prophylactic HIV-1 vaccine have generated much interest in HIV-specific cellular immune responses. Recent technical advances have yielded a tremendous increase in our understanding of HIV-1-specific immunity, as well as HIV-1 replication dynamics and host cell factors that shape the course of acute and chronic infection. Unfortunately, due to small sample volumes and technological limitations, the study of HIV-1-specific T cell immunity in infants and children has been difficult. An improved understanding of the timing, specificity, and intensity of pediatric HIV-specific T cell responses would contribute to the development of a HIV-1 vaccine for use in regions of the developing world without access to antiretroviral therapeutics. In the small number of published studies investigating pediatric HIV-specific immunity, T cell responses were uncommonly detected in infants. It remains unclear, however, whether the lack of HIV-specific T cells is an accurate reflection of the in vivoimmune state in vertically-infected infants, or rather is a consequence of reagents and assays ill-suited to the detection of low-level and/or diverse T cell responses in pediatric subjects. In the present dissertation, several methodologies were used to investigate HIV-specific T cell responses in vertically-infected infants and children. HIV-specific CD8+ T cell responses were infrequently detected in a cohort of young infants, but are commonly detected in older infants and children. Interestingly, CMV-specific CD8+ T cell responses were detected in several young infants that lacked HIV-specific responses, suggesting a specific defect in the ability of some infants to generate HIV-specific CD8+ T cell responses. Further experiments characterizing detectable HIV-1-specific CD8+ T cell responses found that the HIV-1 accessory proteins may be important targets of the immune response during early vertical infection. The role of HLA class I genotype and viral sequence are also explored in a pair of vertically-infected twins with discordant CD8+T cell responses. Finally, viral isolates from an infant with a marked shift in gag-specific epitope usage during infancy are analyzed for the presence of escape mutations. Gag-specific CD4+ T cell responses were commonly detected in a large cohort of vertically-infected children. A linear relationship between HIV-1 replication and the presence and intensity of HIV-specific CD4+ T cell responses was found, but ongoing HIV-1 replication appeared to blunt CD4+T cell proliferation. The data presented in this dissertation describe pediatric T cell immune responses and how they relate to HIV-1 replication. This information may be useful to the design of a prophylactic or therapeutic HIV-1 vaccine for vertically-infected infants and children.

Itk is a member of the Tec family of non-receptor tyrosine kinases. It is expressed in T cells, NK cells, and mast cells. The purpose of this study was to determine the role of Itk in T cell development. Previous work from our lab and others has demonstrated that Itk is involved in signaling downstream of the T cell receptor and initial analysis of Itk-deficient mice revealed that these mice had some defects in T cell development. There are two stages of T cell development, the pre-T cell stage and the CD4+ CD8+ double positive stage, at which signals downstream of the T cell receptor are important. At the CD4+ CD8+ double positive stage, these signals direct two concurrent, but distinct processes known as repertoire selection and CD4/CD8 lineage commitment/differentiation. I show that there are only slight defects in development at the pre-T cell stage, presumably due to reduced TCR signaling. However these results clearly demonstrate that Itk is not essential at this stage of development. In contrast, repertoire selection, in particular positive selection, is significantly affected by the absence of Itk. Similarly, I show that Itk plays a role in lineage differentiation, although commitment to the appropriate lineage occurs normally in the absence of Itk.

Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

This thesis investigates the role of B cells as antigen-specific antigen-presenting cells (APC) in self tolerance to low concentrations of soluble self proteins and in acquired tolerance to low doses of soluble foreign protein antigens. Experiments were performed in normal and B cell-deficient animals, and tolerance induction was measured by T cell proliferation assays. T cell proliferation was reduced in B cell-deficient mice, indicating that B cells may be involved in efficient activation of naive T cells in response to protein antigen both in vivo and in vitro. To study acquired tolerance induced by low doses of soluble foreign protein antigen, normal and B cell-deficient adult mice were injected intravenously with repeated low doses (10 μg) of deaggregated ovalbumin (OVA), and then challenged with OVA in complete Freund's adjuvant. In animals treated with deaggregated OVA, the in vitro proliferative responses of LN T cells to OVA were significantly reduced, and production of the Th1 cytokine, IFN-γ, in response to OVA was lost. This occurred in both normal and B cell-deficient treated animals, indicating that B cell antigen presentation was not required for this phenomenon. B cells were also unnecessary for self tolerance of T cells to the transgenic self antigen, hen egg lysozyme (HEL), in a transgenic mouse strain with very low serum lysozyme concentration. Partial low zone tolerance induced by deaggregated, low-dose OVA was selective for the Th1 response, as measured by in vitro proliferation and IL-2 and IFN-γ production, because antibody responses of normal mice to this T cell-dependent antigen were largely unaffected. Both treated and untreated animals produced equivalent titers of anti-OVA antibodies, predominantly of the IgG1 and IgG2b isotypes, following challenge with OVA in complete Freund's adjuvant. Tolerance to low levels of the transgenic HEL self protein in mice expressing different MHC molecules was also addressed. Transgenic mice that were H-2b/b in the class II region were not tolerant to the transgenic self protein, whereas transgenic mice of the H-2b/k were tolerant.