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.

Orientador: Fábio Roberto Chavarette / Resumo: Este trabalho é dedicado ao desenvolvimento de uma metodologia baseada no monitoramento da integridade estrutural em aeronaves com foco em técnicas de computação inteligente, tendo como intuito detectar, localizar e quantificar falhas estruturais utilizando os sistemas imunológicos artificiais (SIA). Este conceito permite compor o sistema de diagnóstico apto a aprender continuamente, contemplando distintas situações de danos, sem a necessidade de reiniciar o processo de aprendizado. Neste cenário, foi empregado dois algoritmos imunológicos artificiais, sendo o algoritmo de seleção negativa, responsável pelo processo de reconhecimento de padrões, e o algoritmo de seleção clonal responsável pelo processo de aprendizado continuado. Também foi possível quantificar o grau de influência do dano para as cinco situações de danos. Para avaliar a metodologia foi montada uma bancada experimental com transdutores piezelétricos que funcionam como sensor e atuador em configurações experimentais, que podem ser anexadas à estrutura para produzir ou coletar ondas numa placa de alumínio (representando a asa do avião), sendo coletados sinais na situação normal e em cinco situações distintas de danos. Os resultados demonstraram robustez e precisão da nova metodologia proposta. / Abstract: This work is dedicated to the development of a methodology based on the monitoring of structural integrity in aircraft with a focus on intelligent computing techniques, aiming to detect structural failures using the artificial immune systems (AIS). This concept allows to compose the diagnostic system capable of learning continuously, contemplating different situations of damages, without the need to restart the learning process. In this scenario, two artificial immunological algorithms were employed, the negative selection algorithm, responsible for the pattern recognition process, and the clonal selection algorithm responsible for the continuous learning process. It was also possible to quantify the degree of influence of the damage for the five damage situations. To assess the methodology, an experimental bench was mounted with piezoelectric transducers that act as sensors and actuators in experimental configurations, which can be attached to the structure to produce or collect waves on an aluminum plate (representing the wing of the airplane), being collected signals in the normal situation and in five different situations of damages. The results demonstrate the robustness and accuracy of the proposed new methodology. / Doutor

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.

[pt] Este trabalho investiga métodos de detecção de falhas baseados em sistemas imunológicos artificiais, especificamente aqueles baseados no algoritmo de seleção negativa (NSA) e em outras técnicas de reconhecimento próprio/nãopróprio. Inicialmente, foi proposto um esquema de representação baseado em hiperesferas com centros e raios variáveis e três modelos capazes de gerar detectores, com esta representação, de forma eficiente. O primeiro modelo utiliza algoritmos genéticos onde cada gene do cromossomo contém um índice para um ponto de uma distribuição quasi-aleatória que servirá como centro do detector e uma função decodificadora responsável por determinar os raios apropriados. A aptidão do cromossomo é dada por uma estimativa do volume coberto através uma integral de Monte Carlo. O segundo modelo utiliza o particionamento Quadtree para gerar o posicionamento dos detectores e o valor dos raios. Este modelo pode realizar o particionamento a partir de uma função de detecção ou através de divisões recursivas de um detector inicial que ocupa todo o espaço. O terceiro modelo é inspirado nas redes imunológicas. Neste modelo, as células B representam os detectores e a rede formada por eles dá a posição e o raio de cada detector. Experimentos com dados sintéticos e reais demonstram a capacidade dos algoritmos propostos e que eles apresentam melhorias nos aspectos de escalabilidade e desempenho na detecção de falhas. / [en] This work investigates fault detection methods based on Artificial Immune Systems, specifically the negative selection algorithm (NSA) and other self/nonself recognition techniques. First, there was proposed a representation scheme based on hyperspheres with variable center and radius, and three models, which are very capable to generate detectors, based on that representation scheme, in an effective way. The first model employs Genetic Algorithms where each chromosome gene represents an index to a point in a quasi- random distribution, that will serve as a detector center, a decoder function will be responsible to determine the appropriate radius. The chromosome fitness is given by a valuation of the covered volume, which is calculated through a Monte Carlo integral. The second model uses the Quadtree space partition technique to generate the detectors positions and their radius. The space partition could be done by using a detection function or by recursive divisions of an initial detector that occupies the whole space. In third model, inspired on immune networks, the B cells represent the detectors and the network that is established by them gives the location and radius of each detector. Experiments with syntetic and real data show that the proposed algorithms improve scalability and perform better in fault detection.

The popularity of e-commerce sites and applications that use recommendations and user modeling is increased recently. The development and contest in tourism calls attention of large-scale IT companies. These companies have started to work on recommendation systems and user modeling on tourism sector. Some of the clustering methodologies, neighboring methods and machine learning algorithms are commenced to use for making predictions about tourist&rsquo / s interests while he/she is traveling around the city. Recommendation ability is the most interesting thing for a tourist guide application. Recommender systems are composed of two main approaches, collaborative and content-based filtering. Collaborative filtering algorithms look for people that have similar interests and properties, while contentbased filtering methods pay attention to sole user&rsquo / s interests and properties to make recommendations. Both of the approaches have advantages and disadvantages, for that reason sometimes these two approaches are used together. Chosen method directly affects the recommendation quality, so advantages and disadvantages of both methods will be examined carefully. Recommendation of locations or services can be seen as a classification problem. Artificial intelligent systems like neural networks, genetic algorithms, particle swarm optimization algorithms, artificial immune systems are inspired from natural life and can be used as classifier systems. Artificial immune system, inspired from human immune system, has ability to classify huge numbers of different patterns. In this paper ESGuide, a tourist guide application that uses artificial immune system is examined. ESGuide application is a client-server application that helps tourists while they are traveling around the city. ESGuide has two components: Map agent and recommender agent. Map agent helps the tourist while he/she interacts with the city map. Tourist should rate the locations and items while traveling. Due to these ratings and client-server interaction, recommender agent tries to predict user interested places and items. Tourist has a chance to state if he/she likes the recommendation or not. If the tourist does not like the recommendation, new recommendation set is created and presented to the user.

Epstein-Barr virus (EBV) is associated with a spectrum of benign and malignant lymphoproliferative disorders, including acute infectious mononucleosis (IM), Burkitt's lymphoma (BL) and immunosuppression-associated B cell lymphoproliferative disease (LPD). Immunosurveillance mediated by virus-specific cytotoxic T lymphocytes is believed to protect immunocompetent hosts from EBV-associated lymphoma and LPD. Due to the lack of an adequate animal model, however, the precise immunologic mechanisms which provide this protection have not been directly demonstrated in vivo. Human peripheral blood mononuclear cell-reconstituted C.B.-17-scid/scid mice (hu-PBMC-SCID mice) develop EBV-positive LPD following intraperitoneal injection of PBMC from EBV-seropositive donors. The SCID mouse disease mirrors human EBV-associated LPD in morphology, presence of the EBV genome, clonality, and patterns of expression of latent viral cellular differentiation antigens. The hu-PBMC-SCID mouse provides a unique small animal model of EBV+ LPD, and it was used in this study to examine the role of CD8+ CTL in controlling LPD. Survival time increase significantly when EBV-specific cytotoxic T-cell lines (CTL) are adoptive transferred into hu-PBMC-SCID mice, demonstrating suppression of LPD in vivoby a CTL-mediated virus-specific mechanism. Survival time also increases significantly with administration of alloreactive CTL lines, suggesting that a non-virus-specific mechanism also contributes to control of EBV-associated LPD by CTL. NOD-SCID mice reconstituted with PBMC from donors with latent EBV infection develop EBV+ LPD with significantly less frequency than do C.B.17-SCID mice reconstituted with PBMC from the same donors. Administration of anti-CD8 mAb to these mice depletes human CD8+ cells and increases the incidence of LPD to 100%, demonstrating that CD8+ T cells are neccessary for protection from EBV-associated LPD. Adoptive transfer of human CD8+ T cells, but not CD4+ T cells, prevents LPD in CD8-depleted NOD-SCID mice. In vivo depletion of CD4+ T cells prevents engraftment of human T cells, and LPD does not develop in most mice after CD4+ cell depletion. These studies are the first to directly demonstrate both the protective role of CD8+ T cells and a requirement for CD4+ T cells in EBV -associated LPD in an in vivo model.

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.