It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

Influenza A virus infections are a major cause of morbidity and mortality in the United States and throughout the world. The current vaccine elicits primarily a humoral response that is specific for the external glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, these are the viral proteins that are most susceptible to antigenic shift and drift, and can evade the humoral response. Cytotoxic T lymphocytes (CTL) recognize and lyse virus-infected cells and are important in clearing influenza A virus infections. CTL can recognize epitopes on both the external glycoproteins and the more conserved internal viral proteins. This thesis investigates the hypothesis that there is a broad CTL memory response in humans, and, if boosted by vaccines, these CTL may help clear influenza A virus strains of different subtypes. The CTL repertoire specific for influenza A viruses reported in inbred mice is extremely limited and has focused on a few immunodominant epitopes. We perfonned preliminary bulk culture chromium release assays using human peripheral blood mononuclear cells (PBMC) stimulated with influenza virus strain A/PR/8/34 (H1N1) in vitro. CTL activity was observed against autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia constructs that expressed several influenza A viral proteins, including nucleoprotein (NP), matrix (M1), nonstructural 1 (NS1) and polymerase (PB1). This was more diverse than the limited response reported in inbred mice. To further characterize the CTL repertoire in humans, PBMC from healthy adult donors were stimulated and CTL were cloned by limiting dilution. Isolated cell lines were further characterized by their CD4/CD8 surface expression, histocompatibility leukocyte antigen (HLA) restriction, cross-reactive or subtype-specific influenza A subtype recognition, and epitope recognition. CTL lines isolated from three donors recognized epitopes on many different influenza virus proteins. The ELISPOT assay was used to identify the number of IFN-γ- secreting cells and determine the precursor frequency of the CTL specific for the epitopes that were mapped. The precursor frequency of IFN-γ producing CTL ranged from 1 in 4,156 PBMC to 1 in 31,250 PBMC. The precursor frequency for one epitope was below the level of detection of this assay, but most of the memory CTL were readily detected. The cross-reactive or subtype-specific recognition of various human influenza A subtypes by these T cell lines was determined by chromium release assays. Most of the CTL lines recognized B-LCL infected with any of the three influenza A subtypes that have caused epidemics in the last century (H1N1, H2N2, and H3N2) and recognized epitopes on conserved internal influenza viral proteins. Most of the subtype-specific cell lines recognized the surface HA or NA glycoproteins, which are not well conserved between influenza subtypes. Although most of the T cell lines that were characterized were cross-reactive with influenza viruses of human origin, infection of humans with a divergent swine or avian derived strain could cause a global pandemic. To study the human CTL responses to non-human influenza viruses, B-LCL were infected with an Hsw1N1 influenza A virus of swine origin, and cell lines were tested for recognition of these targets in a chromium release assay. Most cell lines lysed the targets infected With the Hsw1N1 subtype to the same degree as targets infected with the human H1N1 strain. Two influenza viruses of duck origin were also tested and were recognized by many of the cell lines. The subtypes of these duck strains were Hav1N1 and H5N2. The isolates of influenza A virus from the Hong Kong outbreak of 1997 were also used to infect targets and analyze recognition by these CTL. We found that approximately 50% of the human T cell lines tested recognized both of the Hong Kong isolates, 25% recognized at least one isolate, and 25% recognized neither isolate to the same degree as the A/PR/8/34 (H1N1) virus. We analyzed the amino acid (aa) changes in the epitopes of the T cells lines from the 25% of cell lines that did not recognize either Hong Kong virus isolate. Non-conservative mutations were found in all of the epitopes that lost recognition by the human CTL lines. Bulk cultures of PBMC from three donors that were stimulated with A/PR/8/34 (H1N1) influenza A virus of human origin recognized all of the non-human virus strains tested. Thus, humans have memory CTL that recognize influenza viruses of avian and swine species. This may provide a second line of defense against influenza infection in case of exposure to a novel influenza A virus derived from these species. These results made it clear that humans have broad CTL memory to influenza A virus. In order to determine whether these T cells could be boosted in a vaccine, immune-stimulatory complexes (Iscom) incorporating inactivated influenza particles were tested in vitro. Iscoms containing inactivated influenza A vaccine (Flu-Iscom) were used to pulse autologous B-LCL overnight that were then used as targets in chromium release assays with human CTL lines as effectors. A CD8+ HA-specific CTL line lysed these targets, but not targets pulsed with Iscoms alone or with inactivated influenza A vaccine alone. An NS1-specific cell line recognized targets pulsed with NS1 protein and Iscoms, but not targets pulsed with Iscoms or NS1 protein alone. Therefore, CTL could recognize in vitrotarget cells that were exposed to the Iscom vaccines containing their specific epitope. Flu-Iscom and Iscom mixed with inactivated influenza virus particles (Flu-Iscomatrix) were then used as vaccines in a clinical trial to test CTL and neutralizing antibody induction against influenza. Fifty-five donors were bled pre-vaccination, and on days 14 and day 56 post-vaccination. Bulk culture chromium release assays were performed using targets infected with live vaccine strain viruses. There were significantly more increases in the influenza A specific CTL activity in the PBMC of donors that were vaccinated with the Flu-Iscom and Flu-Iscomatrix vaccines than in recipients of the standard vaccine. In order to determine whether these increases in cytotoxicity were due to an increase in the precursor frequency of influenza specific CTL, the PBMC were used in ELISPOT assays to assess the changes pre-and post-vaccination. When there was an increase in the level of cytotoxicity detected in bulk culture CTL, there was often also an increase in the precursor frequency of influenza-specific CTL. Peptide-specific increases in the number of CTL that recognize epitopes such as M1 aa 58-66 were detected in several donors confirming the increase in influenza-specific CTL post-vaccination. Another type of T cell that may be involved in defense against viruses is the γδ T cell. T cells expressing the γδ T cell receptor (TCR) have been found extensively in mucosal tissues in mice and humans. Influenza A viruses enter via the airway tract, infecting the epithelial cells at the mucosal surface. These epithelial cells have been shown in vitro to be targets for influenza-specific cytolytic recognition of αβ T cells. To analyze whether γδ T cells can respond to influenza A-infected APCs, PBMC were stimulated with influenza A virus. Intracellular IFN-γ staining was used to determine whether γ/δ T cells can secrete IFN-γ in response to the influenza A virus infection. We observed an increase in the percentage of γ/δ T cells secreting IFN-γ post-influenza A virus infection of PBMC compared to uninfected or allantoic fluid-stimulated cultures. These T cells also upregulated CD25 and CD69 in response to live influenza A virus. We focused on the responses in the CD8- population of γδ T cells, which are the majority of γδ T lymphocytes. Furthermore, the increases in IFN-γ production and activation marker expression were much more clear in the CD8- γδ+ T cells. The level of CD8- γδ T cell activation with inactivated influenza A virus was much less, and in some cases no higher than uninfected PBMC. The CD8+ αβ and γδ responses could be partially blocked by anti-class I antibodies, but the CD8- γδ responses could not. Vaccinia virus infection did not activate the CD8- γδ T cells to the same degree as influenza virus infection. γδ T cells are thought to have a regulatory role that includes the secretion of cytokines and epithelial growth factors to help restore tissue back to health. Humans have broad multi-specific T lymphocyte responses by αβ T cells to influenza A viruses and those responses are cross-reactive with human, avian, and swine virus strains. These CTL can be activated in vitro and boosted in number in vivo by Iscom incorporating vaccines. There is also a population of γδ+ T lymphocytes in humans that responds to influenza virus infection by producing cytokines and becoming activated. Increasing memory CTL as a second line of defense against influenza A viruses may be important in future vaccine development.

Orientadoesr: Fernando José Von Zuben, Leandro Nunes de Castro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-17T00:46:41Z (GMT). No. of bitstreams: 1 Knidel_Helder_M.pdf: 7578387 bytes, checksum: d653b5bd7e4bf2758525ced52374d42d (MD5) Previous issue date: 2006 / Resumo: Esta dissertação propõe a extensão e desenvolvimento de ferramentas imuno-inspiradas. As ferramentas desenvolvidas destinam-se à resolução de problemas de agrupamento e classificação de dados com atributos binários ou reais. Inspirados em idéias advindas do sistema imunológico, os algoritmos propostos apresentam robustez e soluções parcimoniosas. Uma característica comum presente nas ferramentas desenvolvidas é a definição automática do número de protótipos por meio de estágios de clonagem e poda. Baseado na projeção de protótipos, empregando uma técnica de escalonamento multidimensional, desenvolveu-se uma ferramenta de visualização de redes imunológicas com dados numéricos multivariados, com o propósito de obter uma descrição da estrutura global dos grupos, visualizar a presença e forma de grupos, descobrir protótipos pouco representativos e identificar outliers. Por fim, a aplicação de um algoritmo proposto em conjunto com uma heurística desenvolvida e um algoritmo de busca local iterativa solucionou de forma inovadora um problema relacionado à área de equalização de canais em telecomunicações / Abstract: This thesis considers the extension and development of immune-inspired tools. The developed tools are devoted to the resolution of clustering and classification problems with binary or real-valued data attributes. Inspired by ideas of the immune system, the considered algorithms have produced robust and parsimonious solutions. A common feature in the developed tools is the automatic definition of the number of prototypes by means of cloning and pruning stages. Based on the projection of prototypes, using a technique of multidimensional scaling, a visualization tool of immune networks with multivariate numerical data was developed, making it possible to get a description of the global structure of the groups, to visualize the presence and form of groups, to discover low representative prototypes and to identify outliers. Finally, a device composed of one of the tools considered above, a dedicated heuristic and an algorithm for iterative local search was developed. The application of this device solved in an innovative way a problem related to channel equalization / Mestrado / Engenharia de Computação

Made available in DSpace on 2016-03-15T19:37:52Z (GMT). No. of bitstreams: 1 Danilo Souza da Cunha.pdf: 1082171 bytes, checksum: 4d2c64017c5641baf212b0fe377da373 (MD5) Previous issue date: 2013-10-23 / Fundo Mackenzie de Pesquisa / E-commerce has been growing rapidly over the past years. Various products, services, and information are constantly offered to millions of internet users. Defining an adequate strategy to offer a product to a customer is the main goal of a recommender system. To do so, the items to be offered have to take into account the interests of each customer. This association of items is a data mining task, more specifically a task called association rule mining. This dissertation investigated the use of bioinspired algorithms, particularly evolutionary and im-mune algorithms, to build associations among items of a database. Three sets of experiments were performed: an investigation into the influence of different selection and crossover mech-anisms in an evolutionary algorithm for association rule mining; the use of a probabilistic selection in the immune algorithm; and a comparison of the bioinspired algorithms with the standard deterministic algorithm called Apriori. The data bases for comparison were taken from real e-commerce applications. The results allowed the identification of a suitable combi-nation of the selection and crossover mechanisms for the evolutionary algorithm, and to iden-tify the strengths and weaknesses of all approaches when applied to real-world recommender systems. / O comércio eletrônico vem crescendo rapidamente ao longo dos últimos anos. Produtos, serviços e informações dos mais variados tipos são oferecidos todos os dias para milhares de usuários na Internet. Definir uma estratégia adequada para oferecer um produto a clientes é o objetivo dos sistemas de recomendação. Para isso leva em conta itens que podem ser ofertados considerando o interesse de cada cliente. Essa associação entre itens é uma tarefa que recai sobre a competência da mineração de dados, mais especificamente a área chamada de mineração de regras de associação. Esta dissertação investigou o uso de algoritmos bioinspirados, mais especificamente algoritmos evolutivos e imunológicos, a fim de construir associações entre os itens de uma base de dados. Foram feitos três estudos: a influência de diferentes mecanismos de seleseleção e cruzamento no algoritmo evolutivo; o uso de seleção probabilística no algoritmo imunológico; e a comparação dos algoritmos bioinspirados com o algoritmo determinístico clássico aplicado a essa tarefa, chamado de Apriori. As bases de dados para efeitos comparativos foram coletadas em lojas nacionais de comércio eletrônico. Os resulta-dos apresentados permitiram identificar uma combinação adequada dos mecanismos de sele-ção e cruzamento do algoritmo evolutivo, assim como identificar os pontos fortes e fracos dos algoritmos bioinspirados quando comparados ao algoritmo tradicional.

Made available in DSpace on 2016-03-15T19:38:14Z (GMT). No. of bitstreams: 1 Ederson Borges.pdf: 626219 bytes, checksum: d83887c1b3e2287f434525ac9701f0c7 (MD5) Previous issue date: 2010-01-27 / Clustering is an important data mining task from the field of Knowledge Discovery in Databases. There are several algorithms capable of performing clustering tasks, and the most popular ones involve the calculation of a similarity or distance measure among objects from the database. Many algorithms can perform clustering in a simple and efficient manner, but have drawbacks as a way to get the optimal number of partitions and the possibility of getting stuck in local optima solutions. To try and reduce these drawbacks this dissertation proposes a new clustering algorithm based on Artificial Immune Systems. This algorithm is characterized by the generation of multiple simultaneous high quality solutions in terms of the number of partitions (clusters) for the database and the use of a cost function that explicitly evaluates the quality of partitions, minimizing the inconvenience of getting stuck in local optima. The algorithm was tested using four databases known in the literature and obtained satisfactory results in terms of the diversity of solutions, but has a high computational cost compared to other algorithms tested. / Agrupamento de dados é uma importante tarefa da mineração de dados e descoberta de conhecimentos em bases de dados. Existem diversos algoritmos capazes de realizar a tarefa de agrupamento de dados, sendo que os mais populares envolvem o cálculo de similaridade ou distância entre objetos da base de dados. Boa parte dos algoritmos pode agrupar os dados de forma simples e eficiente, mas possui inconvenientes como a forma de obter o número ótimo de partições e a possibilidade de ficar preso em ótimos locais. Para tentar diminuir estes inconvenientes essa dissertação propõe um novo Algoritmo Imunológico para Agrupamento de Dados baseado em Sistemas Imunológicos Artificiais. Esse algoritmo é caracterizado pela geração de múltiplas soluções simultâneas de boa qualidade no que tange o número de partições (grupos) para a base de dados e uma função de custo que avalia explicitamente a qualidade dessas partições, minimizando o inconveniente de ficar preso em ótimos locais. O algoritmo foi testado utilizando quatro bases de dados conhecidas na literatura e obteve resultados satisfatórios no que tange a diversidade das soluções encontradas, mas apresentou um custo computacional elevado em relação a outros algoritmos testados.

Orientadores: Romis Ribeiro de Faissol Attux, Jugurta Rosa Montalvão Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-23T23:31:44Z (GMT). No. of bitstreams: 1 Silva_DanielGuerreiroe_D.pdf: 5960509 bytes, checksum: febb8228109537e82dfcce66fca8aae8 (MD5) Previous issue date: 2013 / Resumo: Esta tese de doutorado possui como tema geral o desenvolvimento de algoritmos de Aprendizado de Máquina Baseado na Teoria da Informação (ITL - Information Theoretic Learning). O paradigma de ITL propõe o uso de critérios de treinamento baseados em medidas como entropia e informação mútua, em substituição aos tradicionais critérios baseados em estatísticas de segunda ordem. Os problemas de inversão cega de sistemas de Wiener e separação cega de sinais em corpos de Galois são os objetos de estudo e desenvolvimento dessas ferramentas. Estes problemas apresentam características marcantes quanto à necessidade de descritores estatísticos de ordem superior, por isso, apresenta-se uma série de contribuições que se baseiam em critérios de ITL e empregam algoritmos imuno-inspirados (ou heurísticas de busca) para adaptar os parâmetros dos modelos envolvidos. As propostas desenvolvidas abrem a perspectiva de futuras aplicações em áreas como genômica, codificação e geofísica. Espera-se também que os resultados apresentados fortaleçam um entendimento mais amplo de ITL, a fim de abranger estratégias mais genéricas de busca, de estimação de informação e de modelagem de dados / Abstract: The main theme of this thesis is the development of Information Theoretic Learning (ITL) algorithms. The ITL paradigm proposes the adoption of training criteria based on information measures such as entropy and mutual information, instead of traditional criteria based on second order statistics. The problems of blind inversion of Wiener systems and blind separation of signals over Galois fields are the tasks over which these tools are applied. Such problems present key aspects that establish a demand for higher order statistics, hence we present several contributions that are based on ITL criteria and employ immune-inspired algorithms (or heuristic-based methods) to perform the adaptation of the parameters of each related model. The developed proposals open the perspective of future applications in genomic, coding theory and geophysics. Furthermore, we expect that the presented results support a wider understanding of ITL, in order to comprise more general strategies of search, information estimation and data modeling / Doutorado / Engenharia de Computação / Doutor em Engenharia Elétrica

The immune systems of cnidaria are important to study for two reasons: to gain a better understanding of the evolution of immune responses, and to provide a basis to partially redress the precipitous world-wide die-offs of reef corals, some of which have been attributed to diseases and stress. Many immune responses share ancient evolutionary origins and are common across many taxa. Using Swiftia exserta, an azooxanthellate ahermatypic local octocoral, as a proxy model organism to study aspects of innate immunity in corals and cnidaria allows us to address both of the reasons listed above while not using endangered species. Utilizing a coral that does not contain symbiotic dinoflagellates (zooxanthellae) simplifies the system by restricting the source of proteins to a single genome. The lack of zooxanthellae in Swiftia exserta also allows the animal’s simple adaptation to lab settings. This study of the innate immune system of an octocoral demonstrates: 1) a novel understanding of the microanatomy of octocoral tissues; 2) that Swiftia exserta has at least two cell types that function as constitutive immunocytes; and 3) the presence of two potent antibacterial peptides, one with a mass between 4694 and 4696 Daltons. My report on the microanatomy of the coenenchyme, the tissue between polyps, advances the understanding of octocoral anatomy by systematically comparing histology sections with electron micrographs. Applying various techniques of enzyme histochemistry, coupled with cryo-preservation, to the coenenchyme I have identified at least two populations of constitutive immunocytes in Swiftia exserta. Two antibacterial proteins are identified by protein purification and antimicrobial testing techniques. The more active protein is partially characterized with modern hyphenated mass-spectrometry techniques, and can be the focus of future study.

Previously, our lab demonstrated that heterologous immunity could result in either gain or loss of protective immunity and alteration in immune pathology following infection by a second un-related pathogen. One of the prototypical models to study T cell-mediated heterologous immunity involves two distantly related arenaviruses, namely lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). Each virus encodes a cross-reactive CD8 epitope that has six out of eight in amino acid (aa) similarity with respect to its counterpart at the position 205-212 of the nucleoprotein (NP205). Heterologous challenge between LCMV and PV results in 1) expansion of the cross-reactive NP205-specific CD8 T cell responses and alteration of the immunodominance hierarchy and 2) partial protective immunity (heterologous immunity). Our lab showed that cross-reactive NP205-specific CD8 T cell receptor (TCR) repertoires become extremely narrowed following a heterologous challenge between LCMV and PV. Therefore, I questioned if LCMV NP205 epitope escape variants could be isolated during a dominant but narrowed crossVI reactive NP205-specific CTL response. In the first part of my thesis, I describe the isolation of a LCMV NP-V207A CTL escape variant in vivo using PV-immune animals challenged with LCMV clone 13. The LCMV NP-V207A variant contains a point mutation, which results in the switching of valine to alanine at the third non-anchoring residue of the LCMV NP205 CD8 epitope. Immunization of mice with the LCMV NP-V207A variant results in a significantly diminished cross-reactive NP205-specific CD8 T cell response. This suggests that the point mutation is responsible for the loss in the immunogenicity of the LCMV NP205 CD8 epitope. In addition, an in vitrorescued(r) recombinant LCMV variant (r/V207A) that encodes the original mutation also induces a highly diminished cross-reactive NP205-specific CD8 T cell response in mice. In agreement with the result obtained from the intracellular cytokine assays (ICS), MHC-Ig dimers loaded with the LCMV NP205 (V-A) peptide could only detect a minute population of cross-reactive NP205-specific CD8 T cells in mice infected with r/V207A variant virus. All the data indicate that the point mutation results in a significant loss in immunogenicity of the LCMV NP205 CD8 epitope. So far, no direct link between the cross-reactive NP205-specific CD8 T cells and heterologous immunity had been established in this system. Therefore, we immunized mice with either LCMV WT or the LCMV NP-V207A variant virus and showed that a significant loss of heterologous immunity is associated with the group immunized with LCMV NP-V207A variant virus. Again, r/V207Aimmune animals also displayed a significant loss in heterologous immunity following PV challenge. This suggests that the cross-reactive NP205-specific CD8 T cells mediate the majority of heterologous immunity between LCMV and PV in vivo. In comparison to the PV-immune control group, PV clearance kinetics mediated by the cross-reactive NP205-specific CD8 T cells were significantly delayed. Finally, these data also suggest that bystander activation plays very little role in heterologous immunity between LCMV and PV. Many studies in murine systems and humans suggest that cross-reactive T cells are often associated with immune pathology. We showed that in mice that were sequentially immunized with PV and LCMV (PV+LCMV WT double immune mice), there was a development of a high incidence and high level of immune pathology known as acute fatty necrosis (AFN) following a final PV challenge. The data suggest that these cross-reactive NP205-specific CD8 T cells might play an important role in immune pathogenesis. Therefore, we asked if the cross-reactive NP205-specific CD8 T cells play a role in immune pathogenesis by comparing the incidence of AFN between the (PV+LCMV WT) and the (PV+LCMV NP-V207A) double immune mice following a final PV challenge. In agreement with our hypothesis, the result showed the (PV+LCMV NP-V207A) double immune mice developed a significantly lower incidence of AFN compared to the (PV+LCMV WT) double immune mice. However, linear correlation studies comparing the frequency of different antigen-specific CD8 T cell populations within the (PV+LCMV WT) double immune mice before challenge and the severity of AFN following the PV challenge suggest that two opposing antigen-specific CD8 T cell populations are involved in determining the final outcome of the immune pathology. The PV NP38-45-specific CD8 T cell response (PV NP38) appears to be more protective than the cross-reactive NP205-specific CD8 T cell response. In addition, a positive linear correlation between the ratio of cross-reactive NP205 to PV NP38 and the severity of AFN seem to suggest that these cross-reactive populations are important contributors to immune pathogenesis. Peptide titration studies examining the functional avidities to different antigenic specificities suggest that both populations consist of high avidity TCR and peptide MHC (TCR:pMHC) interactions. However, skewing within the cross-reactive NP205 specific CD8 T cell response towards the LCMV NP205 epitope response in one of the (PV+LCMV WT) double immune mice suggests that cross-reactive NP205 specific CD8 T cells could constitute a sub-optimal response to a PV challenge. In summary, I questioned what might be some of the immunological consequences of heterologous immunity in this model. First of all, we have established a direct link between the cross-reactive NP205-specific CD8 T cell response and heterologous immunity in LCMV and PV. Second of all, I demonstrated that a LCMV NP205 epitope escape variant could be selected in vivo under the conditions of heterologous immunity. In addition, I showed that PV clearance kinetic was significantly delayed in cross-reactive NP205-mediated heterologous immunity as compared to homologous challenge. Finally, we demonstrated that cross-reactive NP205-specific CD8 T cells could play an important role in immune pathogenesis in this model. However, correlation data indicate that two opposing antigen-specific CD8 T cell populations could ultimately decide the outcome and magnitude of immune pathology in each individual mouse. All the data presented above strongly suggest that the cross-reactive NP205 CD8 T cells play a crucial role in immune pathology in this model system by 1) interfering with the regular establishment of immunodominance hierarchy orders, or 2) exhibiting a sub-optimal protective immunity due to the nature of the cross-reactive epitope.

The Tec family tyrosine kinase, Itk, plays an important role in signal transduction following T cell receptor engagement. Several prior studies have established the importance of Itk in immune system processes, such as T cell development and T cell activation. Additional biochemical studies have found that Itk specifically functions within a multi-molecular signalosome complex, which ultimately functions to provide a platform by which Itk can phosphorylate and activate PLC-γ1, a crucial step in T cell activation. To further study how Itk regulates distinct immune outcomes via T cell effector processes within the peripheral immune system, and to further understand how Itk functions in T cells in response to a physiological ligand-receptor interaction, I crossed Itk-deficient mice to mice transgenic for a TCR specific for a moth cytochrome C peptide. My studies have established a unique role for Itk in several important aspects of T cell function. Following T cell activation, I identified an imperative role for Itk in activation-induced cell death via FasL, a mechanism of immune homeostasis. Furthermore, I found Itk plays a unique role in the process of T cell differentiation, where Itk positively regulates the induction of cytokine genes, such as IL-4, while negatively regulating the induction of T-bet, a transcription factor important for Th1 differentiation. Lastly, following T cell differentiation, I found that Itk mRNA and protein are up-regulated during Th2 differentiation, while Rlk, a related Tec kinase, disappears rapidly from Th2 cells, indicating a critical role for Itk in Th2 cell function. Collectively, my thesis work has more clearly defined an important function for Itk not only in TCR signaling, but also in immune processes such as T cell differentiation and activation-induced cell death that are required for proper immune function.

Itk and Rlk are members of the Tec kinase family of non-receptor protein tyrosine kinases that are preferentially expressed in T cells. Numerous previous studies have demonstrated that these proteins play an important a role in the regulation of signalling processes downstream of TCR activation in CD4+ T cells, particularly in the phosphorylation of PLCγl. In addition, Itk and Rlk have both been shown to be important for CD4+ T cell development, differentiation, function and homeostasis following TCR activation. In the absence of Itk and Rlk, CD8+ SP thymocytes and T cells develop a memory/previously activated phenotypic profile, however, very little is known about the influence of Itk and Rlk on CD8+ T cell development and function. This study illustrates a previously unappreciated role for Itk and Rlk in the regulation of cytokine signals during CD8+ SP thymocyte maturation, and in the development of the memory CD44hi profile of Itk -/- and Itk -/- Rlk -/- CD8+ SP thymocytes and CD8+ T cells. This study also provides the first detailed study of the role of loss of Itk and particularly both Itk and Rlk in CD8+ signalling and function and shows that these Tec kinase family members play an important role in the maintenance of CD8+ T cell fitness and function, particularly in the ability of CD8+ T cells to accumulate in response to infection. Collectively, my studies demonstrate a critical role for Itk and Rlk in the generation of optimal CD8+ T cell responses. They also raise the novel observation that these proteins may be involved on the regulation of cytokine signals in T cells.