Thesis (Ph.D. in Immunology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 180-202). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The Tec family tyrosine kinases Itk, Tec and Rlk are expressed in T cells. Previous studies have established that these kinases are critical for TCR signaling, leading to the activation of PLCγ1. To further understand the functions of Tec kinases in T cell activation, we took three different approaches. First, we performed a thorough analysis of CD28-mediated signaling events and functional responses with purified naïve T cells from Itk-/- mice and a highly controlled stimulation system. Data from this set of studies definitively demonstrate that CD28 costimulation functions efficiently in naïve CD4+ T cells in the absence of Itk. Second, in order to further study the functions of Tec kinases in vivo, we generated transgenic mouse lines expressing a kinase-dead (KD) mutant of Tec on the Itk-/-Rlk-/- background, hoping to study mice that are functionally deficient for all three Tec kinases. The results hint the importance of the Tec kinases in T cell development and/or survival. Finally, in order to identify potential transcriptional targets of Itk, we used microarray technology to compare global gene expression profiles of naïve and stimulated Itk-/- versus Itk+/- CD4+ T cells. This analysis provided a short list of differentially expressed genes in Itk-/- versus Itk+/- CD4 T cells, providing a starting point for further studies of Itk in T cell activation. Collectively, these studies clarified the role of Itk in CD28 signaling, revealed some unexpected aspects of Tec family kinases in T cells, and indicated potential targets of Itk-dependent signaling pathways in T cells.

A person is exposed to many pathogens throughout their lifetime, and with the resolution of each infection, there remains a pool of pathogen-specific immune cells that protect that person from re-infection with the same pathogen. However, there is a great deal of evidence to suggest that the pool of pathogen-specific memory cells can also participate in the immune response to future infections with unrelated pathogens. Many believe T cells to be cross-reactive in nature because of their interaction with self antigens during development in the thymus and their interaction with foreign antigens once in the periphery. There are many features of the interaction between a T cell and its ligand that facilitate this cross-reactive nature. Based on solved crystal structures, relatively few contacts are required for a stable interaction, and that interaction is often mediated by the flexible CDR3 loops of the T cell receptor that accommodate ligands of various structure. There is also evidence in the murine and human systems that subsets of virus-specific memory CD8 T cells take on an activated phenotype upon infection with an unrelated virus. In murine models, these memory T cell subsets could kill target cells, secrete several cytokines, and proliferate in response to a cross-reactive stimulation, suggesting that a cross-reactive T cell response could impact the outcome of a viral infection. In fact, upon heterologous infection, mice immune to a previous virus were often protected, having lower titers of the second unrelated virus, their epitope-specific and T cell receptor repertoires were often skewed, and they were more prone to immune-mediated pathologies. All of these observations coincided with the presence of cross-reactive T cell responses. Thus, we define heterologous immunity as changes in viral replication and the disease pathology associated with that viral infection as a result of the host's history of infection, and this can be mediated, in part, by cross-reactive CD8 T cell responses. Since many human viral infections are associated with a wide range of disease states, we questioned whether cross-reactive CD8 T cell responses occurred as commonly as they appeared to occur in the murine models and whether they influenced the outcome of such infections. Epstein-Barr virus (EBV) infects over 90% of the U. S. population and has a large genome with the capacity to encode a multitude of T cell epitopes. The first part of this thesis research focuses on the identification of cross-reactive CD8 T cell responses with specificity for known epitopes derived from EBV, a common human virus. We directed our study to HLA-A2-restricted responses because of the common expression of this MHC Class I allele in the U. S. population. This study resulted in the detection of cross-reactive responses with five different specificities that involved either the immunodominant lytic EBV-BMLF1280 epitope or the latent EBNA 3A596epitope. Three of the cross-reactive responses had specificity for epitopes derived from another unrelated, but common, human virus, influenza A virus (IV). Each of these cross- reactive responses had the potential to participate in the collective immune response to acute EBV infection. EBV is also well-suited as a model system to study heterologous immunity in humans, as infection at an early age is frequently asymptomatic, while the same infection during adolescence often results in an immune-mediated syndrome, infectious mononucleosis (IM). Since older individuals have presumably been exposed to more pathogens in their lifetime and, therefore, would have memory CD8 T cell pools with more extensive specificities, we hypothesized that acute EBV infection activated cross-reactive memory CD8 T cell responses that promoted the development of IM. In order to determine if the cross-reactive responses we identified above contributed to the immune response to acute EBV infection, we first screened the blood of IM patients for cross-reactive T cells with specificity for EBV-BMLFl280 and IV-M158. The total number of M1-specific T cells of 5 of 8 patients was increased at presentation with IM, which was suggestive of their specific activation during the EBV infection since a bystander mechanism would have resulted in 8 out of 8 patients having increased numbers of M1-specific T cells. Our hypothesis was further supported by the fact that we clearly detected cross-reactive T cells capable of recognizing both BMLF1 and M1 epitopes in the blood of 2 of the 5 IM patients with an augmented M1-specific T cell frequency. Furthermore, the M1-specific TCR repertoires of those two patients were dramatically skewed, which was an indication of cross-reactive M1-specific T cell expansions and, therefore, participation in the lymphoproliferation characteristic of IM. In addition, T cell lines derived from 3 out of 8 healthy donors with previous exposure to both viruses contained a subset of T cells that responded to both BMLF1 and M1 epitopes, suggesting that these cross-reactive cells are often maintained in memory. These cross-reactive T cells were cytotoxic and produced MIP-1β, IFNγ, and TNFα, functions which could potentially promote the symptoms of IM and, indeed, may have been contributed to the severe case of IM noted in one patient. The final part of this thesis research focused on defining the structure of the cross-reactive TCR that recognized both BMLF1 and M1 epitopes, which have only 33% sequence similarity. In addition, we examined the cross-reactive TCR repertoire organization of multiple individuals to determine the breath and, therefore, the likelihood that this cross-reactive T cell response will occur. These studies revealed that a wide range of Vα and Vβ families can mediate interaction with both epitopes and that the cross-reactive TCR repertoire was unique to each individual, relying heavily on the T cell clones present in that individual's private BMLF1- and M1-specific repertoires. We also observed an increased frequency of TCRs with longer CDR3 regions within the cross-reactive repertoire, which were often extended by non-bulky amino acid residues that could provide these TCRs with more flexibility in order. to accommodate the two different epitope structures. Given that we detected a cross-reactive T cell response with specificity for two immunodominant epitopes derived from two of the most common human viruses among people that share one of the most common MHC Class I alleles in the U. S. population, we predict that cross-reactive T cells are common components of human immune responses. The variability in the magnitude and specificity of each cross-reactive T cell response is dependent on each individual's unique history of infection and th,eir unique TCR repertoire, and such responses likely represent one of many factors that could explain the individual variability in disease severity associated with EBV and many other human viral infections.

Made available in DSpace on 2015-10-06T13:03:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-08-14. Added 1 bitstream(s) on 2015-10-06T13:18:24Z : No. of bitstreams: 1 000819084.pdf: 1209964 bytes, checksum: 04874bbac5638856dedf456279299e45 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A leishmaniose visceral é uma doença imunomediada que pode causar alterações na população de linfócitos T em diferentes órgãos e tecidos, e também aumento de células inflamatórias no encéfalo de cães infectados. O objetivo deste estudo foi avaliar e caracterizar as lesões encefálicas por meio de análise de cortes histológicos do encéfalo e as subpopulações de linfócitos T CD4+, CD8+, duplo-positivos (DP), duplo-negativos (DN), TCRs αβ e δγ e células dendríticas (cd11c+/MHC-II+) provenientes de linfonodos regionais da cabeça e do linfonodo poplíteo de cães com leishmaniose visceral utilizando citometria de fluxo. Os resultados mostram que encéfalos de cães com LV podem apresentar infiltrados inflamatórios em região de leptomeninges, plexo coroide e região subependimária. Entre os linfonodos, não houve diferença entre as subpopulações de linfócitos T, porém, observamos redução dos linfócitos T CD8+ e aumento dos T DN e TCR δγ enquanto que os CD4+ e DP e TCR αβ mantiveram-se próximos a valores de cães saudáveis, sugerindo assim que os linfócitos DN e TCR δγ podem estar relacionados com a resposta imunológica de cães sintomáticos com LV. Todos esses achados descartam a hipótese de que os linfonodos regionais da cabeça poderiam apresentar um padrão de resposta imune específica relacionada com as lesões encefálicas dos cães / Visceral leishmaniasis (VL) is an immune-mediated disease which may cause changes in T lymphocytes population in different organs and tissues, and also an increase in the inflammatory cells population in the brain of infected dogs. The aim of this study was to evaluate the encephalic lesions in brain sections, and to characterize the T lymphocytes subsets CD4+, CD8+, double-positive (DP), double-negative (DN), the TCRs αβ and δγ, and the dendritic cells (CD11c+/MHC-II+) from the regional lymph nodes of the head and the popliteal lymph node using flow cytometry. The results evidenced that the brain of dogs with VL may present inflammatory infiltrates in leptomeninges, choroid plexus and subependymal area. Between the lymph nodes, there was no difference among the T cell subsets, nevertheless, there was noticed a reduction in the CD8+ lymphocytes and an increase in the DN subset as well as the TCR δγ, while CD4+, DP and TCR αβ were detected in amounts closely related to described reference ranges, which suggests that the DN and TCR δγ lymphocytes may be related to the immune response in symptomatic dogs. Altogether, these findings discard the hypothesis that the regional lymph nodes of the head may present a specific pattern of immune response related to the brain lesions in dogs with VL.

Resumo: A leishmaniose visceral é uma doença imunomediada que pode causar alterações na população de linfócitos T em diferentes órgãos e tecidos, e também aumento de células inflamatórias no encéfalo de cães infectados. O objetivo deste estudo foi avaliar e caracterizar as lesões encefálicas por meio de análise de cortes histológicos do encéfalo e as subpopulações de linfócitos T CD4+, CD8+, duplo-positivos (DP), duplo-negativos (DN), TCRs αβ e δγ e células dendríticas (cd11c+/MHC-II+) provenientes de linfonodos regionais da cabeça e do linfonodo poplíteo de cães com leishmaniose visceral utilizando citometria de fluxo. Os resultados mostram que encéfalos de cães com LV podem apresentar infiltrados inflamatórios em região de leptomeninges, plexo coroide e região subependimária. Entre os linfonodos, não houve diferença entre as subpopulações de linfócitos T, porém, observamos redução dos linfócitos T CD8+ e aumento dos T DN e TCR δγ enquanto que os CD4+ e DP e TCR αβ mantiveram-se próximos a valores de cães saudáveis, sugerindo assim que os linfócitos DN e TCR δγ podem estar relacionados com a resposta imunológica de cães sintomáticos com LV. Todos esses achados descartam a hipótese de que os linfonodos regionais da cabeça poderiam apresentar um padrão de resposta imune específica relacionada com as lesões encefálicas dos cães / Abstract: - Visceral leishmaniasis (VL) is an immune-mediated disease which may cause changes in T lymphocytes population in different organs and tissues, and also an increase in the inflammatory cells population in the brain of infected dogs. The aim of this study was to evaluate the encephalic lesions in brain sections, and to characterize the T lymphocytes subsets CD4+, CD8+, double-positive (DP), double-negative (DN), the TCRs αβ and δγ, and the dendritic cells (CD11c+/MHC-II+) from the regional lymph nodes of the head and the popliteal lymph node using flow cytometry. The results evidenced that the brain of dogs with VL may present inflammatory infiltrates in leptomeninges, choroid plexus and subependymal area. Between the lymph nodes, there was no difference among the T cell subsets, nevertheless, there was noticed a reduction in the CD8+ lymphocytes and an increase in the DN subset as well as the TCR δγ, while CD4+, DP and TCR αβ were detected in amounts closely related to described reference ranges, which suggests that the DN and TCR δγ lymphocytes may be related to the immune response in symptomatic dogs. Altogether, these findings discard the hypothesis that the regional lymph nodes of the head may present a specific pattern of immune response related to the brain lesions in dogs with VL / Orientador: Gisele Fabrino Machado / Banca: Wagner Luis Ferreira / Banca: Antonio Carlos Alessi / Mestre

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A pandemia Hiv/ Aids já resultou em mais de 34 milhões de pessoas infectadas pelo vírus no mundo até o momento. Causada pelo HIV, de caráter crônico que evolui para um quadro clínico de imunodeficiência (Aids), pode tornar o indivíduo susceptível a infecções oportunistas potencialmente letais. Diferentes fatores foram identificados por ativar o sistema imune, incluindo genótipos do hospedeiro (HLAB-27, HLA-B57, CCR5delta32), co-infecções (GBV-C) e alguns fatores virais como a capacidade de replicação (fitness) e tropismo celular. O HIV-1 possui diversidade genética extensa e dinâmica. Considerando a variabilidade genética dentro do cenário da epidemia no Brasil, as clades do HIV-1 predominantes são B, F e C, além de formas recombinantes. Contudo, ainda não foi completamente estabelecido se essa diversidade genética possa influenciar o curso clínico da doença. O objetivo deste trabalho foi avaliar o perfil de ativação celular induzido encontrado em indivíduos infectados por subtipos virais C e Não- C do HIV-1, durante o primeiro ano de infecção (analisando as fases recente e estabelecida). A análise comparativa dos dois grupos (subtipos C vs. Não-C), identificou no grupo do subtipo-C uma maior frequência de células T CD4+ totais ativadas, como também uma maior frequência e ativação nas subpopulações de células T CD4+ de memória, principalmente memória efetora e efetora terminal, na fase estabelecida. Em relação às células T CD8+, deparamos na fase estabelecida com uma maior frequência de células T CD8+ de memória efetora e ativação das mesmas no grupo do subtipo-C em relação ao grupo do subtipo Não-C. Investigamos também a presença de células T CD4+ que se diferenciaram em células T reguladoras, e foi encontrada uma frequência diminuída dessas células no grupo do subtipo C em relação ao Não- C tanto na fase recente como na fase estabelecida. Na análise comparativa das fases recente e estabelecida, o grupo do subtipo Não-C apresentou um declínio tanto na quantidade de células T CD4+ como na frequência de células T CD8+ ativadas após um ano de infecção. Com base nos resultados encontrados, os dois grupos apresentaram perfis de ativação e diferenciação celular diferentes no primeiro ano de infecção pelo HIV-1, o que aponta para diferentes histórias naturais quando comparamos infecção por clades virais distintas / The Hiv/ Aids pandemic has affected more than 34 million people worldwide, reaching men, women and children. Caused by the HIV virus, a chronic infection that develops into a clinical picture of immunodeficiency (Aids), it can make the individual susceptible to opportunistic infections and result in death. Different factors were identified by activating the immune system, including host genotypes (HLAB-27, HLA-B57, CCR5delta32), co-infections (GBV-C) and some viral factors such as fitness and cellular tropism. The HIV-1 presents an extensive and dynamic genetic diversity, favoring the production of variants with molecular differences. Considering the genetic variability within the scenario of the epidemic in Brazil, the predominant subtypes of HIV-1 are B, F and C. However, it has not yet been completely established if this genetic diversity can impact the clinical course of the disease. The objective of this study was to evaluate the induced cellular activation profile found in HIV-1 C and non-C viral subtypes groups in the first year of infection (analyzing the recent and established phases). The comparative analysis of the two groups (subtypes C vs. Non-C) identified a higher frequency of activated CD4+ T cells in the C-subtype group, as well as a higher frequency and activation in CD4+ T-cell subsets of memory, mainly effector memory and terminal effector on the established phase. About CD8+ T cells, we found in the established phase a higher frequency and activation in the effector memory subset in the C- subtype group compared to the non- C subtype group. We also investigated the presence of CD4+ T cells differentiated into regulators T cells, and a decreased frequency of these cells was found in the subtype C group over non- C in both the recent and established phases. In the recent and established phase comparative analysis evidenced that the non-C subtype group presented a decline in both the number of CD4+ T cells and the CD8+ T-cell activated frequency after 1 year of infection, however, it presented a positive correlation between the viral load and frequency of activated CD4+ and CD8+ T cells in both phases. Based on the results found, the two groups presented different activation and differentiation profiles in the first year of HIV-1 infection, which points to different natural histories when comparing infection with different viral clades

A pandemia Hiv/ Aids já resultou em mais de 34 milhões de pessoas infectadas pelo vírus no mundo até o momento. Causada pelo HIV, de caráter crônico que evolui para um quadro clínico de imunodeficiência (Aids), pode tornar o indivíduo susceptível a infecções oportunistas potencialmente letais. Diferentes fatores foram identificados por ativar o sistema imune, incluindo genótipos do hospedeiro (HLAB-27, HLA-B57, CCR5delta32), co-infecções (GBV-C) e alguns fatores virais como a capacidade de replicação (fitness) e tropismo celular. O HIV-1 possui diversidade genética extensa e dinâmica. Considerando a variabilidade genética dentro do cenário da epidemia no Brasil, as clades do HIV-1 predominantes são B, F e C, além de formas recombinantes. Contudo, ainda não foi completamente estabelecido se essa diversidade genética possa influenciar o curso clínico da doença. O objetivo deste trabalho foi avaliar o perfil de ativação celular induzido encontrado em indivíduos infectados por subtipos virais C e Não- C do HIV-1, durante o primeiro ano de infecção (analisando as fases recente e estabelecida). A análise comparativa dos dois grupos (subtipos C vs. Não-C), identificou no grupo do subtipo-C uma maior frequência de células T CD4+ totais ativadas, como também uma maior frequência e ativação nas subpopulações de células T CD4+ de memória, principalmente memória efetora e efetora terminal, na fase estabelecida. Em relação às células T CD8+, deparamos na fase estabelecida com uma maior frequência de células T CD8+ de memória efetora e ativação das mesmas no grupo do subtipo-C em relação ao grupo do subtipo Não-C. Investigamos também a presença de células T CD4+ que se diferenciaram em células T reguladoras, e foi encontrada uma frequência diminuída dessas células no grupo do subtipo C em relação ao Não- C tanto na fase recente como na fase estabelecida. Na análise comparativa das fases recente e estabelecida, o grupo do subtipo Não-C apresentou um declínio tanto na quantidade de células T CD4+ como na frequência de células T CD8+ ativadas após um ano de infecção. Com base nos resultados encontrados, os dois grupos apresentaram perfis de ativação e diferenciação celular diferentes no primeiro ano de infecção pelo HIV-1, o que aponta para diferentes histórias naturais quando comparamos infecção por clades virais distintas / The Hiv/ Aids pandemic has affected more than 34 million people worldwide, reaching men, women and children. Caused by the HIV virus, a chronic infection that develops into a clinical picture of immunodeficiency (Aids), it can make the individual susceptible to opportunistic infections and result in death. Different factors were identified by activating the immune system, including host genotypes (HLAB-27, HLA-B57, CCR5delta32), co-infections (GBV-C) and some viral factors such as fitness and cellular tropism. The HIV-1 presents an extensive and dynamic genetic diversity, favoring the production of variants with molecular differences. Considering the genetic variability within the scenario of the epidemic in Brazil, the predominant subtypes of HIV-1 are B, F and C. However, it has not yet been completely established if this genetic diversity can impact the clinical course of the disease. The objective of this study was to evaluate the induced cellular activation profile found in HIV-1 C and non-C viral subtypes groups in the first year of infection (analyzing the recent and established phases). The comparative analysis of the two groups (subtypes C vs. Non-C) identified a higher frequency of activated CD4+ T cells in the C-subtype group, as well as a higher frequency and activation in CD4+ T-cell subsets of memory, mainly effector memory and terminal effector on the established phase. About CD8+ T cells, we found in the established phase a higher frequency and activation in the effector memory subset in the C- subtype group compared to the non- C subtype group. We also investigated the presence of CD4+ T cells differentiated into regulators T cells, and a decreased frequency of these cells was found in the subtype C group over non- C in both the recent and established phases. In the recent and established phase comparative analysis evidenced that the non-C subtype group presented a decline in both the number of CD4+ T cells and the CD8+ T-cell activated frequency after 1 year of infection, however, it presented a positive correlation between the viral load and frequency of activated CD4+ and CD8+ T cells in both phases. Based on the results found, the two groups presented different activation and differentiation profiles in the first year of HIV-1 infection, which points to different natural histories when comparing infection with different viral clades

A dengue é uma doença infecciosa aguda causada pelo vírus DEN do gênero Flavivirus e é transmitida pela picada de um mosquito vetor, principalmente o Aedes aegypti. Existem quatro sorotipos do vírus da dengue (DEN-1, DEN-2, DEN-3 e DEN-4) e sua incidência tem aumentado dramaticamente nos últimos 50 anos, inclusive no Brasil. O objetivo deste trabalho é caracterizar subpopulações de linfócitos T, principalmente linfócitos T CD8+, provenientes de pacientes infectados quanto a sua capacidade proliferativa, seu estado de ativação e memória celular. Os pacientes foram recrutados no Hospital Ana Costa de Santos, SP, no ano de 2010, após assinarem o Termo de Consentimento Livre e Esclarecido. O diagnóstico de dengue foi realizado utilizando o teste rápido Dengue Duo e os parâmetros imunológicos foram analisados no citômetro de fluxo. Foi coletado sangue periférico para criopreservação de células mononucleares e separação de soro para detecção da carga viral. Pacientes com dengue apresentaram maior proliferação de linfócitos T CD8+ quando comparados com indivíduos saudáveis. Foi ainda observado que tal proliferação celular foi evidente nos dias cinco e seis de sintomas. Quando marcadores de ativação celular foram analisados por citometria de fluxo, observou-se um aumento de linfócitos T CD8+ expressando CD38 e HLA-DR nos pacientes, quando comparados com indivíduos saudáveis. Da mesma forma, a ativação celular também aumentou com o passar dos dias de sintomas com destaque para o quinto e sexto dia. Este aumento na ativação das células juntamente com os dias de sintomas, foi igualmente observado em várias subpopulações de células T de memória Além disso, foi observada uma correlação negativa entre o número absoluto de linfócitos T CD8+ e a carga viral. Juntos, os resultados desse estudo sugerem que a infecção por DEN leva a um aumento da ativação de células T CD8+ / Dengue is an acute disease caused by DEN, a Flavivirus transmitted by a mosquito vector, primarily Aedes aegypti. There are four serotypes of dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) and their incidences have increased dramatically over the past 50 years, including in Brazil. The goal of this study is to characterize subpopulations of T lymphocytes, mainly CD8+ T cells, from infected patients. Status of cell activation, and memory cell profiles were assessed. Patients were recruited at Hospital Ana Costa, Santos-SP, Brazil, in 2010, after having signed an informed consent form. The serologic diagnosis of dengue was carryied out using a rapid test and immunological parameters were analyzed in flow cytometer. Peripheral blood was collected for cryopreservation of mononuclear cells and separation of serum for viral load testing. Dengue patients showed higher proliferation of CD8+ T cells compared to healthy subjects. Cell proliferation was more evident in the fifth and sixth days of symptoms. We observed increased frequency of CD8+ T cells expressing activation markers CD38 and HLA-DR in patients, when compared to healthy subjects. Similarly, T cell activation also increased along with the passing days of symptoms hitting on the fifth and the sixth days. Such augment in cellular activation along with the days of symptoms was equally observed in the several memory T cell compartments. Furthermore, we observed a negative correlation between the absolute number of CD8+ T lymphocytes and viral load. Together, the results of this study suggest that dengue virus infection leads to an increased activation of CD8+ T cells