O câncer cervical é o segundo maior responsável por mortes atribuídas a câncer em mulheres e dados epidemiológicos tem demonstrado a associação entre a infecção do HPV e o desenvolvimento da neoplasia. Sabe-se que num dado momento da infecção pelo HPV, ocorre integração do genoma viral ao genoma da célula hospedeira e consequente hiperexpressão de dois oncogenes virais, E6 e E7, o que contribui fortemente para a transformação celular. O presente trabalho propõe o uso de vacinas terapêuticas expressando a oncoproteína E7 do HPV-16 geneticamente fusionada à porção amino terminal da flagelina FliCd de Salmonella enterica sv müenchen; e verificação de seu possível papel adjuvante. Vacinas de DNA foram construídas de modo a direcionar as proteínas hibridas ao espaço intracitoplasmático. A verificação da expressão in vitro foi feita utilizando transfecções de culturas celulares, seguida de imunofluorescência e imunodetecção. Em seguida, essas construções vacinais foram administradas em camundongos C57BL6. Ensaios de ELISPOT foram feitos para mensuração o nível de linfócitos T secretores de IFN-g. Os dados de imunofluorescência e imunodetecção demonstraram a correta expressão da proteína. Ensaios de proteção realizados com 5 x 106 células TC-1 administrada por via subcutânea mostraram 80% de proteção em camundongos que haviam recebido previamente 4 doses das vacinas de DNA por biobalística. Ensaios de ELISPOT mostraram em média 9 células do baço secretoras de IFN-g por 106 células do baço (INF-g+/106) responsivas ao peptídeo CD8 / E7 de forma específica. Nossos dados sugerem que a formulação vacinal possui um efeito terapêutico significativo frente ao desafio com as células tumorais TC-1. Em paralelo, foi construída a cepa de salmonela vacinal SL FlaE7 que não mostrou efeito protetor frente ao desafio com células TC-1. / The cervical cancer is the second major responsible for deaths attributed to cancer in women and epidemiologic data have been demonstrating the association between the infection of HPV and the development of this illness. It is known that in a certain moment of the infection with HPV, occurs the integration of the viral genome in the genome of the host cell and consequent over expression of two oncogenes, E6 and E7, it strongly contributes to the transformation of that cell. The present work proposes the use of DNA vaccines expressing the oncoprotein E7 of HPV-16 genetically fused to the portion A-terminal of the flagellin FliCd of Salmonella enterica sv müencheun and verification of your possible performance as adjuvant. The DNA vaccines were constructed in expression vector for eukaryotic to address the hybrid proteins to the intracitoplasmatic space. The verification of the expression in vitro was made using transfections of cellular cultures (COS-7) followed by immunofluorescence and western blot analyses. Soon after, those vaccines were injected in mice C57BL6 that were challenged then with 5^105 tumor cells TC-1 subcutaneous. ELISPOT assays were performed for measure the level of spleen cells secreting interferon g. The immunofluorescence and Western blot data are complementary demonstrating the correct expression of the protein. Protection assays showed 80% of protection in mice that had previously received 4 doses of the DNA vaccines in gene gun form. Assays of ELISPOT show 9 cells of the spleen secreting of IFN-g (average) for 106 cells of the spleen (INF-g /106). Our data suggest that the formulation of vaccines possesses a significant therapeutic effect front to the challenge with the tumor cells TC-1 accompanist splenocyte IFN-g secretory.

CD8 T cells play a critical role in the clearance of pathogenic bacteria, viruses, and protozoan parasites. Upon encountering their cognate antigen through either infection or vaccination, naïve CD8 T cells undergo robust proliferative expansion, which is followed by contraction and the formation of a memory population. Memory CD8 T cells are long-lived, and because they persist in increased numbers and possess enhanced functional abilities compared to naïve CD8 T cells, they are able to provide the host with increased protection following re-infection. Because of these properties, vaccines designed to elicit memory CD8 T cells have the potential to reduce health care burdens related to infection with pathogens including human immuno deficiency virus (HIV), malaria, influenza, and hepatitis virus. However, stimulating protective CD8 T cell responses against these pathogens through vaccination has proven challenging. Therefore, a better understanding of the properties of memory CD8 T cells generated following vaccination, and the characteristics of memory CD8 T cells best suited for providing protection against diverse pathogens is needed. While memory CD8 T cells can be maintained for as long as the life of the host, evidence suggests that their properties change with time after infection. Because CD8 T cell-mediated protection is based upon both the numbers and quality or functional abilities of memory cells present at the time of re-infection, changes in memory CD8 T cell function over time could impact their ability to provide protection upon re-infection. Therefore, a better understanding of how memory CD8 T cells change with time after infection is needed. As part of the studies presented in this thesis, I found that the phenotype and function of memory CD8 T cells including localization, interleukin (IL)-2 cytokine production, responsiveness to homeostatic cytokines, metabolic capabilities, and proliferation and secondary memory generation potential change with time after infection. Interestingly functional changes could not be completely explained by changes in subset composition that occur with time, as changes over time were also seen in defined CD62Lhi subsets. Importantly, functional changes of memory CD8 T cells that occurred with time led to an increased ability to provide protection against a chronic viral infection. These data improve our knowledge of the capabilities of memory CD8 T cells generated following infection, and suggests that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations will depend upon the timing between antigen encounters. Following re-infection, memory CD8 T cells become activated and produce effector cytokines and cytolytic molecules that aid the host in clearing invading microbes. Activation can be triggered not only through cognate antigen recognition, but also by antigen-independent cytokine driven signals. However, our knowledge of how antigen-dependent and –independent signals contribute to CD8 T cell activation and protection following infection is incomplete. In the second part of my thesis, I show that the ability of memory CD8 T cells to become activated in response to inflammation decreases with time after infection, that antigen and inflammation act synergistically to induce activation of memory CD8 T cells, that the presence of cognate antigen enhances activation of memory CD8 T cells that contribute to clearance of infection, and that bystander memory CD8 T cell responses following unrelated bacterial infection do not provide the host with a protective benefit. Together, the data in this thesis further our understanding of memory CD8 T cells generated following infection and/or vaccination, and the properties of memory CD8 T cells important for providing protection upon re-infection with invading pathogens.

During hematopoiesis, multilineage progenitor cells and the precursors are committed to individual hematopoietic lineages. In normal myelopoiesis, the immature myeloid cells (IMCs) differentiate into macrophages, neutrophils or dendritic cells. However, under tumor burden, these IMCs differentiate into myeloid derived suppressor cells (MDSCs) result in an up-regulation of immune suppressive factors and pro-tumor effect. The development of normal or malignant is tightly controlled by endogenous signals such as transcription factors and epigenetic regulations. HDAC11 is the newest identified members of the histone deacetylase (HDAC) family. Previous study in our group had identified HDAC11 as a negative regulator of interleukin 10 (IL-10) production in antigen-presenting cells (APCs). However, the mechanisms of HDAC11 in regulating myeloid cells differentiation and function remained unclear. We have uncovered for the first time that in the absence of HDAC11, upon LPS stimulation, neutrophils isolated form mice displays an over-production of pro-inflammatory cytokines such as TNF-alpha and IL-6. Strikingly, these HDAC11KO neutrophils showed a significantly higher migratory and phagocytosis activity, resulting from an overexpression of the migratory receptor and cytokine CXCR/L2. We have performed Chromatin Immunoprecipitation (ChIP) analysis on the neutrophils and discovered that HDAC11 was recruited to the promoter regulatory region of these genes we have identified. This part of data will be discussed mainly in chapter 2. Not only does HDAC11 plays a crucial role in the neutrophil function, our group have also found out that lacking of HDAC11 result in an increased suppressive activity of the Myeloid-derived Suppressor Cells (MDSCs). The previous publication of our group had shown that the tumor bearing mice experienced a much more aggressive growth pattern in the HDAC11 KO mice compare with C57BL/6 wild type control. MDSCs isolated from mice lacking HDAC11 appeared to gain increased capability to suppress the function of antigen-specific CD8+ T cells in vitro. Followed by this initial study, in chapter 3, we observed an up-regulation of both expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function. The aberrant enzymatic activity of Arg1 and Nos2 in HDAC11KO MDSCs is possibly result from an over-expression of the lineage-specific transcription factor C/EBPβ, which is previously proved to be essential for the differentiation of functional MDSCs. Furthermore, our ChIP data confirmed that HDAC11 may play as an negative regulator of C/EBPβ. Recently, our lab had demonstrated that T cells lacking HDAC11 gained a hyperactive phenotype and anti-tumor effect, indicating that HDAC11 may play a dual role in the host immune system. We further performed an adoptive transfer therapy to C57BL/6 tumor bearing mice. Our data showed that the additional administration of HDAC11KO MDSCs could eliminate, at least partially, the anti-tumor effect by adoptive transfer of HDAC11KO T cells. Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator in the myeloid cells differentiation and function. Based on our data and previous work from our lab, we propose a dual role of HDAC11 played in the host immune system. In the absence of HDAC11, host defenders such as neutrophils and T cells are functionally more aggressive against intruders such as pathogen and cancer. However, the immune suppressors such as MDSCs became more suppressive. The contradictory role HDAC11 played in the immune system may provide some insights for the assessment of the pharmacological value of HDAC11 and contribute to the development of novel immunotherapeutic strategies.

<p>Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is crucial in linking TCR engagement to downstream signaling events, such as calcium flux and Ras-MAPK pathway. Following TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediates the binding of Grb2/Sos, PLC-&#61543;1, and GADS/SLP-76 complexes. This multi-protein signaling complex initiates signaling cascades eventually leading to the activation of transcription factors that regulate the genes required for T cell proliferation and effector functions. The indispensable role of LAT in thymocyte development has been evidenced as LAT-deficient mice completely lack peripheral T cells. To study the function of LAT in mature T cells, our lab previously generated a conditional knock-in mouse line in which the lat gene can be deleted by Cre recombinase. Deletion of LAT in mature T cells revealed the critical role of LAT in T cell activation. Here, we used this inducible LAT deletion mouse line crossed with the OT-I transgenic mice to study the role of LAT in mature CD8 T cells. </p><p>To analyze the contribution of LAT in CD8 T cells during the course of pathogen infection, we infected mice with Listeria monocytogenes-expressing Ova to elicit activation of antigen-specific CD8 T cells, and then inducibly deleted LAT in these cells at different stages of infection under the control of tamoxifen treatment. We show that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction. In addition, memory CD8 T cell can persist in the absence of LAT, suggesting that LAT-signaling is not necessary for memory maintenance. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1lowIL-7RhighCD62Lhigh memory T cells. Together, these data demonstrate that, while it is dispensable for contraction and memory maintenance, LAT-signaling regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.</p><p>The fundamental activity of CD8 T cells is to elicit cytotoxicity toward target cells that express foreign antigens, and this is mediated through granule-dependent and Fas ligand-dependent mechanisms. The signaling events that regulate these processes remain unclear. We showed that LAT-deficient cytotoxic T cells (CTLs) failed to upregulate FasL and produce IFN-&#61543; after engagement with target cells. Moreover, they displayed reduced granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT-deficiency led to altered synapse formation, subsequently causing unstable T cell:APC conjugates. MTOC polarization and granule reorientation were also impaired by LAT-deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and PLC activity. This revealed an unexpected finding that CTL function is not entirely dependent on LAT. Collectively, these data indicate that the signaling circuits governing CTLs are programmed to adopt multiple pathways, allowing CTLs to effectively eliminate various pathogens during adaptive immune responses.</p><p>&#8195;</p> / Dissertation

There is an urgent need for the development of an effective therapeutic vaccine against cancer caused by human papilloma virus (HPV). We focused on HPV-induced malignancies because of their high worldwide prevalence (e.g., cervical carcinoma and head & neck cancer). A successful therapeutic vaccine could prevent the 250 000 deaths/year worldwide and the 2.25 billion dollars that are expended in related care in the US. We used an HPV-induced mouse cancer model to test vaccines composed of a CD8 T cell peptide epitope administered with potent adjuvants designed to generate vast numbers of high avidity cytotoxic T lymphocytes specific for the HPV16-E7 antigen. One vaccination strategy (TriVax) consists of intravenous administration of synthetic peptide HPV16-E749-57 administered together with a Poly-IC (a TLR3 agonist) and anti-CD40 monoclonal antibody(αCD40 mAb) while the second more simple strategy (BiVax) comprises solely of peptide plus Poly-IC. We used an E7 peptide as antigen in the vaccination strategies because expression of the viral E6 and E7 proteins is required to maintain oncogenic phenotype and because normal cells do not express E6/E7, therefore a therapeutic vaccine targeting these proteins has several advantages: a) a strong immune response can be induced since immune tolerance to these foreign antigens does not exist and b) the strong immune response should not inflict damage to normal cells. TriVax and BiVax generate a high number of antigen specific CD8 T cells capable clear subcutaneous tumors and prevent recurrences, both vaccines are efficient through the i.v. and i.m. route. TriVax (prime-boost) clears tumor in 100% of mice while BiVax clears tumor in 50% of mice, this differential effect is due to the number of antigen specific CD8 T cells and increasing the number of booster shot makes BiVax as immunogenic and efficient in clearing tumors. In the absence of type-I IFN signaling (in IFNαΒR KO mice), TriVax is less effective in generating sufficient numbers of CD8 T cells that could be necessary for total disease eradication. We observed a significant anti-tumor effect of TriVax in the absence of interferon gamma, however the cytokine may play some role in the overall effectiveness of TriVax to completely reject the tumors. Immune responses produced by BiVax are highly dependent on the simultaneous administration of peptide and Poly-IC, on the peptide composition, vaccine formulation and route of administration. The magnitude of the response is dependent on the expression of the Poly-IC receptors TLR3 and melanoma differentiation-associated protein 5 (MDA5). Interestingly, the magnitude and duration of the CD8 T cell responses generated by peptide and Poly-IC mixtures does not rely on the presence of CD4 T cells, scavenger receptor-A (SR-A) or type-I IFN signals and was minimally affected by the absence of CD40 signaling. The present findings could facilitate the development of simple and effective subunit vaccines for diseases where CD8 T cells may hold a therapeutic benefit.

CD8+ T cells responses are critical in the immune defense against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection. A major challenge for vaccine development is that HIV/SIV can rapidly mutate to escape containment by the CD8+ T cell response. Therefore, optimal virus control by a vaccine will likely require clonally diverse CD8+ T cells capable of recognizing mutant viruses. Mucosal tissues play a fundamental role in early HIV/SIV pathogenesis by serving as the site for viral entry, CD4+ T cell depletion, and a reservoir for viral replication. Vaccine strategies that induce effective mucosal immunity will likely be critical for protection against HIV/SIV. We examined the SIV Gag p11C-specific CD8+ T cell responses in peripheral blood, gastrointestinal (GI) mucosal and lung mucosal tissues of rhesus monkeys expressing the MHC class I molecule Mamu-A*01. We first investigated the clonal composition of this cell population during the acute and chronic phases of SIVmac infection. We showed that there is a narrowing of the clonal repertoire from acute to chronic infection and the same clonal populations of virus-specific CD8+ T cells are present in the systemic and mucosal compartments of chronically SIV-infected animals. These data indicated that virus-specific CD8+ T cells establish broadly distributed immune responses. Next, we examined the clonal diversity of systemic and mucosal p11C-specific CD8+ T cells induced by prime-boost vaccination. We found that systemic prime-boost vaccination induced clonally diverse p11C-specific populations in mucosal tissues. There were high levels of clonal sharing between systemic and mucosal compartments soon after vaccination. However, later following vaccination there was decreased clonal sharing between the GI mucosa and the systemic circulation. We showed that this was due to limited trafficking of p11C-specific CD8+ T cells to the GI mucosa following vaccination. Overall, these studies indicate that following SIV infection and systemic vaccination the same p11C-specific clones are present in mucosal and systemic compartments. Moreover, the apparent immune compartmentalization is a consequence of differences in cell trafficking between systemic and mucosal CD8+ T cells. These observations have important implications for the design of HIV vaccines that generate effective mucosal immunity.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Irregular screening to identify infected individuals and a lack of sterilizing immunity to C. trachomatis has led to a dramatic increase in the number of reported C. trachomatis infections over the last twenty years. Repeated infections with C. trachomatis lead to serious sequelae such as pelvic inflammatory disease and ectopic pregnancy, which can result in infertility.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

O câncer cervical é o segundo maior responsável por mortes atribuídas a câncer em mulheres e dados epidemiológicos tem demonstrado a associação entre a infecção do HPV e o desenvolvimento da neoplasia. Sabe-se que num dado momento da infecção pelo HPV, ocorre integração do genoma viral ao genoma da célula hospedeira e consequente hiperexpressão de dois oncogenes virais, E6 e E7, o que contribui fortemente para a transformação celular. O presente trabalho propõe o uso de vacinas terapêuticas expressando a oncoproteína E7 do HPV-16 geneticamente fusionada à porção amino terminal da flagelina FliCd de Salmonella enterica sv müenchen; e verificação de seu possível papel adjuvante. Vacinas de DNA foram construídas de modo a direcionar as proteínas hibridas ao espaço intracitoplasmático. A verificação da expressão in vitro foi feita utilizando transfecções de culturas celulares, seguida de imunofluorescência e imunodetecção. Em seguida, essas construções vacinais foram administradas em camundongos C57BL6. Ensaios de ELISPOT foram feitos para mensuração o nível de linfócitos T secretores de IFN-g. Os dados de imunofluorescência e imunodetecção demonstraram a correta expressão da proteína. Ensaios de proteção realizados com 5 x 106 células TC-1 administrada por via subcutânea mostraram 80% de proteção em camundongos que haviam recebido previamente 4 doses das vacinas de DNA por biobalística. Ensaios de ELISPOT mostraram em média 9 células do baço secretoras de IFN-g por 106 células do baço (INF-g+/106) responsivas ao peptídeo CD8 / E7 de forma específica. Nossos dados sugerem que a formulação vacinal possui um efeito terapêutico significativo frente ao desafio com as células tumorais TC-1. Em paralelo, foi construída a cepa de salmonela vacinal SL FlaE7 que não mostrou efeito protetor frente ao desafio com células TC-1. / The cervical cancer is the second major responsible for deaths attributed to cancer in women and epidemiologic data have been demonstrating the association between the infection of HPV and the development of this illness. It is known that in a certain moment of the infection with HPV, occurs the integration of the viral genome in the genome of the host cell and consequent over expression of two oncogenes, E6 and E7, it strongly contributes to the transformation of that cell. The present work proposes the use of DNA vaccines expressing the oncoprotein E7 of HPV-16 genetically fused to the portion A-terminal of the flagellin FliCd of Salmonella enterica sv müencheun and verification of your possible performance as adjuvant. The DNA vaccines were constructed in expression vector for eukaryotic to address the hybrid proteins to the intracitoplasmatic space. The verification of the expression in vitro was made using transfections of cellular cultures (COS-7) followed by immunofluorescence and western blot analyses. Soon after, those vaccines were injected in mice C57BL6 that were challenged then with 5^105 tumor cells TC-1 subcutaneous. ELISPOT assays were performed for measure the level of spleen cells secreting interferon g. The immunofluorescence and Western blot data are complementary demonstrating the correct expression of the protein. Protection assays showed 80% of protection in mice that had previously received 4 doses of the DNA vaccines in gene gun form. Assays of ELISPOT show 9 cells of the spleen secreting of IFN-g (average) for 106 cells of the spleen (INF-g /106). Our data suggest that the formulation of vaccines possesses a significant therapeutic effect front to the challenge with the tumor cells TC-1 accompanist splenocyte IFN-g secretory.