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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
71

The Role of γ<sub>с</sub> Cytokines in T Cell Development, T Cell Homeostasis and CD8+ T Cell Function: A Dissertation

Gozalo, Sara 24 May 2004 (has links)
T lymphocytes are essential components of the immune system and as such are continually regulated by a variety of factors. Every step of their development, survival and function is tightly monitored to ensure their ability to recognize most foreign agents and mount adaptive immune responses during pathogenic infections, while remaining tolerant to self-antigens. Among the many factors that participate in the regulation of T cell development and function are the cytokines. Cytokines that signal through the common gamma (γс) chain and the Janus kinase 3 (Jak3) include IL-2, -4, -7, -9, -15, and -21 and have been implicated in the regulation of every stage in the life of a T cell. Therefore, it is not surprising that mutations in the γс chain or Jak3 lead to a SCID condition in humans and mice. Specifically, Jak3-deficient mice are characterized by a reduction in thymic cellularity and dysregulated T cell homeostasis. They have an expansion of memory-like CD4+ mature T cells and an almost complete absence of mature CD8+ T cells. By investigating the TCR repertoire of CD4+ T cells in the thymus and spleen of Jak3-/- mice, I deduced that the CD4+ T cell activation and expansion is TCR-specific and takes place in the periphery of the mice. After crossing Jak3-deficient mice to Bcl-2 transgenic mice I showed that the developmental block observed in Jak3-/- mice could not be rescued by the anti-apoptotic factor, despite the fact that its expression did increase, slightly, the total numbers of developing thymocytes. The enforced expression of Bcl-2 was also not sufficient to revert the dysregulation of T cell homeostasis in Jak3-/- mice. Finally, in order to further understand the role played by γс cytokines during T cell function, I investigated the ability of mature Jak3-/- CD8+ T cells to become activated and differentiate into effector cells in response to a viral infection. My results indicate that CD8+ T cells are activated and proliferate in response to a viral infection, but their survival, as well as their ability to proliferate and differentiate into effector cells are greatly impaired, resulting in the inability of Jak3-deficient mice to mount a protective response.
72

Molecular Dissection of the Cellular Reponse to Dengue Virus Infection

Warke, Rajas V. 14 April 2008 (has links)
The immune response to viral infection involves a complexity of both innate and adaptive pathways at the cellular and the molecular level. There are many approaches to begin to define the pathways at work to control viral pathogenesis. The approach favored in this thesis was to conduct a broad screen of the innate immune response at the gene expression level of infected cells. The innate immune response is critical to the control of viral infections. Type I interferons (IFN), IFNα and IFNβ, are antiviral proteins that are an integral part of the innate immune response. Furthermore, by virtue of their effects on maturation and activation of antigen-presenting cells, IFNs are a pivotal link between the innate and adaptive immune systems. Most cell types produce type-I IFN when exposed to viruses. However, viruses have evolved multiple strategies to suppress IFN production or signaling. It is imperative to understand the virus-host interaction at the molecular level in order to identify as yet unknown mechanisms of the host antiviral response; these additional pathways may be useful in counteracting the viral suppression of IFN. Type-I IFNs regulate expression of at least five hundred genes, suggesting a complex network of signaling pathways. Depending on the cell type different proteins regulate the induction of IFN or the expression of IFN-inducible genes. Identification of proteins that induce selected IFN-inducible genes may provide synergistic activity with or may have an advantage over type-I IFN for anti-viral therapy in the future. Many diseases are untreatable if identified late in their progression. In resource-limited countries, many diseases are diagnosed clinically, which can lead to incorrect or delayed diagnosis and treatment. The identification of biomarkers of disease has the potential to guide the correct therapy in a timely fashion. The objective of this thesis was to identify novel anti-viral therapies and disease biomarkers for dengue virus (DENV) infection. DENV is a mosquito-borne positive-sense single-stranded RNA virus, which causes an estimated 50 million infections annually. Most DENV infections result in a febrile illness called Dengue fever (DF). Less frequently, infections cause Dengue hemorrhagic fever (DHF), a potentially fatal vascular leakage syndrome associated with the production of pro-inflammatory cytokines. At present patients infected with DENV can only be treated by intravenous fluid support to prevent hypovolemia and hypotensive shock. This treatment is less effective in severe cases if the diagnosis is delayed. Identification of therapeutics with both antiviral and immune-modulatory activity may lower patient mortality and reduce the burden of DENV on society. DENV infection is cleared in most individuals after a short period of viremia {Libraty, 2002 #2225}. Based on in vitro and mouse models, type-I and type-II IFN signaling pathways are thought to be critical in the regulation of DENV infection. Higher serum levels of type I and type II IFNs during acute DENV infection in patients lend support to the above hypothesis {Kurane, 1993 #2152; Libraty, 2002 #2225}. To understand the DENV-human host cell interaction at the molecular level, we performed global gene expression analysis on DENV-infected primary human cells using Affymetrix GeneChips (HG-U133A). We studied dendritic cells (DC), monocytes, B cells and human umbilical vein endothelial cells (HUVECs), all of which are known to be permissive to DENV infection. We first identified genes commonly regulated in multiple cell types in response to DENV infection; we hypothesized that understanding this common gene expression profile would identify signaling pathways involved in regulation of viral spread, activation of immune cells or induction of inflammation. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the 23 common response genes, was identified as a key link between type I and type II interferon response genes. Pretreatment of cells with recombinant TRAIL (rTRAIL) inhibited DENV replication in monocytes, B cells, HUVECs and DCs. Using the DC infection model, we showed that this inhibition of viral replication was apoptosis-independent. Type-I IFN receptor (IFNR) blocking experiments showed that signaling through the type-I IFN receptor played an important role in the antiviral activity of exogenous rTRAIL. Furthermore, TRAIL also significantly reduced the expression of mRNA and protein of pro-inflammatory cytokines (TNFα, MIP-1β and IFNα) and chemokines (MCP-2, IP-10 and IL-6) in response to DENV infection. The data that TRAIL inhibits both viral replication and pro-inflammatory cytokine production suggest that TRAIL has therapeutic value in dengue. The endothelial cell is the site of pathology in DENV infection in vivo (vascular permeability and plasma leakage). To understand the direct effect of DENV infection on endothelial cells and its role in the induction of genes regulating vascular permeability, we compared gene expression in DENV-infected HUVECs to that of uninfected cells and cells infected with other RNA and DNA viruses, including flaviviruses (West Nile, yellow fever, and Japanese encephalitis viruses), bunyaviruses (Sin Nombre and Hantaan viruses), Epstein-Barr virus and vaccinia virus. Among the genes confirmed for their differential expression, ST2 (Interkeukin-1 receptor-like-1 protein-IL1RL1) and indoleamine 2,3-dioxygenase (IDO) were identified to be upregulated specifically in response to DENV infection. Higher serum soluble ST2 (sST2) levels were detected in DENV-infected patients than in patients with other febrile illnesses (OFI) at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). In addition, patients with secondary DENV infections had higher serum sST2 levels compared with patients with primary DENV infections (p=0.047 at the last day of fever and p=0.030 at defervescence). Higher levels of IDO activity (pIn conclusion, global gene expression analysis identified novel proteins with promising characteristics for the treatment and/or diagnosis of DENV infection. Although further studies will be needed to validate the clinical utility of TRAIL, sST2, and IDO, these studies demonstrate the utility of this unbiased genomics approach to identify therapies to currently incurable diseases.
73

Critical Molecular Pathways in Cancer Stem Cells of Chronic Myeloid Leukemia: A Dissertation

Chen, Yaoyu 11 May 2011 (has links)
Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.
74

Primary and Secondary Immune Responses During Sequential West Nile Virus and Japanese Encephalitis Virus Infections: A Dissertation

Trobaugh, Derek W. 14 February 2012 (has links)
Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.
75

CD8+ T Cell Serotype-Cross-Reactivity is a Predominant Feature of Dengue Virus Infections in Humans: A Dissertation

Friberg-Robertson, Heather L. 30 November 2010 (has links)
The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.
76

Regulation of Humoral Immunity by Pim Kinases: A Dissertation

Willems, Kristen N. 16 June 2011 (has links)
Pim (Provirus Integration site for Moloney murine leukemia virus) kinases are a family of three serine/threonine kinases involved in cell cycle, survival and metabolism. These kinases were first identified in malignant cells and are most often associated with their role in cancer. Their role in immunity and lymphocytes is less well known. To date, it has been shown that Pim 1 and/or Pim 2 are important for T lymphocyte survival and activation when the Akt signaling pathway is inhibited by rapamycin. In addition, our laboratory has shown that Pim 2 is critical for BLyS-mediated naive B lymphocyte survival in the presence of rapamycin. This thesis extends the role(s) for Pim 1 and/or 2 to include functions during B cell activation and the generation of immune responses. We found that during in vitro activation of purified resting splenic B cells from wild type mice with a variety of activators that use multiple signaling pathways, including the BCR, TLR and CD40 receptors, both Pim 1 and 2 kinases were induced by 48 hours post-activation, suggesting that they could play a role in B cell activation and differentiation to antibody secreting or memory B cells. Immunization of Pim 1-/-2-/- knockout mice with T cell dependent antigens showed impairment in antibody and antibody secreting cell generation as well as lack of germinal center formation clearly demonstrating an involvement of Pim 1 and/or 2 in the immune response. FACS examination of B cell populations from naive Pim 1-/-2-/- knockout mice revealed normal levels of splenic marginal zone and follicular B cells and T cells, however, decreased numbers of all peritoneal B cell populations and decreased B cells in Peyer's Patches was seen. An examination of serum antibody found in naive Pim 1-/-2-/- knockout mice showed decreased levels of natural antibody, which is likely due to loss of the peritoneal B1 cells but does not explain the significantly decreased TD immune response. To determine whether the defect was B cell intrinsic or a more complex interaction between B and T cells, we determined whether Pim 1-/-2-/- mice would respond to T cell independent, TI-1 and TI-2, antigens. Antibody production and antibody secreting cell formation were also significantly decreased in these mice supporting our notion of a B cell intrinsic defect. To further examine the B cell response problem, we attempted to establish chimeric mice using either bone marrow derived cells or fetal liver cells from WT or Pim 1-/-2-/- donors so that the B cells were derived from Pim 1-/-2-/- mice and the T cells would be WT. Unfortunately, we were not able to consistently engraft and develop mature Pim 1-/-2-/- B cells, which indicate that there is a stem cell defect in these knockout mice that requires further investigation. Because one of the major failures in activated Pim 1-/-2-/- B cells is the generation of antibody secreting cells, an analysis of the expression of transcription factors IRF-4 and BLIMP-1, known to play a role in this process was carried out. Although IRF-4 induction was not affected by the loss of Pim 1 and 2, the number of cells able to increase BLIMP-1 expression was significantly decreased, revealing a partial block in the generation of ASCs. Taken together the data presented in this thesis reveals a new and critical role for Pim 1 and 2 kinases in the humoral immune response.
77

Regulation of Early T Cell Activation by TNF Superfamily Members TNF and FASL: A Dissertation

Priyadharshini, Bhavana 08 September 2010 (has links)
The instructive signals received by T cells during the programming stages of activation will determine the fate of effector and memory populations generated during an immune response. Members of the tumor necrosis factor (TNF) superfamily play an essential role in influencing numerous aspects of T cell adaptive immune responses including cell activation, differentiation, proliferation, survival, and apoptosis. My thesis dissertation describes the involvement of two such members of the TNF superfamily, TNF and FasL, and their influence on the fate of T cells early during responses to viral infections and to the induction of transplantation tolerance. TNF is a pleiotropic pro-inflammatory cytokine that has an immunoregulatory role in limiting the magnitude of T cell responses during a viral infection. Our laboratory discovered that one hallmark of naïve T cells in secondary lymphoid organs is their unique ability to rapidly produce TNF after activation and prior to acquiring other effector functions. I hypothesized that T cell-derived TNF will limit the magnitude of T cell responses. The co-adoptive transfer of wild type (WT) P14 and TNF-deficient P14 TCR transgenic CD8+ T cells, that recognize the GP33 peptide of lymphocytic choriomeningitis virus (LCMV), into either WT or TNF-deficient hosts demonstrated that the donor TNF-deficient P14 TCR transgenic CD8+ T cells accumulate to higher frequencies after LCMV infection. Moreover, these co-adoptive transfer experiments suggested that the effect of T cell-derived TNF is localized in the microenvironment, since the TNF produced by WT P14 TCR transgenic CD8+ T cells did not prevent the accumulation of TNF-deficient P14 TCR transgenic CD8+ T cells. To determine if T cell-produced TNF is acting on professional APC to suppress the generation of virus-specific T cell responses, I performed co-adoptive transfer experiments with WT P14 TCR transgenic CD8+ and TNF-deficient P14 TCR transgenic CD8+ T cells into TNFR1/2 (1 and 2) deficient mice. These experiments demonstrated that the absence of TNFR1/2 signaling pathway in the host cells resulted in a greater accumulation of WT P14 TCR transgenic CD8+ T cells, thereby considerably diminishing the differences between donor WT P14 TCR transgenic CD8+ and donor TNF-deficient P14 TCR transgenic CD8+ T cells. The increased frequency and absolute numbers of WT P14 TCR transgenic CD8+ T cells in TNFR1/R2 deficient recipients suggests that one mechanism for the suppressive effect of T cell-derived TNF on antigen-specific T cells occurs as a result of TNFR signaling in the host cells. However, the donor TNF-deficient P14 TCR transgenic CD8+T cells still accumulated to higher frequency and numbers compared to their donor WT transgenic counterparts. Together, these findings indicate that T cell-produced TNF can function both in an autocrine and a paracrine fashion to limit the magnitude of anti-viral T cell responses. Given the immunoregulatory role of TNF and the ability of peripheral naïve T cells to produce this cytokine, I questioned at what stage of development do T cells become licensed to produce this cytokine. The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. I hypothesized that naïve T cells emigrating from the thymus will be competent to produce TNF only after undergoing a maturation process in the periphery. To test this hypothesis, I compared cytokine profiles of CD4+ and CD8+single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics with respect to the upregulation of CD25 and CD69 following stimulation. The reduced ability of SP thymocytes to produce TNF correlated with a decreased level of detectable TNF message following stimulation when compared to splenic counterparts. Stimulation of SP thymocytes in the context of a splenic environment did not fully enable TNF production, suggesting an intrinsic defect in their ability to produce TNF as opposed to a defect in antigen presentation. Using a thymocyte adoptive transfer model, I demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs identified by the expression of green fluorescent protein (GFP) (NG-BAC transgenic mice), showed a significantly enhanced ability to express TNF relative to SP thymocytes, but not to the extent of MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs. This highlights the functional heterogeneity of the naïve T cell pool (with respect to varying degrees of TNF production) during early T cell activation that can contribute to the many subsequent events that shape the course of an immune response. The productive activation of naïve T cells requires at least initial two signals; the first being through the TCR and the second is the engagement of co-stimulatory molecules on the surface of the T cells. T cells activated in the absence of co-stimulation become anergic or undergo cell death. Agents that block co-stimulation of antigen-specific T cells are emerging as an alternative to immunosuppressive drugs to prolong allograft survival in transplant recipients. Targeted blockade of CD154-CD40 interactions using a αCD154 monoclonal antibody (MR1) with a simultaneous transfusion of allogeneic splenocytes (donor specific transfusion or DST) efficiently induces tolerance to allografts. This co-stimulation blockade-induced tolerance is characterized by the deletion of host alloreactive T cells within 24 hours of treatment. Toll-like receptor (TLR) agonists abrogate tolerance induced by co-stimulation blockade by impairing the deletion of host alloreactive T cells and resulting in allograft rejection. The goal of my study was to determine the underlying molecular mechanisms that protect host alloreactive T cells from early deletion after exposure to TLR agonists. I hypothesized that TLR ligands administered during co-stimulation blockade regimen differentially regulate the expression of pro- and anti-apoptotic molecules in alloreactive T cells, during the initial stages of activation thereby preventing deletion. To test this hypothesis, I used syngeneic bone marrow chimeric mice containing a trace population of alloreactive KB5 TCR transgenic CD8+ T cells (KB5 Tg CD8+ T cells) that recognize H-2Kb as an alloantigen. I show here that KB5-CD8+ T cells downregulate CD127 (IL-7R!) and become apoptotic as early as 12 hrs after co-stimulation blockade. In contrast, KB5 Tg CD8+ T cells from mice treated with bacterial lipopolysaccaride (LPS) during co-stimulation blockade failed to become apoptotic, although CD127 was downregulated. Examination of the mRNA expression profiles of several apoptotic genes in purified KB5 CD8+ T cells from mice treated with DST+anti-CD154 for 12 hrs revealed a significant upregulation of FasL mRNA expression compared to the untreated counterparts. However, in vitro FasL blockade or in vivo cytotoxicity experiments with mice deficient in Fas or FasL indicated that the Fas-FasL pathway might not be crucial for tolerance induction. Another pro-apoptotic molecule BIM was upregulated in alloreactive T cells during co-stimulation blockade. This suggests that both the Fas pathway and BIM may be playing complementary roles in inducing deletional tolerance. Although FasL expression was diminished in alloreactive T cells in the presence of LPS, BIM expression was not diminished, suggesting that alloreactive T cells may still be vulnerable to undergo apoptosis. Concomitantly, I also found that LPS treatment during co-stimulation blockade resulted in non-specific upregulation of Fas expression in alloreactive T cells and non-transgenic T cells (CD4+ and CD8+). I demonstrate here that treatment with Fas agonistic antibody in vitrofor 4 hours can selectively induce apoptosis of alloreactive T cells that were believed to be refractory to apoptosis during LPS treatment. I speculate that under these conditions, deletion may be occurring due to the involvement of both Fas and BIM. Further, the mRNA expression profile revealed interleukin-10 (IL-10) as a molecule induced in alloreactive T cells during LPS treatment. Analysis of serum confirmed the systemic expression of IL-10 protein in mice treated with LPS during co-stimulation blockade. I hypothesized that LPS-induced IL-10 can have an anti-apoptotic role in preventing the deletion of alloreactive T cells and mediating allograft rejection. Contrary to my hypothesis, I found that IL-10 KO mice rejected allogeneic target cells similar to their WT counterparts, suggesting that IL-10 may not be required for LPS-mediated abrogation of tolerance induction. In addition to the systemic induction of IL-10, LPS also induced cytokines such as interleukin-6 (IL-6), TNF and interferon-γ (IFN-γ). These findings suggest that both Fas-FasL and BIM mediated apoptotic pathways may play complementary roles in inducing the early deletion of activated alloreactive T cells during tolerance induction. On the other hand, the mechanism of LPS mediated abrogation of tolerance induction can not be attributed to IL-10 alone as it may be playing a synergistic role along with other proinflammatory cytokines that may in turn result in the prevention of alloreactive T cell death during this process. Most importantly, these findings indicate that despite emerging from a pro-inflammatory cytokine milieu, alloreactive T cells are still susceptible to undergo Fas-mediated apoptosis during the first 24 hours after co-stimulation blockade and LPS treatment. Therefore, targeting the Fas-FasL pathway to induce deletion of alloreactive T cells during the peri-transplant period may still be a potential strategy to improve the efficacy of co-stimulation blockade induced transplantation tolerance during an environmental perturbation such as inflammation or infection.
78

Molecular Studies of T Cell Recognition and Cross-Reactivity: A Dissertation

Shen, Zu T. 27 July 2012 (has links)
Intracellular pathogens are recognized by a specialized subset of lymphocytes known as CD8+ T cells. Pathogen recognition by CD8+ T cells occurs through binding of T cell receptors (TCR) to processed antigens in complex with major histocompatibility complex (MHC) class I proteins. TCR engagement of antigens in complex with MHC class I typically lead to cytotoxic CD8+ T cell responses, which result in pathogen clearance. Due to the large number of foreign antigens that might be encountered by any given host a diverse repertoire of TCRs must be available for immune recognition. The main source of TCR diversity is generated by somatic recombination of the TCR genes. However, it has been suggested that selection eliminates so many recombined TCR sequences, that a high degree of TCR cross-reactivity must occur for the immune system to be able to recognize a large set of foreign pathogens. The work presented in this thesis was directed towards the understanding of the molecular mechanisms of CD8+ T cell recognition and cross-reactivity. Chapter I of this thesis gives an overview of the immune system, with a focus on CD8+ T cells. Chapter II of this thesis describes the development of novel bi-specific MHC heterodimers that are specific towards cross-reactive CD8+ T cells. Classically, MHC tetramers have been used for phenotypic characterization of antigen-specific T cells. However, identification of cross-reactive T cells requires the simultaneous use of two MHC tetramers, which was found to result in MHC tetramer cross-competition. For this reason, we generated bi-specific MHC heterodimers, which would not be affected by the affinity between the component peptide-MHC complexes for TCR. We generated T cell lines, which cross-react with antigens from lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VV), to test our bi-specific MHC heterodimers. We show that the heterobifunctional cross-linking utilized to generate bi-specific MHC heterodimers does not affect specific binding onto cross-reactive CD8+ T cells. Chapter III describes a mechanism for a cross-reactive CD8+ T cell response between the disparate antigens, lymphocytic choriomeningitis virus (LCMV)-GP34 (AVYNFATM) and vaccinia virus (VV)-A11R (AIVNYANL), which share the three underlined residues. The recognition determinants for LCMV-GP34 and VV-A11R were compared by an alanine/lysine scanning approach for both epitopes. Functional analysis of the mutated peptides clearly indicates that the shared P4N residue between LCMV-GP34 and VV-A11R is an important TCR contact for the recognition of both epitopes. In addition, we determined the crystal structures of both Kb-VV-A11R and Kb-LCMV-GP34. Structural analysis revealed that the two complexes are nearly identical structural mimics, which was unexpected due to the primary sequence disparity. Together with the functional studies, our results highlight that structural similarities between different peptide-MHC complexes can mediate cross-reactive T cell responses. Chapter IV of this thesis includes additional discussion, overall conclusions and future directions. Chapter V includes the protocols and the gene constructs that were used in this work. Also included in Chapter V are results from two unrelated incomplete projects which have yielded significant findings.
79

Drosophila piRNA Function in Genome Maintenance, Telomere Protection and Genome Evolution: A Dissertation

Khurana, Jaspreet S. 26 October 2010 (has links)
Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.
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The Role of ITK in the Development of Gamma Delta NKT Cells: A Dissertation

Yin, Catherine C 08 August 2012 (has links)
The immune system is a complex network of interacting cells and tissues that is designed to protect the body from pathogens and other foreign substances. T cells are a major component of the immune system and consist of two distinct lineages distinguished by the expression of αβ or γδ T cell receptors (TCR). The Tec family kinase, Itk is an important mediator of signaling downstream of the TCR. Past studies on Itk has focused on how Itk regulates development, activation and differentiation of conventional αβ T cells and more recently how Itk regulates the development of innate-like αβ T cells. However, very little is known about the influence of Itk on γδ T cells. My studies show a previously unknown role for Itk in the development and function of γδ T cells. We report in the absence of Itk, γδ T cells were responsible for the spontaneously elevated levels of serum IgE and Itk-/- mice γδ T cells produced high levels of TH2 cytokines. Furthermore, there was an increase in γδ T cells specifically in the Vγ1.1+Vδ6.3+ (V6) subset that represents the dominant population of γδ NKT cells in Itk-/- mice. In addition, the V6 subset had increased expression of PLZF, a transcription factor normally required for αβ iNKT cell development. We further show that V6 cells develop and mature similar to αβ iNKT cells. Similar to defects previously seen in the terminal differentiation of Itk-/- αβ iNKT cell, V6 cells also had impaired maturation in the thymus in the absence of Itk. This data demonstrates a previously unknown role of Itk for the terminal maturation of V6 cells that has been shown to be the cell population that led to spontaneous dermatitis in mice. Given that drug companies have targeted Itk as a potential allergy drug due to Itk’s role in TH2 development and function, our data suggests that further studies on Itk are warranted.

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