1991 |
Berg, Leslie J. |
R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Function of Mhc Class Ii Mutants
This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.
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0.936 |
1994 — 2003 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Thymic Selection and T Cell Lineage Commitment @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION: (Adapted from the applicant's abstract and Specific Aims.) The generation of the T cell receptor repertoire is a critical factor in determining immune responses. A system for studying T cell development and the selection mechanisms shaping the T cell repertoire using 2B4 T cell receptor (TCR) transgenic mice have been developed. In these mice, over 95 percent of the developing T cells express a single TCR specificity (cytochrome c plus I-Ek). The previous genetic experiments using this system have demonstrated that T cell maturation requires an interaction between the TCR and I-Ek molecules present on thymic stromal cells. Furthermore, this interaction determines the differentiation of immature thymocytes into mature CD4+ helper T cells, rather than CD8+ cytotoxic T cells. Using site directed mutagenesis and an in vitro functional assay, two independent sites for CD4 interaction on the beta chain of the MHC class II-Ek molecule have been recently identified. A double mutant carrying both of these alterations will be introduced into the germline of mice. Transgenic mice carrying the mutant E-beta gene will be crossed to the 2B4 TCR transgenic mice to assess the role of MHC class II/CD4 interaction during positive and negative selection. In addition, the role of this interaction in the commitment of differentiating T cells to the helper versus cytotoxic lineage will be evaluated. T cell development in vitro has been studied in thymic organ cultures derived from the 2B4 TCR transgenic mice. It was found that both the interactions resulting in the elimination of self-reactive T cells (negative selection) and the differentiation of immature thymocytes (positive selection) can be faithfully reproduced in this in vitro system. Using this approach, the goal is to understand which intercellular interactions can induce differentiation, which can induce cell death, and whether specific thymic stromal cell types are necessary for each of these interactions. Thymic organ cultures derived from I- E-2B4 TCR transgenic mice will be supplemented with exogenously-derived cells, or cells plus peptides, in an attempt to generate the missing signal required to induce T cell differentiation or cell death. Specifically, Specific Aim 1 proposes to analyze the role of MHC class II/CD4 interactions during the thymic selection process. Specific Aim 2 proposes to study the thymocyte/antigen presenting cell interactions responsible for inducing positive and negative selection. Specific Aim 3 proposes to study the coordinate regulation of helper versus cytotoxic T cell function with CD4 versus CD8 lineage commitment. This information could enhance our understanding of T cell recognition and the establishment of self-tolerance.
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0.936 |
1995 — 1998 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
T Cell Development and the Tyrosine Kinase, Tsk @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (Adapted from the applicant's abstract): T lymphocyte development in the thymus is dependent on interactions with the T cell antigen receptor (TCR). Early in development, thymocytes which fail to rearrange a functional TCR beta chain are arrested at the CD4-8- stage. Subsequently, CD4+8+ thymocytes, which are the first cells in the thymus to express a complete TCR alphabeta/CD3 complex on their cell surface, undergo two selection processes: positive selection and clonal deletion. Together these two selection processes create a TCR repertoire which is self-MHC-restricted and self-tolerant. In an effort to understand the signal transduction pathways underlying these thymic selection events, a PCR based molecular screen for novel tyrosine kinases expressed in the thymus was initiated. One of the genes identified in the screen, called TSK for T cell specific kinase, is the subject of this proposal. The expression pattern of TSK (also called ITK or EMT by other workers) is highly suggestive of a role in T cell development. Analysis of RNA isolated from a wide range of mouse tissues and cell lines indicates that TSK is expressed only in T cells. In addition the levels of TSK mRNA are approximately tenfold higher in the thymus than in resting peripheral T cells. TSK is expressed throughout fetal thymic ontogeny, starting as early as fetal day 14, but is not expressed in bone marrow stem cells. TSK, BTK, and a third protein, TECA, define a new family of tyrosine kinases. Mutations in the BTK gene have been shown to be responsible for the human immunodeficiency disease, X-linked agammaglobulinemia (XLA), and for the xid deficiency in mice. Besides a causative role in immune deficiency diseases, tyrosine kinases are involved in the decisions leading to proliferation as opposed to differentiation of developing cells in many lineages, and have been implicated in a variety of cancers. The elucidation of signal transduction pathways in T lymphocytes will also promote our understanding of the factors leading to autoimmune diseases. To examine the role of TSK in T cell development, biochemical and genetic approaches will be taken. Preliminary results indicating an interaction between the TSK-SH2 domain and Syk in activated thymocytes will be extended and applied to the analysis of TSK Zap-70 complexes. Whether TSK itself is tyrosine phosphorylated and/or activated in thymocytes stimulated by a number of surface receptors including the TCR/CD3 complex will be investigated. In addition, the function of TSK in vivo will be studied by analyzing transgenic mice overexpressing wild type or kinase-inactive TSK. A TSK-deficient mouse line is being generated by gene targeting in embryonal stem cells. The T cell-restricted expression pattern of TSK, together with its high level of expression in the thymus, strongly suggest a role for the TSK kinase in a signaling pathway unique to cells of the T lymphocyte lineage.
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0.936 |
1999 — 2003 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
T Cell Development and the Tyrosine Kinase, Itk/Tsk @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (Adapted from Investigator's Abstract): This is a competitive renewal of a previously funded application from an established investigator. T cell development and activation are dependent on multiple independent receptor signaling pathways. Since tyrosine kinases are known to play a critical role in receptor proximal signaling events, Dr. Berg previously set out to identify novel tyrosine kinases expressed in T cells. These efforts led to the cloning of Itk, a member of the Tec family of tyrosine kinases expressed only in T cells and NK cells. Btk, a close relative of Itk expressed in B cells and mast cells, has been shown to be the gene defective in human and mouse immunodeficiencies (X-linked agammaglobulinemia and XID, respectively). To examine the role of Itk in T cell signaling, Itk-deficient mice were generated and characterized. T cells from these mice are defective in TCR-induced calcium mobilization and cytokine production. A profound defect in positive selection in the thymus was also found. Using T cells from the Itk-deficient mice and retroviral gene transfer techniques, Dr. Berg proposes to define the protein domains and phosphorylation sites of Itk that are necessary for its role in TCR-induced calcium mobilization and cytokine production. She will characterize the biochemical defects in the Itk-deficient T cells, focusing on the transcription factors involved in IL-2 gene induction and the role of Itk in PLCgamma1 activation, and on CD28 signaling. Finally, she will address the role of Itk in T cell development by crossing the Itk-deficient mice to three different TCR transgenic lines, and then assess positive and negative selection in these mice. Together, these studies will provide important information on the role of Itk in both T cell development and T cell activation. As tyrosine kinases have been implicated in numerous human immunodeficiency diseases and cancers, and are involved in decisions leading to the proliferation versus differentiation in many cell lineages, these experiments will provide information relevant to an understanding of oncogenesis, autoimmunity, as well as genetic immunodeficiences.
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0.905 |
2000 — 2004 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Immunobiology of Jak3-Deficient Mice @ Univ of Massachusetts Med Sch Worcester
T cell development and activation are dependent on multiple independent signaling pathways. These include signals through T cell antigen receptors (TCRs), co-receptors, co-stimulatory receptors, cytokine receptors, adhesion molecules, and others. As tyrosine kinases are known to play a critical role in receptor proximal signaling events, we set out to identify novel tyrosine kinases expressed in T cells. This effort led to the cloning of Jak3, which was shown to be critical for signaling through gammac-containing cytokine receptors, including the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. To examine the role of Jak3 during lymphocyte development and activation, we generated Jak3-deficient mice, and have shown that T cell maturation and function are grossly aberrant in these mice. The importance of Jak3 in the immune system is also highlighted by the identification of human severe combined immunodeficiency patients whose disease is caused by mutations in the Jak3 gene. We propose to use the Jak3-deficient mice as a model system to investigate the precise role of Jak3 in the immune system. First, we will test the hypothesis that Jak3 plays a critical role in the development and/or survival of T cell progenitors in the thymus, fetal liver, or bone marrow. We will also examine the role of Jak3 in mature peripheral T cells, and test the idea that Jak3 is necessary for long-term T cell survival, responsiveness to antigenic stimulation, and activation-induced cell death. Finally, we will use a gene-targeting "knock-in" strategy to perform structure/function analysis of Jak3 in vivo, allowing us to assess the activity of specific Jak3 mutants in primary cells. As tyrosine kinases have been implicated in numerous human immunodeficiency diseases and cancers, and are involved in decisions leading to proliferation versus differentiation in many cell lineages, these experiments will provide information relevant to an understanding of oncogenesis, autoimmunity, as well as genetic immunodeficiencies.
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0.905 |
2000 — 2002 |
Berg, Leslie J. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core--Transgenic/Knockout Animal Facility @ Univ of Massachusetts Med Sch Worcester
Description: (Taken directly from the application) The techniques of modern mouse genetics have provided extremely important tools in biomedical research. The generation of transgenic mice has allowed investigators to examine the roles of genes involved in a wide variety of disease processes. The ability to generate specific mutations in genes of interest, including both knockouts and knock-ins, has expanded the power of this approach, allowing the identification of gene regulating immune responses and autoimmunity, as well as genes involved in susceptibility to disease. In addition, these techniques have provided a straightforward means to generate mouse models of important human diseases. The Transgenic/Knockout Mouse Core facility at UMMS currently generates transgenic mice for the analysis of gene function and regulation in intact animals. Transgenic animals are generated by pronuclear injection of DNA constructs into fertilized one-cell mouse embryos, followed by oviduct transfer of the injected embryos into foster mothers. The facility is currently expanding to provide gene targeting (knockout and knock-in) services. As of July 1998, the facility began providing the services of embryonic stem cell injections into 2.5 day mouse blastocysts, followed by uterine transfer of injected embryos into foster mothers. In the near future (by July 1999), the facility will begin providing embryonic stem cell culture services, assisting investigators in growing, transfecting, selecting, and cloning embryonic stem cells for gene targeting purposes.
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0.905 |
2005 — 2009 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tec Family Tyrosine Kinases in T Cell Signaling @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Protective immunity to pathogenic infection requires a complex set of effector responses mediated by multiple cell types. Effector CD4+ T cells are critical producers of IFN-gamma during infections by intracellular bacteria and protozoa (Th1 cells) and of IL-4 and IL-5 in response to parasites (Th2 cells). While much is known about the environmental effects of cytokines on T helper cell differentiation, the role of T cell intrinsic signaling pathways in this process is poorly understood. Tec-family tyrosine kinases are important regulators of phospholipase C-gamma activation following T cell antigen receptor stimulation. The goal of this proposal is to address the role of Tec kinases, Itk and Rlk, and Tec kinase-dependent signaling pathways, in Th1 vs. Th2 differentiation. Previous studies indicate that Itk-deficient mice are unable to mount protective immune responses to pathogens that require Th2 effector functions; in contrast Itk/Rlk-deficient mice regain this ability. Our recent findings provide insight into these data, by demonstrating that Itk signaling is required to inhibit T-bet induction following TCR stimulation; thus, in the absence of Itk, CD4+ T cells preferentially differentiate into Th1 effector cells. We have also found that Itk is upregulated in Th2 cells compared to Th1 cells, whereas Rlk is selectively lost from Th2 cells. On the basis of these data, we hypothesize that Itk and Rlk play distinct roles in regulating Th2 and Th1 differentiation and effector functions, respectively. We also hypothesize that Itk promotes Th2 differentiation via the negative regulation of T-bet expression. To address these hypotheses, we first propose to determine the mechanism by which Itk regulates T-bet, and to test the in vivo relevance of our findings using a T cell adoptive transfer lung airway inflammation (Th2) model. We will also determine whether constitutive Itk or Rlk expression in T cells interferes with, or biases, normal Th1 or Th2 differentiation, respectively. To determine the underlying basis for the paradoxically normal Th2 responses in Itk/Rlk-deficient mice, we will use a carefully controlled in vitro CD4+ T cell differentiation assay to examine the mechanisms regulating cytokine production and differentiation of Itk/Rlk-deficient cells. These studies will provide important information for future efforts to modulate Th1 vs. Th2 effector responses in vivo by manipulating distinct T cell signaling molecules and pathways.
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0.905 |
2006 — 2010 |
Berg, Leslie J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Immunobiology of Jak3 Deficient Mice @ Univ of Massachusetts Med Sch Worcester
[unreadable] DESCRIPTION (provided by applicant): Jak3 is a protein kinase required for signaling through cytokine receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies have shown that mutations in Jak3 result in a severe combined immunodeficiency syndrome (SCID) in humans, indicating that Jak3 plays an absolutely essential role in the function of the immune system. In addition, pharmacological inhibitors of Jak3 are currently being developed for use as immunosuppressants. Nonetheless, the precise role of Jak3 in T cell activation, differentiation, and homeostasis is currently uncertain, as are the consequences of long-term inhibition of Jak3 activity. We have developed an animal model using Jak3-deficient mice that will allow us to address the role of Jak3- dependent cytokine signaling in normal cells and in intact animals. Our recent data, based on findings from gene expression profiling and cytokine analysis experiments, indicate that the CD4+ T cells present in Jak3-/- mice closely resemble regulatory T cells of the Treg-1 (Tr1) phenotype. These cells are non- proliferative in vitro, and secrete large amounts of IFN-g, IL-10, and TGF-? following TCR stimulation, but no IL-2 or IL-4. In addition, they have high constitutive levels of the inhibitory surface receptor, PD-1. Preliminary data also suggest that these Jak3-/- T cells can suppress proliferative responses of wild type T cells in vitro. Our hypothesis is that Jak3-dependent cytokine signals are critical for the differentiation of activated T cells into functional, conventional effector T cells, and that in the absence of these signals, activated T cells differentiate into Tr1 regulatory T cells. We also hypothesize that the "activation" of Jak3-/- CD4+ T cells results from the homeostatic proliferation of these cells in the lymphophenic environment of the Jak3-/- host. To test these hypotheses, we propose to determine whether Jak3 -/- CD4+ T cells function as regulatory T cells, and the role(s) of IL-10, TGF-b, and PD-1 in this function. Second, we will examine the differentiation pathway of naive Jak3-/- CD4+ T cells following antigen stimulation in vitro and in vivo, and following homeostatic expansion in a lymphopenic wild type host. Finally, we will determine the role of Stat5 as a downstream mediator of Jak-3 dependent cytokine signals in regulating effector T cell versus regulatory T cell differentiation pathways. [unreadable] [unreadable] [unreadable]
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0.905 |
2006 — 2009 |
Berg, Leslie J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tec Kinases Ltk and Rlk in Mast Cell Signaling @ Univ of Massachusetts Med Sch Worcester
Allergic asthma is a complex disease involving a localized inflammatory response mediated by lymphocytes, eosinophils and mast cells. One critical component of this response is the T helper type 2 (Th2) cytokines, including IL-4, IL-5, IL-9, and IL-13, produced by T cells and mast cells. The Tec family tyrosine kinases, Itk and Rlk, play important roles in activating phospholipase C-y1 downstream of the T cell receptor. Itk/Rlk- deficient T cells show impaired TCR signaling, leading to substantially reduced cytokine production and defects in clearing pathogenic infections. Interestingly, unimmunized Itk-/- and Itk-/-Rlk-/- mice exhibit abnormalities suggestive of excess Th2-type cytokine production, such as germinal center hyperplasia in the spleen, eosinophilia, and elevated levels of serum IgE antibodies. These data, together with the fact that Itk and Rlk are also expressed in mast cells, suggest the following hypothesis: Itk and/or Rlk may be critical in T cells for IL-4 production leading to Th2 cell differentiation and cytokine production, but may also play a role in mast cell cytokine production. To test this hypothesis, we propose to determine whether the abnormalities seen in unimmunized Itk-/- and Itk-/-Rlk-/- mice are due to a T cell-intrinsic defect, a defect in mast cells, or both. Second, we find that Itk-/- bone marrow-derived mast cells secrete enhanced levels of IL-4, IL-6, and IL-13 following FceRI stimulation. Based on these data, we hypothesize that Itk is required for optimal activation of the SHIP1/Dok-1 pathway, known to attenuate mast cell responses. To test this idea, we will examine Itk-/- mast cells for alterations in degranulation and cytokine production in response to stimulation with IgE alone, IgE plus antigen, SCF, as well as to FceRI and FcgRII co-aggregation. Biochemical analyses will directly address the role of Itk in the SHIP1-dependent pathway. The overall goal of these studies is to further our understanding of signaling pathways that may contribute to the development of allergic diseases, and to suggest mechanisms for the manipulation and/or prevention of allergic responses.
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0.905 |
2010 — 2013 |
Berg, Leslie J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Conventional Versus Innate Cd8+ T Cell Development @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Regulation of conventional versus innate CD8+ T cell development in the thymus is now known to produce a wide array of distinct T cell lineages. Many of these T cell subsets play key roles in regulating immune responses, both to self as well as to pathogens. In addition to the conventional CD4+ and CD8+ ???T cells that are key components of the adaptive immune response, there are regulatory T cells, NKT cells, ?? T cells, and a variety of additional innate T cell subsets. The appropriate balance of T cells developing into each of these lineages is essential to maintain immunological homeostasis, self-tolerance, and the ability to produce both rapid and delayed responses to pathogenic infections. Currently, the molecular mechanisms governing these developmental lineage choices are under intense investigation. Our own studies have identified a signaling pathway involving the Tec family tyrosine kinase, Itk, which determines conventional versus innate CD8+ T cell development. In wild-type (WT) thymocytes with normal Itk function, MHC class I-specific T cells predominantly develop into conventional naive CD8+ T cells, which are precursors of effector cytotoxic T cells. In addition, a very minor subset of cells develop into innate CD8+ T cells that have characteristics of previously-activated memory CD8+ T cells, and exhibit immediate effector function when activated. In contrast to this, MHC class I-specific thymocytes lacking the Tec kinase, Itk, develop nearly exclusively into innate CD8+ T cells that express high levels of the T-box transcription factor, Eomesodermin. These findings indicate that a signaling pathway requiring Itk regulates the lineage decision between conventional and innate CD8+ T cells. As Itk is well known as a component of the TCR signaling pathway leading to phospholipase?-31 activation and actin polymerization, these data also implicate altered TCR signaling as a modulator of these key T cell lineage decisions. To determine the transcriptional regulators of this lineage decision, we performed a microarray experiment to identify factors differentially expressed between WT conventional and Itk-deficient innate CD8+ thymocytes. Interestingly, this analysis indicated that the single most highly up-regulated transcription factor in WT relative to Itk-deficient CD8+ thymocytes is IRF4; further, our preliminary studies indicate that in the absence of IRF4, nearly all CD8+ T cells also develop into the innate lineage. In contrast, the transcription factor most highly expressed in Itk- deficient thymocytes relative to WT is Runx2. We hypothesize that Itk signaling promotes conventional CD8+ T cell development by inducing the transcription of IRF4, and that in the absence of Itk, Runx2 upregulation converts conventional CD8+ T cells into innate T cells, leading to upregulation of Eomesodermin. To determine the importance of these transcription factors in regulating conventional versus innate CD8+ T cell development we propose to examine whether IRF4 is essential for conventional CD8+ T cell lineage commitment. We will also investigate whether IRF4 is sufficient to suppress Eomesodermin expression in CD8+ T cells. Third, we will determine whether different strengths of TCR signaling lead to graded expression of IRF4. Finally, we will examine whether Runx2 is required for innate CD8+ T cell development. PUBLIC HEALTH RELEVANCE: Our immune system protects us against a wide array of pathogens using many different types of white blood cells. The work described in this proposal will investigate how our body produces the correct types of white blood cells in the correct proportions. This understanding will aid in efforts to regulate and control the immune system to fight infections and eradicate cancer cells.
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0.905 |
2010 |
Andreotti, Amy H (co-PI) [⬀] Berg, Leslie J. Kang, Joonsoo |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Screening For Inhibitors of the T Cell Tec Kinase, Ltk @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Autoimmune diseases result from a breakdown of self-tolerance. Disease progression involves several key steps, including the inappropriate activation of autoreactive lymphocytes followed by the infiltration of pathogenic effectors T cells into the target tissues. Our recent studies have focused on a mouse model of multi- organ autoimmune disease that results from the absence of the costimulatory molecule, CTLA-4 (i.e., Ctla4-/- mice). In this model, mice succumb to a rapid and fatal disease that results from massive T cell activation, infiltration into vital organs, and the subsequent failure of those organs. We have found that the Tec family tyrosine kinase ITK plays a critical role in the process of autoreactive T cell migration into tissues in this multi- organ autoimmune disease. Thus, Itk-/-Ctla4-/- mice show unprecedented T cell activation and proliferation, but are protected from the lethal autoimmunity of Ctla4-/- mice due to a failure of these activated effectors T cells to accumulate in non-lymphoid tissues. In addition, we can prevent the onset of severe autoimmune disease in Ctla4-/- mice by treatment with a small molecule ITK inhibitor. ITK is a well-characterized signaling protein that is activated by TCR, CD28, and chemokine receptor stimulation;in turn, ITK activates phospholipase-Cg and induces actin polymerization. These findings indicate that ITK signaling in effectors T cells is critical in regulating T cell infiltration into tissues, and more importantly, that ITK is essential for the pathogenesis of autoimmune T cells. In this project, we propose to establish and validate a biochemical assay to screen for novel small molecule inhibitors of ITK. Although two groups have previously reported the identification of ITK inhibitors, these inhibitors are of relatively low potency and have poor pharmacokinetics in vivo. Moreover, the screens that produced these compounds used the isolated ITK kinase domain and so yielded only ATP-site directed inhibitors. Based on our in depth biochemical investigations of full length ITK and our recent description of a remote ITK substrate docking mechanism, we have begun to establish a greatly improved ITK in vitro kinase assay, in which the Km of ITK for its substrate is >15-fold higher than in previously-established assays. Use of this novel assay will provide a platform for identifying inhibitors that are efficacious at lower concentrations and with higher selectivity for ITK. In the first aim, we will optimize the ITK in vitro kinase assay to maximize reproducibility and to determine assay conditions amenable to HTS. In the second aim, we will establish cell-based and whole animal assays for ITK inhibitors as secondary and tertiary screens, including counter-screens for ITK inhibition. Our overall goal is to establish a robust high-throughput screen to identify novel, selective, and high affinity small molecule inhibitors of ITK, and ultimately, to assess the efficacy of these inhibitors in animal models of organ-specific autoimmune diseases, as well as in the inhibition of human T cell migration and diapedesis. PUBLIC HEALTH RELEVANCE: Autoimmune diseases occur when an individual's immune system attacks his/her own cells and organs. We have identified an enzyme that plays an essential role in this disease process. This proposal aims to develop a screen for inhibitors of this enzyme, which could be used to prevent the onset of autoimmune processes in susceptible individuals and to treat and cure patients with these diseases.
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0.905 |
2011 — 2015 |
Berg, Leslie J. Kang, Joonsoo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Itk: An Emerging Target For Treatment of T Cell-Mediated Autoimmune Disease @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): ITK: an emerging target for treatment of T cell-mediated autoimmune diseases Lymphocyte-mediated autoimmune diseases arise from a breakdown of self-tolerance. One of the key proteins regulating self-tolerance is the inhibitory T cell protein, CTLA-4. When CTLA-4 is absent, mice succumb to a rapid and fatal multi-organ autoimmune disease, and die by three weeks of age. Recent studies from our labs have demonstrated that CTLA-4 has two distinct functions in preventing autoimmunity. First, CTLA-4 is required for the function of FOXP3+ regulatory T cells in maintaining T cell tolerance. Second, CTLA-4 is required in conventional T cells, to block aberrantly activated self-reactive T cell accumulation in tissues under non-inflammatory conditions. It is unknown how costimulatory molecules control aberrantly activated self-reactive T cells from infiltrating and damaging tissues. Based on the observation that the null mutation of the Tec kinase Itk prevents activated T cell migration and autoimmune pathology of Ctla4-/- mice, we propose that ITK activation is critical for T cell-mediated autoimmune diseases. We hypothesize that ITK is required for the synergistic activation of VAV1 and the pathways downstream of VAV1 leading to actin polymerization and cytoskeletal reorganization. Further, we propose that ITK activates VAV1 by phosphorylating and activating the Src family kinase, FYN, which in turn phosphorylates and activates VAV1. We will test this hypothesis by examining the migratory behavior of self-reactive T cells from Itk-/- mice in vitro and in vivo to determine the molecular mechanism by which ITK controls T cell movement into non-lymphoid tissues. We will also determine whether ITK regulates cytoskeletal reorganization in primary T cells by activating FYN, whether FYN is required for actin polymerization and cytoskeletal reorganization in primary T cells and contribute to autoimmune disease progression. Finally, we will investigate the efficacy and mechanism of suppression of Type I diabetes in animals by small molecule inhibitors of ITK, to begin to explore the utility of targeted ITK blockade in clinics to treat various organ-specific autoimmune diseases. PUBLIC HEALTH RELEVANCE: ITK: an emerging target for treatment of T cell-mediated autoimmune diseases occur when an individual's immune system attacks his/her own cells and organs. This proposal addresses one of the basic mechanisms that prevent autoimmunity from occurring in healthy individuals. A better understanding of this mechanism may provide opportunities to prevent the onset of autoimmune processes in susceptible individuals and to treat and cure patients with these diseases.
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0.905 |
2013 — 2016 |
Berg, Leslie J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Plasticity of T Helper Cell Differentiation @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): For protection against a wide array of diverse pathogens, T cells acquire distinct effector functions in response to each infection. For CD4+ T cells, these effector functions are characterized by the predominant cytokines produced by the effector cells. To date, five different subsets of CD4+ effector T cells have been described, and can be generated from na¿ve CD4+ precursors under controlled stimulation conditions. Similar subsets of functionally distinct CD8+ effector T cells have also been described. While early work in this area indicated that effector T cell differentiation was comparable to the terminal differentiation processes that occur during ontogeny, recent evidence indicates that T cell effector subsets are more plastic in their differentiation status. Not only do some T cells exhibit characteristics of more than one effector lineage at the same time, but additional instances of CD4+ and CD8+ effector T cells acquiring new cytokine profiles over the course of a response have also been observed. For instance, under certain conditions, CD4+ Th17 cells will acquire the capacity to produce IFNgamma, and Th1 cells will become IL-21-secreting Tfh cells. These data suggest that T cell responses can evolve over time, leading to alterations in the effector functions that predominate at different stages of an immune response. Importantly, this process is likely to play a key role in the evolution of autoimmune responses and may also contribute to the pathogenesis of chronic inflammatory diseases. We hypothesize that the transcriptional repressor, Blimp-1, is a critical regulator of T cell plasticity. Blimp-1 is upregulated in activatd CD4+ and CD8+ T cells by a specific subset of cytokines, including IL-2, IL-12, and IL- 4; thus, Blimp-1 is expressed in CD4+ effector Th1 and Th2, but not Th17, cells, as well as in Type I effector CD8+ T cells. We find that Th1 cells generated from Blimp-1-deficient na¿ve CD4+ T cells acquire a multi- lineage molecular program, expressing both Th1- and Th17-specific genes; a similar change in gene expression is seen in Blimp-1-deficient CD8+ T cells following LCMV infection. These data suggest that Blimp- 1 normally functions to repress Th17 and Tc17 differentiation, and further, may be required for effector cells to maintain a highly polarized Typ I subset identity. To test this hypothesis, we will first examine the molecular regulation of Blimp 1 transcription by distinct cytokines to determine the pattern of Blimp-1 expression at different stages of the immune response. We will then determine whether graded expression of Blimp-1 and/or Bcl-6 regulate Type I versus Type 17 effector cell differentiation during the development of a Th17-dependent autoimmune disease and during virus infection. Finally, we will determine whether persistent Blimp-1 expression is required to maintain Type I lineage identity, and whether conditional deletion of Blimp-1 in effector T cells alters their effector functions, and promotes their ability to induce autoimmunity. Together, these studies will determine whether the magnitude and duration of Blimp-1 expression are critical in the maintenance of T cell differentiation states, and whether alterations in Blimp-1 expression during an immune response contribute to the plasticity of effector functions. These data will provide important insights into the mechanisms contributing to autoimmune and other diseases caused by pathogenic T cell responses.
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0.905 |
2014 — 2017 |
Berg, Leslie J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Anti-Viral T Cell Responses by Irf4 @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): The generation of protective immunity that reduces or prevents re-infection with the same pathogen is a hallmark of the adaptive immune system. One key component of this process is the formation of memory T cells. Several factors are known to impact the numbers, as well as the effectiveness of the memory T cells generated in response to virus infection. These include antigen abundance and duration, cytokine signals, and costimulatory molecules. However, the precise molecular mechanisms by which the integration of these signals regulate memory cell formation and function are not well understood. In particular, there is remarkably little known about how differences in TCR signaling, due to variations in TCR affinity/avidity for antigen-MHC complexes, impact the differentiation of effector versus memory CD8+ T cells at the molecular level. Our work has focused on the signaling pathways downstream of the TCR. We have found that the transcription factor, IRF4, is upregulated upon TCR stimulation of na¿ve T cells, and that the amount of IRF4 produced in each T cell is dependent on the strength of the TCR signal. We also found that IRF4 upregulation was markedly impaired in T cells lacking the Tec kinase, ITK, a known modulator of TCR signal strength. Stimulation of IRF4- deficient CD8+ T cells induced high levels of Eomesodermin, a transcription factor associated with memory CD8+ T cells. Together, these data lead us to hypothesize that strong TCR signaling in CD8+ T cells responding to a virus infection induces robust ITK activation and high expression of IRF4. In turn, these factors induce high expression of T-bet and Blimp-1, suppress TCF1 and Eomesodermin expression, and thereby promote a vigorous expansion of short-lived effector cells. In contrast, cells receiving weak TCR stimulation would activate little ITK, upregulate low levels of IRF4, and differentiate rapidly into memory precursor cells. To test this hypothesis, we will examine the CD8+ T cell response to LCMV-Armstrong, as well as Influenza A virus, by cells carrying two, one, or zero copies of a functional IRF4 gene (IRF4+/+, IRF4+/-, IRF4-/-). We will also modulate the strength of TCR signaling by infecting wild type mice with LCMV- Armstrong, followed by varying doses of a small molecule inhibitor of ITK. As a third approach, we will use an LCMV-Armstrong variant carrying a point mutation in the GP33 peptide epitope recognized by the P14 TCR, leading to low affinity stimulation of P14 transgenic CD8+ T cells. In the second aim, we will address a set of putative downstream targets of IRF4, and will assess whether TCF1 is involved in the mechanism by which IRF4 represses Eomesodermin expression. In the third aim, we will examine the CD8+ T cell responses of mice carrying heterozygous mutations in ITK, IRF4, or both, to infections of the clone 13 strain of LCMV, a virus that establishes chronic infections in wild type mice. In the fourth aim, we will determine whether IL-4 synergizes with low TCR signal strength to promote memory T cell differentiation in vivo. Together, these studies will provide important insights into the signaling pathways that regulate short-lived effector versus memory precursor T cell formation, and in particular, the role of TCR signaling in this process.
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0.905 |
2020 — 2021 |
Berg, Leslie J Huseby, Eric S. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tcr Signaling Control of Thymic Treg Selection and Immune Homeostasis @ Univ of Massachusetts Med Sch Worcester
Abstract Neonatal thymic-derived Foxp3+ T regulatory cells (tTregs) are required for the development of immune homeostasis and limiting organ specific autoimmune disease. The molecular details of TCR-pMHC interactions, and the specific downstream signaling pathways that allow neonatal tTregs to develop, seed peripheral tissues and regulate acute inflammation are not well understood. We hypothesize that a subset of neonatal tTregs distinguishes health from disease via the expression of TCR with specificity for self-ligands that are upregulated during inflammatory conditions. This tTreg TCR recognition property manifests as graded levels of immune suppression based on the context and magnitude of the inflammatory setting. Preliminary data further suggest that the development of these tTreg clones within the neonatal selection window is temporally constrained by negative selection, and is predicated on kinetic proofreading, with TCR-self-pMHC dwell times within a conventional binding mode as a key to specifying tTreg development. To test our hypothesis, we will first identify endogenous self-ligands recognized by Foxp3+ CD4 tTreg cell subsets. Our approach is based on our proven ability to identify self-ligands recognized by T cells, paired with mass spectrometry of MHC-II bound self-peptides presented on APC isolated from different anatomical locations, as well as high-throughput pipelines for determining recognition properties of individual T cell clonotypes. Using paired sets of TCR-self-pMHC combinations our second aim will directly examine whether neonatal tTreg selection is based the dwell time of the interaction, and assess the influence of ?unconventional? TCR/self- pMHC binding modes in selecting the neonatal tTreg repertoire. Aim 3 will identify synergies between self- pMHC presentation by thymic APCs and the quality of TCR signals generated by thymocytes that define the neonatal tTreg selection window. Signaling by the Tec family kinase, Itk, is proposed to regulate a signaling threshold that separates Foxp3 Treg selection from late stage deletion by amplifying pro-survival TCR signals derived from moderate dwell-time ligands via NFAT and NF-?B signaling pathways. Finally, in mature tTregs, we propose that Itk functions to amplify weak TCR responses, thereby allowing mature Tregs to recognize gradients in self-antigen displayed. These studies will provide important insights into the fine-tuning of T cell responses and the signaling pathways that discriminate effector cells from regulatory cells, leading to rational approaches in the design of therapeutics to manipulate immune responses for treatments of cancer and autoimmune diseases.
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0.905 |