Expression And Function Of Tob In T Lymphocytes
Vassiliki A Boussiotis, Associate Professor
Beth Israel Deaconess Medical Center
Grant 7R01CA104596-04 from National Cancer Institute, IRG: CMI
Abstract: Cellular quiescence is a state characterized by decreased cell size and metabolic activity. Quiescence in naive lymphocytes acts to reduce the resources, energy and space, required to maintain a vast repertoire of T and B cells. Quiescence might also protect cells from accumulating metabolic damage that could result in malignancy. Recent studies have shown that quiescence in lymphocytes is an actively maintained rather than a default state in the absence of a signal. Quiescence factors identified to date, represent potential tumor suppressor genes because alterations in their expression or function contributes to progression of lymphoid malignancies. Thus, strategies to understand and enforce lymphocyte quiescence might be useful in controlling leukemia and lymphoma. Recently, we identified Tob as a gene that mediates quiescence in T lymphocytes. Our studies showed that Tob mRNA is highly expressed in anergic cells. Tob mRNA is also constitutively expressed in unstimulated, primary, peripheral blood T lymphocytes and is downregulated during activation via TCR/CD3 in the presence of costimulation. Forced expression of Tob inhibits transcription of cytokines and cyclins and T cell proliferation. In contrast, suppression of Tob with antisense oligonucleotide augments CDS-mediated responses and abrogates the requirement of costimulation for maximal proliferation and cytokine secretion. In order to understand the functional role of Tob in the immune response of the intact host, we have generated transgenic mice, which constitutively express Tob in their T cells. In vitro, Tob-Tg T cells display reduced proliferation and cytokine production. After in vivo immunization, Tob-transgenic mice display reduced primary responses and abrogated recall T cells responses to antigenic challenge. These results provide evidence that T cell quiescence is not a default state, but an actively maintained gene program that must be suppressed for T cell activation to occur. Our findings indicate that Tob has a critical role in maintaining T cell quiescence. Thus, understanding the biochemistry and logic behind the integrative processes that control Tob expression will illuminate drug targets and approaches to better regulate T cell immune responses either for immunosuppression in autoimmunity, transplantation and allergy or for augmentation in vaccines, chronic infections and cancer. Because Tob represents a potential tumor suppressor and Tob deficient mice develop malignant lymphomas, such studies might also provide insights to the pathophysiology of lymphoid malignancies. Thus, strategies to regulate Tob expression might be useful in controlling leukemia and lymphoma. To achieve these objectives I propose three specific aims to 1) Determine the mechanisms that regulate Tob mRNA expression; 2) Determine the biochemical signaling pathways involved in transcriptional and post-translational regulation of Tob; 3) Determine the role of Tob in regulating T cell immune responses in the intact host, by using transgenic mice for Tob
Keywords: T lymphocyte, cellular respiration, gene expression, helper T lymphocyte, protein structure function, tumor suppressor protein DNA binding protein, binding site, biological signal transduction, cellular immunity, gene expression profiling, genetic regulation, genetic transcription, immune tolerance /unresponsiveness, phosphorylation, posttranslational modification, transcription factor, ubiquitin DNA footprinting, chromatin immunoprecipitation, genetically modified animal, human subject, laboratory mouse
Project start date: 2005-05-01
Project end date: 2010-03-31
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Grants awarded to Vassiliki A Boussiotis
The Role Of Rap1 In T-Cell Responses
Vassiliki A Boussiotis, Associate Professor
Massachusetts General Hospital
Grant 5R01AI043552-09 from National Institute Of Allergy And Infectious Diseases, IRG: TTT
Abstract: The long-term goal of this proposal is to dissect the functions of Rap1 and its molecular targets in the regulation of T cell responses. Rap1, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, adherens junction positioning and adhesion. In T lymphocytes transient activation and localization of Rap1 at the immunological synapse is one of the physiologic consequences of TCR ligation. In contrast, sustained increase of active Rap1 is detected in anergic cells. Forced expression of active Rap1 inhibits T cell activation and IL-2 transcription. Our studies proposed here are based on preliminary results generated during the previous funding period of this application 1) Using the yeast two-hybrid system we detected that Rap1-GTP interacts with AF-6 in T cells. AF-6 regulates actin remodeling proximal to junctional complexes and promotes clustering of its associated proteins at cellular junctions. Thus, AF-6 is an attractive candidate to mediate the effects of Rap1 on integrin activation and immunological synapse formation. 2) By suppression subtractive hybridization, we determined that Rap1-GTP induces a distinct gene expression pattern in T cells. Among Rap1-GTP-induced genes were MKP-7 dual specificity phosphatase, and CD103. MKP-7 has selective activity for MAP kinases p38 and JNK that are required for induction and activation of NFAT and AP-1 transcription factors. CD103 defines a unique subset of Treg, which are the most potent immunosuppressors in vivo. 3) We have generated Rap1-GTP (E63)-Transgenic mice. These mice have increased CD103+CD4+CD25+ Treg and display impaired recall T cell responses to antigenic stimulation. These exciting results will become the basis of the studies proposed in this application. Our Specific Aims are 1. To investigate the role of the Rap-1 associated molecule AF-6 and the Rap1-induced molecules MKP-7 and CD103 in T cell responses in vitro. We will examine the involvement of AF-6 in formation of the immunological synapse, localization of Rap1 at the synapse and in Rap1-mediated integrin activation. We will examine the effects of MKP-7 on activation of p38 and JNK, expression and transactivation of NFAT, phosphorylation of c-Jun, transactivation of AP-1, and transcription of IL-2. Finally, we will determine the role of CD103 on T cell activation and cytokine production in a cell autonomous and non-autonomous manner. 2. To investigate the role of Rap1 in T cell responses in vitro and in vivo by using mice transgenic for constitutively active Rap1. Our preliminary data with Rap1-GTP-Tg mice point to a negative role of Rap1-GTP in vivo in regulating effector T cell responses. The studies proposed here will extend our preliminary observations and will examine the effects of Rap1 on the activation and differentiation of na?ve CD4+ cells, function of effector CD4+ T cells, function of CD4+ helper T cells, induction of T cell tolerance in vivo to a peptide antigen and alloantigen, and function of CD8+ cytolytic cells. Thus our in vitro and in vivo studies will complement each other in understanding the effects of Rap1 at the molecular/signaling level and in the intact host
Keywords: T lymphocyte, cytoskeletal protein, guanosine triphosphate, leukocyte activation /transformation, protein protein interaction, protein structure function, transcription factor actin, anergy, cytokine, integrin, phosphorylation, protein localization genetically modified animal, human subject, laboratory mouse
Project start date: 1998-07-01
Project end date: 2009-12-31
5R01AI043552-08 (2007): $324752
5R01AI043552-07 (2006): $334451
2R01AI043552-06A2 (2005): $291125
The Role Of P27kip1 In T-cell Responses
Vassiliki A Boussiotis, Associate Professor
Dana-farber Cancer Institute
44 Binney St
boston, Ma 02115
Grant 1R01AI046548-01A2 from National Institute Of Allergy And Infectious Diseases, IRG: EI
Abstract: Naive antigen-specific T lymphocytes require stimulation via their T cell receptor (TCR) and costimulatory molecules in order to become activated, secrete cytokines, clonally expand, and mount an immune response. When TCR is ligated by antigen alone without costimulation or IL-2, T cells become anergic and are incapable of clonally expanding and transcribing the IL-2 gene. In anergic cells many of the critical signals initiated by T cell activation do not occur, whereas other signals predominate. Anergic cells have defective activation of lck, ZAP 70, Ras, ERK, JKN and Trans activation of AP-1 and NF-AT. In contrast, these cells activate fyn, increase calcium levels, and activate Rap1, indicating that induction of anergy requires active signaling events. We have recently determined that anergizing signals result in increased intracellular cAMP that upregulates the cyclin dependent kinase (cdk) inhibitor p27kipl, rendering cyclin D2 cdk4 defective and preventing progression of T cells through the Gl restriction point of the cell cycle. In contrast, CD28 costimulation prevents p27kipl accumulation by decreasing the levels of intracellular cAMP and promoting ubiquitin-dependent degradation of p27kipl. p27kipl associates with JABi, a coactivator of cJun transcription factor, resulting in its cytoplasmic translocation and defective AP-1 transactivation. Better understanding of the biochemical and molecular basis of T cell anergy will facilitate the reversal of the anergic state of tumor-specific T cells isolated from cancer patients in order to make them capable of clonal expansion prior to the administration of tumor vaccines or immunotherapy. Understanding the molecular basis of T cell anergy will also provide targets to fashion more specific treatment approaches instead of global immunosuppression to prevent graft rejection and GVHD in patients undergoing allogeneic bone marrow and organ transplantation. Although our findings strongly suggest that p27kipl functions as an anergy factor, the regulation of p27kipl expression during T cell activation and the precise mechanism by which it mediates induction and maintenance of the anergic state remain unclear. To achieve these goals I propose three specific aims 1) to identify the biochemical modification of p27kipl in T cell immunity and anergy 2) to identify molecules that associate with p27kipl and study their role in T cell immunity and anergy, and 3) to determine the role of p27kipl in the stimulation of naive and activated antigen-specific T cells in vitro and in vivo
Keywords: T lymphocyte, anergy, cyclin dependent kinase, enzyme inhibitor, leukocyte activation /transformation intermolecular interaction, protein structure function human subject, laboratory mouse
Project start date: 2001-09-15
Project end date: 2004-08-31
1R01AI046548-01A2 (2001): $287511
5R01AI046548-03 (2003): $296682
The Role Of Rap1 In T-Cell Responses
Vassiliki A Boussiotis, Associate Professor
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 2R21AI043552-06 from National Institute Of Allergy And Infectious Diseases, IRG: IMB
Abstract: Maintenance of peripheral tolerance, while retaining the ability for initiation of immune responses is important for immune homeostasis. Induction of T cell tolerance and its in vitro counterpart anergy is an active process but the signaling events that predominate in anergy are different from those that predominate during productive responses. Although activation of Ras is blocked, activation of Rap1 is retained in anergic cells. To determine the mechanisms via which the active, GTP-bound Rap1 might affect the functional program of anergic cells we employed several approaches during the funding period of the last application First, using the yeast two hybrid system we cloned RIAM, a novel Rapl-interacting molecule, which associates selectively with active, GTP-bound Rap1. RIAM provides a link between Rapl-GTP and the cytoskeleton by interacting with Mena, VASP and WASP and the actin regulator profilin. Second, by suppression subtractive hybridization we have determined that Rapl-GTP regulates gene transcription, resulting in a distinct gene expression pattern, which affects the function of T cells. Third, we observed that Rapl-GTP mediates cell spreading and adhesion to extracellular matrix via the Pl3 kinase pathway. Fourth, we have generated Rapl-GTP-Tg mice, which display impaired expression of T cell activation markers and defective T cell responses. These exciting results have initiated intensive in vitro and in vivo studies and have provided the experimental foundation for more than one research project. As a renewal of a R29, this grant will be subject to a budget cap that will support the project outlined below, but would not be sufficient for all of the scientific directions that we can now undertake. Therefore, studies on the function of RIAM, the Rapl-GTPIRIAM interactions, their effects on actin reorganization and the role of RIAM in T cell immunity and anergy will be continued in a separate project. For this application, we shall focus only on further exploration of the role of Rap-1 in T cell responses in vitro and in vivo. To achieve this objective two specific aims are proposed First, to identify molecules, which are induced by or associate with Rap1 and study their functional activity in T cell responses in vitro and second, to determine the role of Rap1 in T cell responses in vivo by using mice transgenic for active or dominant negative Rap1.
Keywords: T lymphocyte, actin, anergy, guanine nucleotide binding protein, immunity, protein protein interaction, protein structure function, biological signal transduction, chimeric protein, gene expression, genetic screening, humoral immunity, isoantigen, leukocyte activation /transformation, protein sequence, SDS polyacrylamide gel electrophoresis, human subject, laboratory mouse, mass spectrometry, proteomics, transgenic animal, yeast two hybrid system
Project start date: 1998-07-01
Project end date: 2004-09-14
2R21AI043552-06 (2003): $171000
RAP1 AND ASSOCIATED MOLECULES AND T CELL ANERGY
Vassiliki A Boussiotis, Associate Professor
Dana-farber Cancer Institute
44 Binney St
boston, Ma 02115
Grant 5R01AI043552-04 from National Institute Of Allergy And Infectious Diseases, IRG: IMB
Abstract: For optimal activation and clonal expansion T-cells require both an antigen-specific signal mediated via their T-cell receptor (TCR) and a second signal, provided by accessory molecules and mediated by co-stimulatory pathways. In the absence of costimulation, TCR crosslinking by antigen does not lead to successful activation but instead leads to a state of antigen-specific unresponsiveness, termed anergy in vitro and tolerance in vivo. Mounting evidence demonstrates the biologic significance of costimulation in the antigen-specific response and underscores its role in the generation of tumor immunity, allograft rejection, and autoimmunity. However, the biochemical signals that accompany the generation of anergy and its prevention are not presently understood. Anergic cells are incapable of activating IL-2 transcription when restimulated with antigen even in the presence of costimulation. This event is associated with lack of activation of lck, ZAP-70, Ras, ERK, JNK, and defective transactivation of the IL-2 enhancer elements AP-1 and NF-AT. Therefore, the question arises whether the anergic state results from the absence of sufficient positive signals or, alternatively, from the activation of distinct signaling pathways. Recently, this investigator has identified a novel signaling pathway that results in active blockade of IL-2 gene transcription in T-cell anergy. Anergic cells demonstrate increased phosphorylation of fyn-co-immunoprecipitated cbl, recruitment of crkL/C3G complexes and activation of Rap 1, a competitor of Ras. Transfection of even low levels of the active form of Rap 1 in Jurkat T-cells recapitulates the anergic defect and results in blockade of TCR and CD28 mediated IL-2 gene transcription. These results strongly argue that a distinct signaling pathway is induced by TCR signaling in the absence of adequate costimulation which induces T-cell anergy and active inhibition of IL-2 gene transcription. Understanding of the precise downstream end points of the anergic mediators will provide insights in the potential signaling defects of helper T-cells in tumor bearing hosts which need to be reversed in order to generate tumor-specific immune response. Moreover, such knowledge will facilitate the ability to identify the specific molecular targets of anergy and design novel clinical therapeutic approaches in order to induce antigen-specific transplantation tolerance. To achieve these goals two aims are proposed First, to identify known and yet to be described molecules that are associated with activated Rap 1 and study their role in the induction of T-cell anergy; second, to study the mechanism(s) of how Rap 1-GTP and its associated molecules inhibit transcriptional activation of the IL-2 gene
Keywords: T lymphocyte, anergy, biological signal transduction, guanosine triphosphate, guanosinetriphosphatase T cell receptor, genetic transcription, interleukin 2, leukocyte activation /transformation, protein binding human subject, tissue /cell culture, transfection
Project start date: 1998-07-01
Project end date: 2003-05-31
5R01AI043552-04 (2001): $189668
5R01AI043552-03 (2000): $174059
5R01AI043552-02 (1999): $117600
1R01AI043552-01 (1998): $117425
Expression And Function Of Tob In T Lymphocytes
Vassiliki A Boussiotis, Associate Professor
Beth Israel Deaconess Medical Center
Grant 7R01CA104596-04 from National Cancer Institute, IRG: CMI
Abstract: Cellular quiescence is a state characterized by decreased cell size and metabolic activity. Quiescence in naive lymphocytes acts to reduce the resources, energy and space, required to maintain a vast repertoire of T and B cells. Quiescence might also protect cells from accumulating metabolic damage that could result in malignancy. Recent studies have shown that quiescence in lymphocytes is an actively maintained rather than a default state in the absence of a signal. Quiescence factors identified to date, represent potential tumor suppressor genes because alterations in their expression or function contributes to progression of lymphoid malignancies. Thus, strategies to understand and enforce lymphocyte quiescence might be useful in controlling leukemia and lymphoma. Recently, we identified Tob as a gene that mediates quiescence in T lymphocytes. Our studies showed that Tob mRNA is highly expressed in anergic cells. Tob mRNA is also constitutively expressed in unstimulated, primary, peripheral blood T lymphocytes and is downregulated during activation via TCR/CD3 in the presence of costimulation. Forced expression of Tob inhibits transcription of cytokines and cyclins and T cell proliferation. In contrast, suppression of Tob with antisense oligonucleotide augments CDS-mediated responses and abrogates the requirement of costimulation for maximal proliferation and cytokine secretion. In order to understand the functional role of Tob in the immune response of the intact host, we have generated transgenic mice, which constitutively express Tob in their T cells. In vitro, Tob-Tg T cells display reduced proliferation and cytokine production. After in vivo immunization, Tob-transgenic mice display reduced primary responses and abrogated recall T cells responses to antigenic challenge. These results provide evidence that T cell quiescence is not a default state, but an actively maintained gene program that must be suppressed for T cell activation to occur. Our findings indicate that Tob has a critical role in maintaining T cell quiescence. Thus, understanding the biochemistry and logic behind the integrative processes that control Tob expression will illuminate drug targets and approaches to better regulate T cell immune responses either for immunosuppression in autoimmunity, transplantation and allergy or for augmentation in vaccines, chronic infections and cancer. Because Tob represents a potential tumor suppressor and Tob deficient mice develop malignant lymphomas, such studies might also provide insights to the pathophysiology of lymphoid malignancies. Thus, strategies to regulate Tob expression might be useful in controlling leukemia and lymphoma. To achieve these objectives I propose three specific aims to 1) Determine the mechanisms that regulate Tob mRNA expression; 2) Determine the biochemical signaling pathways involved in transcriptional and post-translational regulation of Tob; 3) Determine the role of Tob in regulating T cell immune responses in the intact host, by using transgenic mice for Tob
Keywords: T lymphocyte, cellular respiration, gene expression, helper T lymphocyte, protein structure function, tumor suppressor protein DNA binding protein, binding site, biological signal transduction, cellular immunity, gene expression profiling, genetic regulation, genetic transcription, immune tolerance /unresponsiveness, phosphorylation, posttranslational modification, transcription factor, ubiquitin DNA footprinting, chromatin immunoprecipitation, genetically modified animal, human subject, laboratory mouse
Project start date: 2005-05-01
Project end date: 2010-03-31
5R01CA104596-03 (2007): $316479
5R01CA104596-02 (2006): $325931
1R01CA104596-01A2 (2005): $333775
Related Publications
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Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo. J Immunol. 2005 Sep 1; 175( 5): 3133-9. PMID: 16116203 CD4+CD25+ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy. Blood. 2005 Nov 1; 106( 9): 3068-73. Epub 2005 Jul 14. PMID: 16020508 Interleukin-7 in T-cell acute lymphoblastic leukemia: an extrinsic factor supporting leukemogenesis? Leuk Lymphoma. 2005 Apr; 46( 4): 483-95. Review. PMID: 16019476 Common gamma chain-signaling cytokines promote proliferation of T-cell acute lymphoblastic leukemia. Haematologica. 2004 Dec; 89( 12): 1459-67. PMID: 15590396 Development and validation of a clinical prediction rule for bone marrow involvement in patients with Hodgkin lymphoma. Blood. 2005 Mar 1; 105( 5): 1875-80. Epub 2004 Nov 9. PMID: 15536150 RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev Cell. 2004 Oct; 7( 4): 585-95. PMID: 15469846 Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev Cell. 2004 Oct; 7( 4): 571-83. PMID: 15469845 Intrinsic and extrinsic regulation of T lymphocyte quiescence. Leuk Lymphoma. 2004 Oct; 45( 10): 1959-67. Review. PMID: 15370239