Yong-Jun Liu, Ph.D. - US grants

Thymic stromal lymphopoietin (TSLP) is a novel IL-7-like cytokine. Human TSLP (hTSLP) activates CD11+ dendritic cells (DCs) both to up-regulate MHC class II and costimulatory molecules CD40, CD80, and CD86 and to secrete the TH2-attracting chemokines TARC and MDC. TSLP-activated DCs induce the most marked antigen-specific CD4+, naive T-cell expansion, after which the T cells differentiate into TH2 effector cells that secrete high concentrations of interleukin (IL)-4, IL-5, IL-13, and tumor necrosis factor-alpha(TNF-alpha). hTSLP is mainly expressed by epithelial cells in allergic inflamed tissues. Our central hypothesis is that allergic insults caused by chemicals, microbes, and allergens initially trigger mucosal epithelial cells or skin keratinocytes, to produce TSLP. TSLP then activates Langerhans' cells (immature dendritic cells in epidermis), which migrate into the draining lymph nodes and prime allergen-specific na'fve T cells to expand and differentiate into proallergic TH2 effector cells. These TH2 effector T cells then produce IL-4, IL-5, and IL-13 that initiate an allergic inflammation by triggering immunoglobulin E (IgE) production, eosinophilia, and mucus production. Further investigation of the molecular mechanisms underlying the regulation of TSLP production by epithelial cells and TH2 differentiation induced by TSLP-activated DCs is essential for understanding the whole pathophysiological process underlying allergic responses, and hence for developing more effective therapies for allergic diseases. The specific aims of this proposal are: 1. To identify the molecular triggers of TSLP production by epithelial cells and the association of these triggers with human allergic diseases; 2. To identify the molecular regulation of TSLP receptor (TSLPR) and IL-7Ralpha co-expression; 3. To elicidate the molecular mechanisms by which TSLP-activated DCs induce TH2 differentiation.

DESCRIPTION (provided by applicant): Type 1 interferon-(alpha, beta, omega)-producing cells (IPC/pDC), or plasmacytoid dendritic cell precursors (pDC) represent 0.2-0.8 of peripheral blood mononuclear cells in both humans and mice. IPC/pDC display plasmacell morphology, have the capacity to produce a large amount of type 1 IFN following viral stimulation, and are able to differentiate into professional antigen presenting dendritic cells (called plasmacytoid DCs). Studies of HIV-infected human subjects and of a murine model of viral infection demonstrated that IPC/pDC are the key effectors in anti-viral innate immunity. In addition, recent studies have shown that patients suffering from systemic lupus erythematosis (SLE) have an elevated level of serum IFN-a, resulting from constitutively activation of IPC/pDC by DNA and anti-DNA antibody complexes in SLE patients, and contribute to the pathological processes of SLE. Therefore understanding the developmental regulation of IPC/pDCs may have important implication in controlling viral infections and autoimmune diseases. We have previously shown that FLT3-L represents a key differentiation factor for IPC/pDC development from hematopoietic stem cells in humans and mice, and developed the in vitro culture system to generate human and mouse IPC/pDC. However, the developmental steps of IPC/pDC from early hematopoietic stem cells, and its lineage relationship to lymphoid and myeloid lineages are poorly defined. The molecular regulation of IPC/Pdc development by other growth factors and signaling pathways are largely undefined. In this grant application, we have three aims: 1) To define the developmental steps and lineage origin of human IPCs;2) to define the developmental steps and lineage origin of mouse IPCs;and 3) to investigate the molecular mechanisms which regulate IPC development.

DESCRIPTION (provided by applicant): T-cell homeostasis is critical for the adaptive immune system to respond to a variety of new pathogens and for maintaining immunological memory. It contributes to the recovery of the peripheral T-cell pool after T cell depletion caused by irradiation or viral infection. In cancer patients, homeostatic expansion of tumor-specific T cells following adoptive T cell therapy in lymphopenic cancer patients induced by chemo or radiotherapy may be beneficial for the patients to control and eliminate cancers. In HIV-infected subjects, impairment of homeostatic proliferation causes severe depletion of CD4 + T cells and disease progression to AIDS. Thymic stromal lymphopoietin (TSLP) is an IL-7-1ike cytokine. We have recently demonstrated that human TSLP strongly activated CD11c+ immature myeloid DCs (TSLP-DC). TSLP-DCs induced a strong allogeneic naive CD4+ T cell proliferation and subsequent differentiation into inflammatory TH2 cells. More recently, we found that TSLP was expressed by epithelial cells of thymus and of tonsils in the absence of allergic inflammation, suggesting that human TSLP might have additional functions. We found that TSLP activated DCs could induce a robust homeostatic proliferation of autologous naive CD4+ T cells in the absence of exogenous antigens and cytokines. This finding suggests that TSLP expressed by epithelial cells in thymus and peripheral lymphoid tissues may play a critical role in DC-mediated T cell homeostasis. The objectives of this proposal are: i) to characterize homeostatic proliferation of human naive CD4+ T cells induced by autologous TSLP-activated DCs; ii) to elucidate the molecular mechanisms underlying the ability of TSLP-activated DCs to induce homeostatic T cell proliferation; and iii) to understand why TSLP-activated DCs induce inflammatory TH2 differentiation in the allogeneic system, but homeostatic T cell proliferation in the autologous system. The specific aims in this proposal determining the function of DCs and hTSLP in the regulation of human peripheral T cell homeostasis may provide new strategies in enhancing immune responses to infectious diseases and cancer, and in controlling autoimmune diseases.

DESCRIPTION (provided by applicant): This is an application for a Texas Medical Center NIH/NIAID Asthma and Allergic Diseases Cooperative Research Center that unites the University of Texas M.D. Anderson Cancer Center and Baylor College of Medicine to investigate the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. The proposed studies will test the overall hypothesis that TSLP is a master switch for developing and maintaining atopy and asthma. TSLP is a hematopoietic cytokine encoded on the human chromosome 5q22 cluster of Th2 cytokine genes, and in humans is produced predominately by epithelial cells and activated mast cells, and is expressed in the context of atopic dermatitis and allergic asthma. Genetically engineered murine models of allergic disease also support a central role for TSLP in the pathobiology of atopy. This proposal integrates four research projects, two research cores and an administrative core focused on TSLP. Project 1 will investigate the mechanisms for induction of TSLP expression by human respiratory epithelial cells and mast cells. Project 2 will determine the mechanisms by which TSLP activates myeloid dendritic cells. Project 3 will elucidate mechanisms by which TSLP activated myeloid dendritic cells propagate Th2 effecter and memory cells. Project 4 will focus on the role of inflammatory T helper cells (Thi) in the upregulation of TSLP production. These projects will be supported by an Administrative Core, an Analytical Research Core, and a Clinical Research Core. The proposed studies should provide new insight into the mechanisms of allergic disease and asthma, and potentially identify new targets for therapeutic intervention. PROJECT 1: Regulation of TSLP Expression (Huston, D.) DESCRIPTION (provided by applicant): This application for a Texas Medical Center AADCRC is an inter-disciplinary, inter-institutional, highly integrated program focused on elucidating the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. Preliminary data support an innate and adaptive two-stage model for TSLP-mediated allergic inflammation. Four integrated Projects and three Cores are proposed to investigate the overall hypothesis that TSLP is a master switch for developing and maintaining the allergic phenotype. Project 1 is focused on mechanisms regulating the expression of TSLP;Project 2 is focused on TSLP activation of myeloid dendritic cells (mDC);Project 3 is focused on mDC propagation of Th2 immunity;and Project 4 is focused on T cell feedback regulation on TSLP expression. Project 1 will test the hypothesis that aeroallergens and respiratory viral pathogens linked to atopy and allergic asthma can stimulate respiratory epithelial cells and mast cells to produce TSLP, and thereby initiate the innate immunologic cascade for allergic inflammation. Aim 1 will investigate the hypothesis that respiratory syncytial virus (RSV), rhinovirus (RV), and aeroallergens induce expression of TSLP by human respiratory epithelial cells and mast cells, in vitro. Aim 2 will investigate the hypothesis that upregulation of TSLP expression in human respiratory epithelial cells and mast cells, utilizes NF?B, AP-1, and STAT6 activation pathways. Aim 3 will investigate the in vivo effects of RSV, RV, and aeroallergens to induce expression of TSLP in patients with and without allergic rhinitis and asthma. Proposed studies will utilize established techniques for: (i) measuring TSLP by real-time PCR, in situ hybridization, immunohistology, ELISA, and bioassays;(ii) culture and analysis of human primary bronchial and nasal epithelial cells, bronchial epithelial cell lines, CD34- derived mast cells, and the LAD2 mast cell line;(iii) RSV and RV expertise through the BCM-NIH Viral Respiratory Pathogens Research Unit;(iv) analysis of nuclear gene activation, using promoter-luciferase constructs;and (v) Clinical Research studies with available patients with and without allergic rhinitis and asthma. Studies will utilize and leverage the expertise in all three Cores. The proposed studies will provide insight into the pathogenic mechanisms of allergic inflammation and define novel targets for developing innovative therapies for allergic asthma and atopy.

DESCRIPTION (provided by applicant): This is an application for a Texas Medical Center NIH/NIAID Asthma and Allergic Diseases Cooperative Research Center that unites the University of Texas M.D. Anderson Cancer Center and Baylor College of Medicine to investigate the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. The proposed studies will test the overall hypothesis that TSLP is a master switch for developing and maintaining atopy and asthma. TSLP is a hematopoietic cytokine encoded on the human chromosome 5q22 cluster of Th2 cytokine genes, and in humans is produced predominately by epithelial cells and activated mast cells, and is expressed in the context of atopic dermatitis and allergic asthma. Genetically engineered murine models of allergic disease also support a central role for TSLP in the pathobiology of atopy. This proposal integrates four research projects, two research cores and an administrative core focused on TSLP. Project 1 will investigate the mechanisms for induction of TSLP expression by human respiratory epithelial cells and mast cells. Project 2 will determine the mechanisms by which TSLP activates myeloid dendritic cells. Project 3 will elucidate mechanisms by which TSLP activated myeloid dendritic cells propagate Th2 effecter and memory cells. Project 4 will focus on the role of inflammatory T helper cells (Thi) in the upregulation of TSLP production. These projects will be supported by an Administrative Core, an Analytical Research Core, and a Clinical Research Core. The proposed studies should provide new insight into the mechanisms of allergic disease and asthma, and potentially identify new targets for therapeutic intervention. PROJECT 1: Regulation of TSLP Expression (Huston, D.) DESCRIPTION (provided by applicant): This application for a Texas Medical Center AADCRC is an inter-disciplinary, inter-institutional, highly integrated program focused on elucidating the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. Preliminary data support an innate and adaptive two-stage model for TSLP-mediated allergic inflammation. Four integrated Projects and three Cores are proposed to investigate the overall hypothesis that TSLP is a master switch for developing and maintaining the allergic phenotype. Project 1 is focused on mechanisms regulating the expression of TSLP;Project 2 is focused on TSLP activation of myeloid dendritic cells (mDC);Project 3 is focused on mDC propagation of Th2 immunity;and Project 4 is focused on T cell feedback regulation on TSLP expression. Project 1 will test the hypothesis that aeroallergens and respiratory viral pathogens linked to atopy and allergic asthma can stimulate respiratory epithelial cells and mast cells to produce TSLP, and thereby initiate the innate immunologic cascade for allergic inflammation. Aim 1 will investigate the hypothesis that respiratory syncytial virus (RSV), rhinovirus (RV), and aeroallergens induce expression of TSLP by human respiratory epithelial cells and mast cells, in vitro. Aim 2 will investigate the hypothesis that upregulation of TSLP expression in human respiratory epithelial cells and mast cells, utilizes NF?B, AP-1, and STAT6 activation pathways. Aim 3 will investigate the in vivo effects of RSV, RV, and aeroallergens to induce expression of TSLP in patients with and without allergic rhinitis and asthma. Proposed studies will utilize established techniques for: (i) measuring TSLP by real-time PCR, in situ hybridization, immunohistology, ELISA, and bioassays;(ii) culture and analysis of human primary bronchial and nasal epithelial cells, bronchial epithelial cell lines, CD34- derived mast cells, and the LAD2 mast cell line;(iii) RSV and RV expertise through the BCM-NIH Viral Respiratory Pathogens Research Unit;(iv) analysis of nuclear gene activation, using promoter-luciferase constructs;and (v) Clinical Research studies with available patients with and without allergic rhinitis and asthma. Studies will utilize and leverage the expertise in all three Cores. The proposed studies will provide insight into the pathogenic mechanisms of allergic inflammation and define novel targets for developing innovative therapies for allergic asthma and atopy.

DESCRIPTION (provided by applicant): This is an application for a Texas Medical Center NIH/NIAID Asthma and Allergic Diseases Cooperative Research Center that unites the University of Texas M.D. Anderson Cancer Center and Baylor College of Medicine to investigate the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. The proposed studies will test the overall hypothesis that TSLP is a master switch for developing and maintaining atopy and asthma. TSLP is a hematopoietic cytokine encoded on the human chromosome 5q22 cluster of Th2 cytokine genes, and in humans is produced predominately by epithelial cells and activated mast cells, and is expressed in the context of atopic dermatitis and allergic asthma. Genetically engineered murine models of allergic disease also support a central role for TSLP in the pathobiology of atopy. This proposal integrates four research projects, two research cores and an administrative core focused on TSLP. Project 1 will investigate the mechanisms for induction of TSLP expression by human respiratory epithelial cells and mast cells. Project 2 will determine the mechanisms by which TSLP activates myeloid dendritic cells. Project 3 will elucidate mechanisms by which TSLP activated myeloid dendritic cells propagate Th2 effecter and memory cells. Project 4 will focus on the role of inflammatory T helper cells (Thi) in the upregulation of TSLP production. These projects will be supported by an Administrative Core, an Analytical Research Core, and a Clinical Research Core. The proposed studies should provide new insight into the mechanisms of allergic disease and asthma, and potentially identify new targets for therapeutic intervention. PROJECT 1: Regulation of TSLP Expression (Huston, D.) DESCRIPTION (provided by applicant): This application for a Texas Medical Center AADCRC is an inter-disciplinary, inter-institutional, highly integrated program focused on elucidating the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. Preliminary data support an innate and adaptive two-stage model for TSLP-mediated allergic inflammation. Four integrated Projects and three Cores are proposed to investigate the overall hypothesis that TSLP is a master switch for developing and maintaining the allergic phenotype. Project 1 is focused on mechanisms regulating the expression of TSLP;Project 2 is focused on TSLP activation of myeloid dendritic cells (mDC);Project 3 is focused on mDC propagation of Th2 immunity;and Project 4 is focused on T cell feedback regulation on TSLP expression. Project 1 will test the hypothesis that aeroallergens and respiratory viral pathogens linked to atopy and allergic asthma can stimulate respiratory epithelial cells and mast cells to produce TSLP, and thereby initiate the innate immunologic cascade for allergic inflammation. Aim 1 will investigate the hypothesis that respiratory syncytial virus (RSV), rhinovirus (RV), and aeroallergens induce expression of TSLP by human respiratory epithelial cells and mast cells, in vitro. Aim 2 will investigate the hypothesis that upregulation of TSLP expression in human respiratory epithelial cells and mast cells, utilizes NF?B, AP-1, and STAT6 activation pathways. Aim 3 will investigate the in vivo effects of RSV, RV, and aeroallergens to induce expression of TSLP in patients with and without allergic rhinitis and asthma. Proposed studies will utilize established techniques for: (i) measuring TSLP by real-time PCR, in situ hybridization, immunohistology, ELISA, and bioassays;(ii) culture and analysis of human primary bronchial and nasal epithelial cells, bronchial epithelial cell lines, CD34- derived mast cells, and the LAD2 mast cell line;(iii) RSV and RV expertise through the BCM-NIH Viral Respiratory Pathogens Research Unit;(iv) analysis of nuclear gene activation, using promoter-luciferase constructs;and (v) Clinical Research studies with available patients with and without allergic rhinitis and asthma. Studies will utilize and leverage the expertise in all three Cores. The proposed studies will provide insight into the pathogenic mechanisms of allergic inflammation and define novel targets for developing innovative therapies for allergic asthma and atopy.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This is an application for a Texas Medical Center NIH/NIAID Asthma and Allergic Diseases Cooperative Research Center that unites the University of Texas M.D. Anderson Cancer Center and Baylor College of Medicine to investigate the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. The proposed studies will test the overall hypothesis that TSLP is a master switch for developing and maintaining atopy and asthma. TSLP is a hematopoietic cytokine encoded on the human chromosome 5q22 cluster of Th2 cytokine genes, and in humans is produced predominately by epithelial cells and activated mast cells, and is expressed in the context of atopic dermatitis and allergic asthma. Genetically engineered murine models of allergic disease also support a central role for TSLP in the pathobiology of atopy. This proposal integrates four research projects, two research cores and an administrative core focused on TSLP. Project 1 will investigate the mechanisms for induction of TSLP expression by human respiratory epithelial cells and mast cells. Project 2 will determine the mechanisms by which TSLP activates myeloid dendritic cells. Project 3 will elucidate mechanisms by which TSLP activated myeloid dendritic cells propagate Th2 effecter and memory cells. Project 4 will focus on the role of inflammatory T helper cells (Thi) in the upregulation of TSLP production. These projects will be supported by an Administrative Core, an Analytical Research Core, and a Clinical Research Core. The proposed studies should provide new insight into the mechanisms of allergic disease and asthma, and potentially identify new targets for therapeutic intervention. [unreadable] [unreadable] [unreadable] PROJECT 1: Regulation of TSLP Expression (Huston, D.) [unreadable] [unreadable] DESCRIPTION (provided by applicant): [unreadable] This application for a Texas Medical Center AADCRC is an inter-disciplinary, inter-institutional, highly integrated program focused on elucidating the role of human Thymic Stromal Lymphopoietin (TSLP) in the development and maintenance of the Th2 immune response that dominates the allergic phenotype. Preliminary data support an innate and adaptive two-stage model for TSLP-mediated allergic inflammation. Four integrated Projects and three Cores are proposed to investigate the overall hypothesis that TSLP is a master switch for developing and maintaining the allergic phenotype. Project 1 is focused on mechanisms regulating the expression of TSLP; Project 2 is focused on TSLP activation of myeloid dendritic cells (mDC); Project 3 is focused on mDC propagation of Th2 immunity; and Project 4 is focused on T cell feedback regulation on TSLP expression. Project 1 will test the hypothesis that aeroallergens and respiratory viral pathogens linked to atopy and allergic asthma can stimulate respiratory epithelial cells and mast cells to produce TSLP, and thereby initiate the innate immunologic cascade for allergic inflammation. Aim 1 will investigate the hypothesis that respiratory syncytial virus (RSV), rhinovirus (RV), and aeroallergens induce expression of TSLP by human respiratory epithelial cells and mast cells, in vitro. Aim 2 will investigate the hypothesis that upregulation of TSLP expression in human respiratory epithelial cells and mast cells, utilizes NF?B, AP-1, and STAT6 activation pathways. Aim 3 will investigate the in vivo effects of RSV, RV, and aeroallergens to induce expression of TSLP in patients with and without allergic rhinitis and asthma. Proposed studies will utilize established techniques for: (i) measuring TSLP by real-time PCR, in situ hybridization, immunohistology, ELISA, and bioassays; (ii) culture and analysis of human primary bronchial and nasal epithelial cells, bronchial epithelial cell lines, CD34- derived mast cells, and the LAD2 mast cell line; (iii) RSV and RV expertise through the BCM-NIH Viral Respiratory Pathogens Research Unit; (iv) analysis of nuclear gene activation, using promoter-luciferase constructs; and (v) Clinical Research studies with available patients with and without allergic rhinitis and asthma. Studies will utilize and leverage the expertise in all three Cores. The proposed studies will provide insight into the pathogenic mechanisms of allergic inflammation and define novel targets for developing innovative therapies for allergic asthma and atopy. [unreadable] [unreadable] [unreadable]

Type 1 interferon-producing cells (IPCs), also known as plasmacytoid dendritic cells (pDCs), have the capacity to rapidly produce a large amount of type-1 IFNs following viral stimulation. pDCs play critical roles in controlling the function of NK cells, myeloid DCs and B cells through type 1-IFNs (innate phase). After the innate phase of immune responses, pDCs undergo a functional switch from professional type 1 IFNproducing cells to professional antigen presenting mature DCs that are able to activate and regulate T cell responses (adaptive phase). Depletion of pDC/IPC was found to be associated with uncontrolled viral infection and over-activation of pDC/IPCs was found to be associated with the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis. Targeting TLR9 or TLR7 expressed on pDC leads to strong pDC activation, which promotes the function of mDC, NK cells and B cells in anti-viral or antitumor immune responses. Although the majority of the current studies on pDCs have been focused on type 1 IFNs, our recent microarray gene expression analysis reveals for the first time that pDCs not only rapidly express more than 20 different type 1 IFN subtypes (Ito T et al, Blood, 2005), but also express high levels of more than 9 different co-stimulatory molecules within the TNF super-family members, including TNF-a, LT-a, OX40L, 4-1BBL, CD27L (CD70), GITRL, TRAIL, BAFF and APRIL. The microarray data also reveal the expression of the corresponding TNFR family members in all human immune cell types, and thus establishes a potentially new immunological network between pDCs to mDCs, pDC to NK cells and pDC to B cells through members of the TNF and TNFR superfamily. The expression of TNF/TNFR family members by human immune cell types at the mRNA levels is now further confirmed at the protein levels by flowcvtometrv. We further show that GITRL expressed by pDCs activated through TLR9 costimulates NK cell activation, a case study demonstrating our ability to translate the microarrav data into protein and functional data (Hanabuchi S. Blood 2006). Our central hypothesis is that type 1 IFNs and members of the TNF superfamily expressed by activated pDCs play distinct and synergistic roles in responding to viral infection. Establishing the molecular mechanisms and networks by which pDC sense viral infection and communicate with other cell types within the immune system will generate great value in targeting pDC either negatively for blocking autoimmune diseases or positively for the development of more effective vaccine for cancer and infectious diseases. Accordingly, the specific aims of this proposal are: >Aim 1. To investigate the molecular interactions between pDC and mDCs in regulating T cell mediated immune responses >Aim 2. To investigate the molecular interactions between pDC and NK cells >Aim 3. To investigate the molecular interactions between pDCs and B lymphocytes

DESCRIPTION (provided by applicant): During the past decade, major efforts using genomic and genetic approaches have identified two major classes of innate immune receptors for sensing microbial nucleic acids: Toll-like receptors (TLR: TLR3, 7, 8, 9) and retinoic acid-inducible gene I-like helicases (RLH: RIG-I, LGP2, MDA-5). However, genomic and genetic approaches have left a major gap in our understanding on how these receptors bind nucleic acids and whether additional receptors or coreceptors exist. We decided to open this "Black Box" by taking a biochemical approach to directly isolate and characterize microbial nucleic acid-binding proteins in human plasmacytoid dendritic cells (pDCs) by CpG-DNA pull down and mass spectrometric peptide sequencing experiments. We identified two novel members of the DExD/H-box helicase family DHX36 and DHX9 that specifically bind CpG- A and CpG-B, respectively, in human pDCs. Knocking down DHX36 expression by siRNA in a pDC cell line was associated with over 50% reduction in type 1 IFN responses and abolished IRF7 nuclear translocation induced by CpG-A and knocking down DHX9 expression was associated with a diminished TNF and IL-6 response and blocking of NF-kB p50 nuclear translocation induced by to CpG-B. The DExD/H helicase family has over 50 members. Based on these preliminary data and the fact that the other viral sensors, including RIG- I-like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9- independent DNA sensors in pDCs;and 2) the DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. ) PUBLIC HEALTH RELEVANCE: By CpG-DNA pull down and mass spectrometric peptide sequencing, we identified two novel DExD/H- box helicase family members, DHX36 and DHX9, that specifically bind and respond to CpG-A and CpG-B, respectively, in human pDCs. Because many other viral sensors, including RIG-I like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9-independent DNA sensors in pDCs;and 2) DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. )

Vaccination represents one of the major successes of medicine as it has spared countless people from polio, tetanus and other acute infections. Yet, improved immunization strategies are needed to make vaccines for microbes that cause considerable morbidity . To identify novel strategies for protective vaccination we will study dendritic cells (DCs) which specialized to capture and process antigens in vivo, presenting the MHC molecules to T cells. DCs also present antigens to B cells. Maturation and subsets allow DCs to control diverse immune responses. Our long-term goal is to develop novel human vaccines based on in vivo DC-targeting. Our hypothesis is that Human Dendritic cells subsets express distinct uptake and signaling receptors that need to be mobilized in concert to provide durable immune responses leading to increased resistance to microbes at the mucosal port of entry. To this end, we have made high affinity monoclonal antibodies against several DC surface molecules and conjugated them to several influenza virus proteins. We have shown that antigens delivered to a single type of human DCs through different surface lectins induce distinct types of antigen-specific CD4+ T cell responses. The current focus is on mucosal immunity because mucosa is a major site of invasion as well as replication of pathogens, including influenza virus. Thus, the induction/activation of two major effectors, B cells and CD8+ T cells, with mucosal homing capacity is expected to limit viral replication, resulting in reduced disease burden. Furthermore, induction of CD4+ T cells with helper functions for B cells or CTLs will enhance the longevity of memory cells and the magnitude and the quality of mucosal homing effectors. We view the candidate vaccine as a bispecific antibody a) binding to two different cell surface antigens, such as specific lectin for antigen delivery and CD40 for activation, or to two different DC subsets, to harness their capacity to induce different type of immune effectors, and in addition b)TLR agonists as DC activators. We propose four projects and two technical development components which will be supported by six cores.

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cell precursors (pDCs) play a key role at the interface of innate and acquired immunity in anti-viral responses by sensing viral infection through TLR7/TLR9 and rapidly producing large amounts of type 1 interferons (IFNs). However, several recent studies have also identified pDC as critical players in oral and airway tolerance. This is consistent with the findings that pDCs are capable of inducing development of regulatory T cells (Treg), known to be immunosuppressive. pDCs infiltrate many human tumors. In breast tumors, pDC infiltration is associated with poor survival. More recent studies showed that pDC infiltration plays a critical role in the induction of immune suppression in ovarian cancer and in promoting tumor cell growth, survival and drug resistance in human multiple myeloma (MM). However, the molecular mechanisms underlying the role of pDCs in the generation and maintenance of immune tolerance and in promoting myeloma cell growth, survival and drug resistance are unknown. We have recently identified a pDC-specific receptor complex ILT7/FceR?1 (pDCR) and its ligand BST2. Signaling through pDCR induces a BCR-like signal cascade that potently inhibits TLR7/9-mediated type 1 IFN responses by pDCs. Interestingly, BST2 is highly expressed by myeloma cells, suggesting that myeloma cells may directly inhibit the innate immune function of pDCs via BST2 and ILT7 interaction. Our hypothesis is that human myeloma cells may inhibit the innate immune function of pDCs via BST2/ILT7 interaction and enhance the tolerogenic function of pDCs within the myeloma tumor microenvironment. We recently generated monoclonal antibodies to BST2, which can be used to directly kill myeloma cells by antibody-mediated cytotoxicity, block the immunosuppressive function of pDCs and promote the innate immune function of pDCs when activated by a TLR9 ligand. We propose the following aims: Aim 1 will determine whether human myeloma-infiltrating pDCs display gene and signaling signatures induced by BST2/ILT7 interaction and have enhanced function for inducing immune tolerance; Aim 2 will establish the role of ILT7 and BST2 interaction in myeloma-induced dysfunction of pDCs in the myeloma microenvironment; and finally, Aim 3 will develop a new strategy to reprogram pDCs from inducing tolerance to inducing anti-viral-like anti-tumor immunity in MM. Such studies will result in the development of novel combinational therapies, such as chemotherapy drugs plus BST2/ILT7 blocking antibodies, for the treatment of myeloma patients; they will also improve our understanding of the mechanisms and significance of simultaneously immunotargeting MM cells and pDCs to restore the immune system and maximize the efficacy of cancer treatments.

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are professional interferon (IFN)-producing cells of the immune system3-8 that are specialized in recognizing viral RNA and DNA via endosomal TLR7 and TLR9, respectively. Among the TLRs that associate with MyD88, TLR7 and TLR9 strictly depend on MyD88 for signal transduction, and reside in the endoplasmic reticulum (ER) in association with UNC93B and gp96. Engagement of TLR9 by CpG-A oligodeoxynucleotide (ODN) in the early endosomes preferentially triggers the TRAF3/IRAK1/IKK1/PI3K/IRF7 signal cascade leading to type 1 IFN responses, whereas engagement of TLR9 by CpG-B ODN in the late endosomes preferentially triggers the TRAF6/BTK/IRAK4/TAK1/IRF5/NF-kB signal cascade leading to the production of proinflammatory cytokines TNF and IL-6. However, the molecular mechanisms underlying the specialization of TLR9-mediated early endosomal IFN responses versus late endosomal proinflammatory cytokine responses are unknown. We discovered that PACSIN1, a member of the PACSIN (protein kinase C and casein kinase substrate in neurons) family, was specifically expressed by pDCs within the immune system. Our preliminary studies showed that knockdown of PACSIN1 expression in human pDCs or knockout of the Pacsin1 gene in mouse pDCs led to a great reduction in the early endosomal IFN response to CpG-A ODN without affecting the late endosomal proinflammatory cytokine response to CpG-B ODN. Our central hypothesis is that the molecular basis for the differential TLR9-mediated early and late endosomal responses to DNA is determined by the use of different adaptor molecules. The first aim will focus on the potential role of in CpG ODN trafficking by using a human pDC cell line with stable knockdown of PACSIN1 expression by shRNA and mouse pDCs derived from PACSIN-deficient mice. The second will focus on identification of PACSIN1-binding proteins by yeast two-hybrid screening and co-IP followed by mass spectrometry. The third aim will focus on further characterization of three potential PACSIN1-interacting molecules including CD2AP, MEKK4 and TRAF4. PUBLIC HEALTH RELEVANCE: This proposal will focus on the characterization of the molecular pathways in TLR9- mediated early endosomal type 1 IFN-responses to viral DNA by plasmacytoid dendritic cells (pDCs). By combining microarray gene expression analysis, yeast two-hybrid screening, co-IP, mass spectrometry and gene targeting technology, four potential molecules including PACSIN1, CD2AP, MEKK4 and TRAF4 will be studied. Understanding pDC type 1 IFN responses to DNA will be highly significant for the development of therapies for viral infectious diseases and autoimmune disorders.