Yiping Yang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Dr. Yang?s long-term career goal is to pursue translational research in the development of novel therapies for the malignancies. His immediate term career objective is to explore antigendefined immunotherapeutic approaches for Epstein Barr virus (EBV)-associated malignancies. EBV(+) tumors offer a unique opportunity to develop antigendefined immunotherapeutic strategies because specific EBV antigens expressed in tumor cells can serve as validated targets for T cell mediated specific tumor killing. The overall objective of this proposal is to study methods of immune manipulation targeting EBV-associated tumors, specifically nasopharyngeal carcinoma (NPC) and Hodgkin?s disease (HD). Strategies to be pursued include: 1) an antigen-specific cell vaccine to induce or enhance specific T cell responses in patients with NPC; 2) ex vivo expansion of antigen-specific T cells from patients with HD or NPC using dendritic cells transduced with self-inactivating recombinant lentiviral vectors of interest; and 3) combination of in vivo vaccination and ex vivo expansion strategies in NPC patients. Success in ex vivo expansion will lead to an adoptive immunotherapeutic trial. These EBV-associated tumors are seen as models for other tumors in which tumor specific antigens have been identified and the development of novel therapeutic approaches.

DESCRIPTION (provided by applicant): Tumor-specific T cell tolerance represents one of the major challenges in cancer immunotherapy. To elicit effective anti-tumor immunity, it is necessary to develop immunotherapeutic strategies capable of overcoming T cell tolerance. In a murine model of A20 lymphoma, we have demonstrated that tumor-specific CDS tolerance is mediated by CD4+CD25+ regulatory T (TReg) cells and that reversal of established TReg cell mediated tumor-specific CDS tolerance by tumor vaccines depends on sustained Toll-like receptor (TLR) signals of innate immunity in vivo. This can be achieved by virus-based vaccines that can provide TLR signals intrinsically or mature DC vaccines with co-administration of a TLR ligand. More importantly, provision of TLR signals significantly enhances the efficacy of tumor vaccines in treating pre-established A20 lymphoma, suggesting an essential role of TLR signals in cancer immunotherapy. In this application, we will investigate the mechanisms underlying TLR-dependent reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo. Specifically, we will pursue the following five aims: 1) To determine if reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo by vaccinia virus vs. adenovirus vaccines is mediated through distinct TLR pathways; 2) To delineate the role of pro-inflammatory cytokines IL-6, IL-12 and/or IL-1 in reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo; 3)To assess the role of Type I interferons (IFNs) in overcoming TReg cell-mediated tumor-specific CD8 tolerance in vivo; 4) To investigate the role of glucocorticoid-induced TNF receptor family-related protein (GITR) and GITR ligand interaction in breaking TReg cell-mediated tumor-specific CDS tolerance in vivo; 5) To evaluate the efficacy of tumor vaccines with TLR signals in treating pre-established A20 lymphoma. The outcome of these studies should have significant impact on the design of effective immunotherapeutic strategies for treating cancer.

DESCRIPTION (provided by applicant): Vaccinia virus (VV) is the live smallpox vaccine responsible for the successful eradication of smallpox worldwide. This unparalleled success is now being threatened by bioterrorists reintroducing smallpox, against which vaccination is no longer routine. The revival of smallpox vaccination has been countermanded by relatively high incidence of severe adverse events associated with the currently used live VV vaccine. Thus, there is an imminent need to develop alternative and safe approaches to control not only the actual smallpox infection, but also potential complications from the smallpox vaccination. Critical for the development of effective strategies for the control of poxviral infections is to better understand the host's innate immune defense mechanism(s) against poxviruses in vivo. Natural killer (NK) cells represent an important component of the innate immune system and play a critical role in the control of poxviruses in vivo. However, how NK cells are activated in response to poxviral infection and the underlying mechanism(s) responsible for NK cell-mediated control of poxviruses remain largely undefined. We have shown that innate immune recognition of VV is mediated by Toll-like receptor 2 (TLR2) and that activation of the TLR2-MyD88 pathway is crucial to the control of VV in vivo. In the preliminary studies, we have further demonstrated that the TLR2-MyD88 pathway is required for the activation of NK cells upon W infection and their trafficking to the site of infection in vivo, and that stimulation of the TLR2 pathway by W directly on NK cells, but not on accessory cells such as dendritic cells (DCs), is necessary for NK cell activation in vitro. Taken together, these observations suggest a central hypothesis that direct stimulation of the TLR2-MyD88 pathway on NK cells by W is required for NK cell activation, recruitment and function in the control of W in vivo. To test this hypothesis, we will pursue the following five aims: 1) To define the role of direct TLR2 signaling in NK cell activation, trafficking, and function in vivo; 2) To determine the mechanisms underlying TLR2-dependent NK cell activation and trafficking upon VV infection; 3) To identify the W ligand(s) that triggers the TLR2 signaling pathway; 4) To investigate the role of TLR2-dependent NK cell activation in adaptive CD8+ T cell response to W infection; and 5) To study the role of the TLR2 pathway in the activation of human NK cells by VV. We expect this work will lead to a better understanding of mechanisms underlying NK cell activation, trafficking and function in response to VV infection in vivo, which in turn will provide important insights into the design; of novel strategies to effectively activate NK cell function for the control of poxviral infections.

DESCRIPTION (provided by applicant): Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for many types of hematologic malignancies. However, graft-versus-host disease (GVHD) mediated by alloreactive T cells remains the most common cause of nonrelapse-related morbidity and mortality after allo-HSCT. Although T cell depletion and other strategies of reducing T cell alloreactivity are effective in the control of GVHD, they are often associated with increased risks of tumor recurrence. Thus, it is compelling to develop strategies for enhancing anti-tumor effect without GVHD. Studies have shown that donor natural killer (NK) cell alloreactivity can exert anti-tumor effect while protecting patients against GVHD. However, it is not clear whether these donor-derived alloreactive NK cells can persist to become long-lived memory cells and protect hosts from tumor recurrence. Although immunological memory has been thought to be a hallmark of adaptive immunity, recent advance has suggested that specific subsets of NK cells can develop into long-lived and specific memory cells in models of hapten-induced contact hypersensitivity and viral infections. In a murine model of allogeneic transplantation, we showed in the preliminary studies that alloreactive NK cells can persist for a long time, possess the properties of memory cells and protect against a secondary tumor challenge. The objective of this application is to understand the mechanisms underlying the formation of alloreactive memory NK cells. We hypothesize that multiple signals are required for the formation of alloreactive NK cell memory. We will test this hypothesis with four specific aims and pursue critical elements required for the formation of alloreactive memory NK cells. By providing molecular and cellular basis for the formation of alloreactive NK cell memory, these studies will have profound implications in the development of new therapeutic strategies for cancer prevention and therapy in the setting of allogeneic stem cell transplantation. Furthermore, the principles learned here can be applied to the NK cell memory field in general for the treatment of other diseases such as pathogenic infections.

One of the major challenges in cancer immunotherapy is tumor-induced immune evasion. The mechanisms underlying this immune evasion include defective tumor-antigen CD4[+]CD25[+] regulatory T (T{Reg}) cells myeloablative chemotherapy with stem cell transplant may offer the best chance of achieving a state of minimal residual disease, and thus minimizing tumor-induced immune evasion. However, T{Reg}-mediated tumor-specific T cell tolerance persists following transplant. We have identified that in vivo stimulation of Toll-like receptors (TLRs) of the innate immunity is required for the reversal of T{Reg}-mediated suppression on tumor-specific CD8[+] T cells post-transplant. This can be achieved by conventional dendritic cell (cDC) vaccines co-administered with a TLR9 ligand, CpG in vivo. We have also discovered that vaccinia virus (W)-based vaccines can even more potently activate tumor-specific CDS T cell response post-transplant due to their unique ability to activate multiple innate immune pathways in vivo: W activates cDCs through TLR2, leading to the production of pro-inflammatory cytokines, and a TLR-independent pathway, resulting in secretion of type I interferons (IFNs); In addition, W also activates plasmacytoid DCs (pDCs) to secrete high levels of type I IFNs in a TLR-dependent manner. Taken together, these observations suggest a central hypothesis that efficient activation of innate immune system is critical for overcoming T{Reg}-mediated suppression on tumor-specific T cells through post-transplant vaccinations. To test this hypothesis, we propose to pursue the following four aims: 1) To determine if pDCs are critical for TLR9-dependent augmentation of anti-tumor CDS immunity post-transplant; 2) to delineate the role of type I IFNs in TLR-dependent reversal of T{Reg}-mediated suppression on tumor-specific CD8[+] T cells post-transplant; 3) To test if activation of multiple TLRs synergistically enhances the efficacy of post-transplant cDC vaccinations and generates long-term, protective anti-tumor immunity; 4) To investigate the role of TLRs in promoting the differentiation of hematopoietic progenitor cells into pDCs and cDCs. We expect that this work will lead to the identification of critical factors for enhancing anti-tumor T cell immunity post-transplant, and in turn will help us design new generations of effective tumor vaccines in the settings of stem cell transplant.

DESCRIPTION (provided by applicant): Vaccinia virus (VV) is the live vaccine responsible for successful eradication of smallpox worldwide. This success has led to development of recombinant VV as a vaccine vehicle for other infectious diseases and cancer. However, the development of VV vaccines for use in humans is limited by concerns about safety. Furthermore, the efficacy of VV vaccines has been limited by the pre-existing neutralizing antibodies in people who have been previously vaccinated against smallpox. Thus, there is a need to understand immunological mechanisms underlying the unique potency of VV vaccines, which in turn will help develop safer vaccines that are highly immunogenic but have minimal pathogenic potential. Recent advances in immunology have suggested a crucial role of the innate immune system in shaping adaptive immunity. We have shown that innate immune recognition of VV is mediated by Toll-like receptor 2 (TLR2) and dependent on MyD88 (a common adaptor for TLR signaling) and that the activation of TLR2-MyD88 pathway is critical for adaptive CD8 T cell immunity to VV in vivo. How activation of innate immunity promotes adaptive CD8 T cell responses to VV remains largely unknown. We have found in the preliminary studies that defective TLR signaling on APCs only moderately affected CD8 T cell priming, but not CD8 memory formation in response to VV infection in vivo and that direct TLR2 signaling on CD8 T cells enhanced their proliferation and survival in vitro. The objective of this application is to test the hypothesis that in addition to previously observed TLR-induced APC maturation, direct TLR2 signaling on CD8 T cells intrinsically is also critical for efficient CD8 T cell priming and formation of long- lived memory cells in vivo. Specifically, we will pursue the following two aims: 1) To test if direct TLR2-MyD88 signaling on CD8 T cells is required for their clonal expansion and effector differentiation following VV infection in vivo;2) To define the role of intrinsic TLR2-MyD88 signaling in regulating the formation of long-lived memory CD8 T cells in response to VV infection in vivo. The outcome of these studies will not only shed light on how innate immunity modulates the formation of long-lived protective immunity, but more importantly, lead to the development of new generations of safer, effective vaccines to combat infectious diseases including smallpox, and cancer. PUBLIC HEALTH RELEVANCE: Vaccinia virus (VV) is the live smallpox vaccine responsible for successful eradication of smallpox worldwide. This success has led to development of recombinant VV as a vaccine vehicle for other infectious diseases and cancer. However, the development of VV vaccines for use in humans is limited by concerns about safety and reduced efficacy due to the pre-existing neutralizing antibodies in people who have been previously vaccinated against smallpox. Thus, there is a need to understand immunological mechanisms underlying the unique potency of VV vaccines, which in turn will help develop safer vaccines that are highly immunogenic but have minimal pathogenicpotential. The proposed work is to investigate immunological basis of live VV vaccines in the generation of effective and long-lasting CD8 T cell immunity, which in turn will lead to the development of new generations of safer vaccines to combat infectious diseases including smallpox, and cancer in humans.

DESCRIPTION (provided by applicant): Recent advances have highlighted an important role for chronic inflammation in promoting tumor development. How chronic inflammation promotes tumor formation during the early stages of tumorigenesis remains unknown. In the preliminary studies, we investigated whether MyD88, a common adaptor for the innate immune signaling pathways, plays a role in the development of inflammation-induced, spontaneously developed hepatocellular carcinoma (HCC). We showed that hepatocyte-intrinsic MyD88 signaling is critical for the development of the precancerous dysplastic nodules and their progression to HCC. The objective of this application is to elucidate critical mechanisms governing the immune evasion by precancerous dysplastic lesions, leading to progression to HCC. Specifically, we hypothesize that precancerous dysplastic hepatocytes evade immune elimination by recruiting immunosuppressive inflammatory monocytes. We will test this hypothesis and pursue the critical tumor-derived factors involved in the recruitment of the immune suppressors. By providing how tumor-derived factors influence the anti-tumor immune response during the early time points of tumorigenesis, these studies will have profound implications in the development of new therapeutic targets for cancer prevention and therapy.

? DESCRIPTION (provided by applicant): An important goal of cancer immunotherapy is to generate long-lived memory T cells to provide long-term immunity against cancer. However, it is virtually unknown what controls the formation of tumor-specific memory T cells. Thus, understanding and exploiting mechanisms underlying the induction of long-lasting memory T cell immunity is crucial to the development of vaccines and immunotherapies for the prevention and treatment of cancers in humans. The objective of this application is to elucidate critical mechanisms governing the formation of long-lived, tumor-specific memory CD8 T cells. Specifically, we hypothesize that NK cells are critical for the generation of long-lived, tumor-specific CD8 memory T cells. We will test this hypothesis with four specific aims and pursue the mechanisms underlying NK cell-dependent formation of long-lived memory CD8 T cells in two different tumor models. By providing molecular and cellular basis for the formation of long- lived, tumor-specific CD8 memory T cells, these studies will substantially increase knowledge of critical factors responsible for the generation of long-lasting anti-tumor immunity. Thus, the outcomes of this proposal will have significant impact on the design of effective vaccine strategies for treating cancer and preventing relapses, leading to the potential cure of cancer. Furthermore, the principles learned here can be applied to the memory T cell field in general for the treatment of other diseases such as pathogenic infections.

Project Summary/Abstract Chronic graft versus host disease (cGVHD) represents the leading cause of non-relapse mortality and morbidity after allogeneic hematopoietic stem cell transplantation (HCT). It is typically clinically manifested as an autoimmune-like syndrome and systemic fibrosis. Recent studies have suggested that macrophages with an anti-inflammatory M2-phenotype are capable of promoting fibrosis in cGVHD. However, it remains largely undefined what drives the polarization of M2 macrophages at sites of cGVHD and how M2 macrophages promote GVHD-related fibrotic changes. The objective of this application is to understand the role of hedgehog (Hh) signaling in the polarization of M2 macrophages and systemic fibrosis. Specifically, we will test the hypothesis that the Hh signaling pathway is critical for M2 macrophage polarization in cGVHD target organs, which in turn promotes fibrosis through the production of fibrogenic factors. We will employ mouse models and human tissues to pursue four specific aims that will allow us to define the role of Hh signaling and the source of sonic hedgehog (SHH) in promoting pro-fibrotic macrophages in cGVHD; we will delineate molecular mechanisms and factors mediated by macrophages that lead to an ongoing pro-fibrotic state in GVHD, and end-organ fibrosis in cGVHD. The long-term objective of this project is to define the role of Hh signaling in cGVHD by providing a mechanistic understanding of its effects on macrophage polarization within GVHD target organs and its contributions to immune cell dysregulations and tissue fibrosis. Our work may lead to the development of novel therapeutic strategies for treating systemic fibrosis in cGVHD.

Project Abstract Tumor-associated macrophages (TAMs), a major component of the tumor stromal mass, demonstrate great phenotypic heterogeneity and diverse functional capabilities under the influence of local tumor microenvironment (TME). These TAMs generally display an anti-inflammatory, M2-type phenotype and can facilitate tumor growth by promoting angiogenesis, invasion and metastasis, as well as immune evasion. However, it remains largely undefined exactly how these TAMs regulate anti-tumor immune responses within the TME. The objective of this application is to understand the role of TAM-derived PD-L1/siglec-15 in inducing intratumoral CD8 T cell dysfunction, to delineate mechanisms underlying PD-L1/siglec-15 upregulation in TAMs as well as to develop novel strategies to promote intratumoral CD8 T cell infiltration and function in favor of enhancing anti-tumor immunity. The long-term objective of this project is to understand signals required for functional polarization of TAMs within the TME, and its contributions to immune cell dysregulations, cancer development and progression, which may lead to the development of novel cancer therapeutic strategies.