ITI Seed Grants 2013-14

The Stanford Institute for Immunity, Transplantation and Infection (ITI)

Announcing the Recipients of the 2013-14 ITI Seed Grants and Young Investigator Awards
funded by:

• The Marion Avery Family Seed Grant Endowment for ITI
• The Dean's office, Stanford School of Medicine
• CHRI Funding
• The Institute for Immunity, Transplantation and Infection (ITI)

2013-14 ITI Seed Grant and Young Investigator Award Abstracts

Faculty Seed Grants 2013-2014

Inflammatory and Mechanical Cues for T Cell Activation in Type 1 Diabetes
Manish Butte, MD, PhD and Paul Bollyky, PhD

Type 1 diabetes (T1D), which affects 1 in 400 children in the United States, arises when T cells destroy insulin-producing cells in the pancreatic islets. Little is understood about the inflammatory environment of the islets and how it affects autoimmune diseases like T1D. We have shown that blocking the synthesis of hyaluronic acid, a tissue mediator of edema, abolishes diabetes in a mouse model of T1D. We hypothesize that the activity of T cells in inflamed tissues is governed by its mechanical stiffness. We propose an interdisciplinary collaboration to investigate how forces and stiffness in the extracellular matrix regulate T-cell activation in T1D. To test this notion, we propose to combine the expertise in atomic force microscopy (AFM) and T-cell immunology from the Butte lab with the expertise in matrix biology and inflammation from the Bollyky’s lab. We will study the biomechanical properties of inflamed tissues and define their effects on T cell function. Overall, we seek to improve therapies for inflammatory diseases by producing deep and fundamentally new insights into the relationships between the immune system and inflammation.

Multiplex viral detection from formalin-fixed paraffin-embedded tissue samples
Benjamin Pinsky, MD, PhD

Transplantation is an increasingly common and successful therapeutic intervention; however, despite its increasing sophistication, dangerous levels of immunosuppression remain a near-universal pre-condition for graft success. As such, both solid organ and hematopoietic stem cell transplantation remain complicated by high rates of transplant-attributable morbidity and mortality from opportunistic infection. In such infections, early and definitive diagnosis is critical to treatment outcome; however, viral etiologies—the most common and clinically problematic—often remain diagnoses of exclusion or inference even after costly and time-consuming diagnostic evaluation. While clinical context can assist in some cases, many cases are diagnosed only retrospectively after empiric increase or decrease in immunosuppression, which often delays treatment, exacerbates disease, and further jeopardizes patient outcomes. This empiric approach is necessary as the available options for viral diagnosis in transplant patients are limited. There is therefore an urgent and unmet clinical need for a highly sensitive, highly specific clinical diagnostic assay capable of detecting a wide range of viruses in clinical specimens, particularly formalin-fixed paraffin-embedded (FFPE) biopsy tissues. In this proposal, we outline the development, validation, and demonstration of clinical feasibility for such an assay using padlock-based capture probes and next-generation sequencing. By matching previously published/validated informatics resources with technology already in use in the Clinical Virology and Molecular Pathology laboratories, this assay can be developed with great speed and low risk and operated clinically at low cost.

Treat Locally & Cure Systemically - A New Paradigm in Cancer Therapy: A Phase I/II Study of Intratumoral Injection of Ipilimumab In Combination with Local Radiation
Holbrook Kohrt, MD PhD, Oncology

Through the support of the ITI Seed Grant, we will perform a first-in-human clinical trial of a novel immunotherapeutic paradigm for the treatment of cancer.  A major limitation of current cancer therapies is tolerability, leading to early discontinuation of treatment and therefore limited efficacy among patients who experience toxicity.  The first approved immunotherapy for melanoma has already demonstrated striking clinical efficacy, with 1 in 10 patients cured. However, up to 1 in 2 patients develop significant toxicity. We will perform the first trial of direct injection of this therapy into the tumor, thus focusing its activity and hopefully limiting systemic toxicity. If successful, this novel approach may be extended to other cancer therapies, thereby improving patient’s quality of life while maintaining the goal of cure.

Nano-Engineered Plasmonic Chip for Point-of-Care Diagnosis of Type 1 Diabetes
Brian Feldman, MD, Pediatrics (Endocrinology)

Diabetes is the second most common chronic disease of children, and the incidences of both type 1 and type 2 diabetes are rising worldwide. The prevalence of autoimmune type 1 diabetes (T1D) in children is predicted to increase by 70% between 2004 and 2020, with twenty-five percent of affected children in South-east Asia. The incidence of type 2 diabetes (T2D) in children, along with the prevalence of obesity, has also been increasing exponentially since the early 1990s. The SEARCH for Diabetes in Youth epidemiologic study in the US demonstrated that about 25% of new diabetics less than 20 years in age have type 2. This concurrent rise has resulted in a worldwide diagnostic dilemma and it is no longer clear what type of diabetes a symptomatic child has at disease presentation. Unfortunately, this testing is cumbersome, is not available in resource-poor settings, and results are not available until days to weeks after a treatment plan has been selected. A simple point-of-care diabetes autoantibody test that is reliable, and easily used and interpreted by staff in any clinical setting, would fundamentally alter the clinical approach to childhood diabetes worldwide. This project is aimed at using advances in plasmonic chips and nanotechnology to develop a novel platform that will meet this important need.

Young Investigator Awards

NOTCH-NOTCH ligand signaling in inflammatory blood vessel disease
Darya Orlova, PhD, Postdoctoral Fellow, Genetics

Inflammatory disease of human blood vessels occurs principally in two forms; atherosclerosis, now recognized as a slowly progressive inflammatory disorder that leads to vascular  remodeling,  stenosis  and  secondary  tissue  ischemia;  and  vasculitis,  a  rapidly progressing inflammatory destruction of the blood vessel wall resulting in life-threatening complications. The most frequent type of large vessel vasculitis is giant cell arteritis which causes blindness, stroke, aortic insufficiency, aortic dissection/rupture, extremity ischemia, hypertension and cardiac insufficiency. The underlying disease process is a granulomatous arteritis with CD4 T cells and macrophages occupying the wall layers of the aorta and its major branches. The overarching goal of this project is to define mechanisms underlying the interaction between T lymphocytes and stromal cells in the tissue microenvironment. Such immune-stromal interactions dictate the tropism of immune-mediated diseases. We will work in a model system of giant cell arteritis, a granulomatous vasculitis that affects the aorta and its large branches displays a stringent tissue tropism and targets only selected territories of the vascular tree. The prototypic vasculitic infiltrates emerge within the wall layers of medium and large arteries where T cells, macrophages, endothelial cells, fibroblasts and vascular smooth muscle cells interact to form granulomatous lesions. How T cells move in the 3-dimensional space of the blood vessel wall and how vascular cells regulate their behavior is currently unknown. The specific aim is to reveal the role of NOTCH receptor-ligand interactions which mediate immuno-stromal communications, amplify immune stimulation, vessel wall damage and intimal hyperplasia. Interfering with NOTCH signaling provides new avenues of suppressing vasculitic T cells and protects vascular cells from maladaptive response patterns; opening novel therapeutic strategies for large vessel vasculitis. We will develop a novel 3D scaffold system to assemble wall structures mimicking human arteries. This model system allows us to bring the study of vasculitis into new territory and quantify the disease process. We have overcome a critical barrier in studying human arteries which, due to diameter and wall structure, are difficult to model in rodents. By bioengineering multiple layers and assembling the wall structure in vitro, we can genetically manipulate the different cell populations and study cross-regulation of immune cells and vascular cells ex vivo. The project bridges translational immunology, molecular biology and biophysics to arrive at a better understanding of how immune cells function in the tissue microenvironment.

Analysis of autoreactive CD8+ T cell ligands relevant to type 1 diabetes!
Ruth Taniguchi, PhD, Postdoctoral Fellow, Microbiology & Immunology

Type 1 diabetes (T1D) is an autoimmune disease that results from T cell destruction of the beta cells in pancreatic islets.  Many important studies have demonstrated that autoreactive CD8+ T cells play a major role in the destruction of islets in T1D.  For this project, I will carry out a comprehensive analysis of the repertoire of ligands that are presented to CD8+ T cells by islets.  MHC-bound peptides eluted from human islet cells will be analyzed by mass spectrometry.  T cell specificity for these antigens in T1D and healthy donors can then be assessed by MHC class I tetramer analysis. By carrying out a comprehensive study of human islet-derived peptides by mass spectrometry, the following goals will be addressed: 1) novel antigens relevant to T1D may uncovered, and 2) differences in the repertoire of class I-bound peptides between individuals with the same HLA-A2 polymorphism will be assessed; i.e. by analyzing islets from different donors, it can be determined if there are some common HLA-A2-bound peptides and some unique to specific individuals.  Overall, these studies will serve as an invaluable resource for the design of antigen specific therapies for T1D.

Notch Signaling in Regulation of the Mucosal Vascular Addressin MAdCAM1
Helena Kiefel, PhD, Postdoctoral Fellow, Pathology

Chronic inflammation and increased immune cell recruitment into the intestine are hallmarks of inflammatory bowel disease (IBD). The exit of immune cells from the circulation is mediated through the interaction with specialized endothelial cells of "high endothelial venules" (HEV). One of the surface proteins on HEV that mediates immune cell binding to HEV in the intestine is MAdCAM1. In the inflamed gastrointestinal tract such as in IBD MAdCAM1 expression is increased, promoting immune cell infiltration. We propose that Notch signaling, a conserved cell signaling system which is involved in the specialization of blood vessels during development, controls the expression of MAdCAM1 by post-capillary venules in lymphoid tissues and in the inflamed intestine in adult mice, and thus controls immune cell trafficking into the gastrointestinal tract. Immune cell trafficking is a critical therapeutic control point in pathologic inflammation and MAdCAM1 is a major determinant of immune cell infiltration in the gut. The proposed studies will provide novel insights into the molecular mechanisms that regulate MAdCAM1 expression and may lead to novel therapeutic approaches to target IBD.

The molecular mechanism of Mycobacterium tuberculosis intracellular viability in CD271+ bone marrow stem cells
Bikul Das, PhD

This project addresses the potential importance of adult stem cell as protective niche for latent/dormant Mycobacterium tuberculosis (Mtb). Nearly 4 billion people harbor dormant Mtb in their body. Reactivation of dormant Mtb lead to the pathogenesis of pulmonary tuberculosis (PTB), a major global health problem. It is not yet clear how Mtb persist in human despite a vigorous immune response and months of anti-tubercular drug treatment. I hypothesized that dormant Mtb may reside intracellular to stem cells to evade immune reaction and anti-TB drug-induced killing. In support of this hypothesis, my colleague and I have recovered viable Mtb from the CD271+ Bone marrow mesenchymal stem cells (CD271+ BM-MSCs) in PTB subjects who completed anti-TB drug therapy  (http://med.stanford.edu/ism/2013/january/tuberculosis.html). The CD271+ BM-MSCs are immunosuppressive and expresses drug efflux pump ABCG2. In this project, I will investigate if the ABCG2 drug efflux pump is involved in the persistence of viable Mtb intracellular to CD271+ BM-MSCs following anti-TB drug therapy. I will also investigate if Mtb initiate a pathogen/host interaction to modulate the stemness of CD271+ BM-MSCs to favor pathogen’s long-term intracellular survival. I will specifically address if Mtb reprogram Mtb to an enhanced stemness reprogramming (ESR) state, a stem cell mechanism of survival that I recently reported in human embryonic stem cells (http://med.stanford.edu/ism/2012/june/altruism.html). The ESR state confers an altruistic phenotype to stem cells and this phenotype is characterized by very high intracellular anti-oxidant, ABCG2 activity and low reactive oxygen species (ROS) level. I speculate that the ESR phenotype of CD271+ BM-MSCs could provide a highly favorable microenvironment for Mtb long-term persistence. The adult stem cell niche could be a clinically relevant reservoir of pathogens including Mtb, and the results and broad conclusions of this project will be highly relevant to our effort to control and eliminate Mtb from their human reservoir.

Role of AP-transcription factors in Systemic Sclerosis, an autoimmune disease
Gerlinde Wernig, Instructor, Pathology

Systemic sclerosis (SSc, scleroderma) is a connective tissue disease characterized byexcessive extracellular matrix deposition in the skin and visceral organs and believed to have an autoimmune component, which is poorly understood. Although the molecular basis for SSc pathogenesis is unknown, hypergammaglobulinemia, polyclonal and memory B-cell hyperactivity, and altered B-cell homeostasis are found in SSc patients, with B-cell-associated transcripts up-regulated in lesional skin. Disease-specific autoantibodies reactive with DNA topoisomerase I, RNA polymerases, fibrillin-1 and further suggest that activated B cells contribute to disease pathogenesis. C-JUN belongs to the AP1 superfamily of basic leucine zipper proteins and numerous studies had described Jun as the paradigm for bZIP transforming transcription factors (1-3). Evidence for its function as an oncogene in human cancer only came in 2007 with the discovery of its amplification and overexpression in aggressive sarcomas and increased levels of c-JUN have been described in a variety of tumors(4-5). So far a comprehensive investigation of its role in reactive possible immune mediated fibrogenesis is yet to be determined (6). In silico data generated in our lab identified c-JUN as a key factor in fibrosing conditions; therefore we propose to fully assess the role of c-JUN and recently generated a novel mouse model with inducible c-JUN/JUNB expression. Our preliminary data reveal that c-JUN expression is sufficient to induce significant multiorgan fibrosis shortly after induction in a pattern reminiscent of systemic sclerosis, suggesting that therapeutic modulation of the AP-1 pathway might be relevant in fibrosing conditions such as scleroderma. Thesignaling pathways through which this rapid fibrogenesis occurs are not established yet, but our preliminary data suggest that several pathways are involved. The overall goal of this proposal is to genetically dissect c-JUN mediated and possible immune mediated fibrogenesis by fully characterizing the murine phenotype, investigating the contribution of autoimmune mediated phenomena and assess its role in human disease. Together, these studies should provide new and important insights into potential strategies for targeting the c-JUN/AP-1 axis in autoimmune mediated fibrosing conditions such as systemic sclerosis.