Awardees and Abstracts

2005 Awardees

Senior Awards

Albert Bendelac, M.D., Ph.D.
University of Chicago/HHMI
Pathology
NKT Cell Regulation of Human Asthma
Dan R. Littman, M.D., Ph.D.
NYU School of Medicine, Skirball Institute of Biomolecular Medicine/HHMI
Pathology and Microbiology
Dendritic Cell Function in Asthma Pathogenesis
Yong-Jun Liu, M.D., Ph.D.
University of Texas M.D. Anderson Cancer Center (Now at Baylor Research Institute)
Immunology
Function of TSLP-activated Dendritic Cells in the Maintenance of Human TH2 Memory T Cells
Deborah A. Meyers, Ph.D.
Wake Forest University Health Sciences
Center for Human Genomics
Gene-gene Interaction in Mouse and Man
Alexander Y. Rudensky, Ph.D.
University of Washington/HHMI (Now at Sloan-Kettering Institute)
Immunology
Genetic Analysis of the Role of Regulatory Foxp3+ T Cells in Asthma
Jenny P-Y Ting, Ph.D.
University of North Carolina at Chapel Hill
Microbiology and Immunology
Innate Immune Genes and Asthma

Junior Awards

K. Christopher Garcia, Ph.D.
Stanford University/HHMI
Microbiology and Immunology
Targeting Interfaces in the IL-4 Receptor Complex: Structure and Design of Asthma Therapeutics
Anne-Laure Perraud, Ph.D.
National Jewish Medical and Research Center
Immunology
The Oxidant and ADP-ribose Gated TRPM2 Ion Channel and the Inflammatory Response in Asthma
Christine Pham, M.D.
Washington University, St. Louis
Rheumatology
Role of Cathespins in Murine Models of Asthma

2005 Awards Project Abstracts

Albert Bendelac, M.D., Ph.D. — 2005 Senior Award

University of Chicago/HHMI

NKT Cell Regulation of Human Asthma

A central pathogenic component of asthmatic disease in humans is the bronchioalveolar infiltration by hemopoietic cell types producing TH2 cytokines and chemokines, which recruit and coordinate the various effector mechanisms involved in asthma pathogenesis. These Th2 cytokineichemokine producing cell types include CD4 T cells as well as eosinophils and basophils. While the causative agents of disease, CD4 Th2 cells are generally thought to include MHC class II-restricted, allergen peptide-specific cells, IL-4/IL-13 producing CD1d-restricted lipidspecific NKT cells have also been considered. Studies in a mouse model of OVA-induced airway hyperreactivity have suggested a crucial role for NKT cells. In humans with asthma, emerging studies have revealed a depletion of NKT cells from peripheral blood and an extraordinary level of NKT cell infiltration among the CD4 T cells recovered from bronchioalveolar lavage (BAL) as well as in situ in bronchial biopsies. These findings combined with the conspicuous lipid transfer properties of many prominent allergenic proteins and their structural homology with lipid transfer proteins involved in CD1d mediated presentation of lipids, suggest the intriguing possibility that lipid disturbances may contribute to asthma through NKT cell recruitment and activation. In this project, we propose to explore the hypothesis that NKT cells are involved in the pathogenesis of human asthma. We have assembled collaborators within the University of Chicago Asthma Research Center and have already obtained preliminary data demonstrating massive infiltration of NKT cells in the human asthmatic airway. We will first perform a general study of asthma patients and controls to determine the degree of airway infiltration by NKT cells, their functional properties and antigenic specificities. Because we have shown that NKT cells recognize the self glycosphingolipid iGb3 we will study the regulation of this NKT cell trigger in vivo and in vitro and the contribution of lung and allergen lipid transfer proteins. Finally, we will study the contribution of NKT cells in the mouse model of OVA-induced airway hyperreactivity. These studies will provide original insights into the pathogenesis of asthma and into the functions of NKT cells.

K. Christopher Garcia, Ph.D. — 2005 Junior Award

Stanford University/HHMI

Targeting Interfaces in the IL-4 Receptor Complex: Structure and Design of Asthma Therapeutics

Asthma is profoundly influenced by the cytokines IL-4 and IL-13, which interact with the IL-4 receptor (IL-4Rα), and subsequently signal through recruitment of either the common gamma chain (γc) or IL-13 receptor (IL-13Rα1). Certain immune cells release IgE antibodies in response to activation of the IL-4/13 receptors, ultimately resulting in the symptoms of allergic asthma. IL-4Rα is the recycled component of this activation system and is therefore a compelling therapeutic target, which would benefit from structural information to guide the design of drugs. The structural basis by which IL-4Rα signals through its tri-molecular receptor complexes is not known. We propose to carry out a comprehensive structural analysis of IL-4Rα complexes, in tandem with combinatorial libraries to probe the newly resolved receptor-cytokine interfaces.

Our aims are to:
  1. Determine the structure of IL-4 in complex with IL-4Rα and gc.
  2. Determine the structure of IL-4 in complex with IL-4Rα and IL-13Rα1.
  3. Determine the structure of IL-13 in complex with IL-13Rα1 and IL-4Rα.
  4. Evolve IL-4Rα antagonists, using yeast display in vitro evolution.

This set of tri-molecular complexes will reveal new receptor-ligand and receptor-receptor interfaces towards which drugs, both protein and small molecule, can be targeted. This enabling component of our proposal is a recent technological breakthrough which has resulted in determining the structure of the quaternary signaling complex of human IL-2 with its α, β and γc receptors. The methodologies developed for biophysical studies of this system will now be applied to IL-4Rα complexes.

Dan R. Littman, M.D., Ph.D. — 2005 Senior Award

NYU School of Medicine, Skirball Institute of Biomolecular Medicine/HHMI

Dendritic Cell Function in Asthma Pathogenesis

Several recent studies have suggested that dendritic cells have key roles in the pathogenesis of asthma. Dendritic cells contribute to activating allergen-specific T helper 2 cells, but they are also thought to activate regulatory T cells that prevent aberrant inflammatory responses to airway antigens. Recent studies have shown that mice with null mutations for the transcription factors Runx3 and CBFβ2, which form heterodimers that regulate numerous target genes, develop airway inflammation characteristic of asthma and display accelerated dendritic cell maturation. We will study how loss of Runx/CBFβ contributes to the development of the asthma-like syndrome and will focus on the following aims: (1) We will generate mouse strains that lack expression of Runx1, Runx3, and CBFβ in distinct myeloid and lymphoid lineages, and will determine which cells are responsible for airway inflammation. (2) We will eliminate regulatory T cells systemically and in lung by administering diphtheria toxin to mice in which diphtheria toxin receptor is expressed exclusively in regulatory T cells, and we will determine if the animals can be tolerized to airway antigen and if they become more susceptible to spontaneous and allergen-induced asthma. The influence of regulatory T cell depletion in mice with Runx/CBFβ deficiencies will be examined. (3) Genetic targets of Runx/CBFβ in different subsets of dendritic cells will be characterized by RNA expression analysis with mutant and wild type animals. We anticipate that this approach will provide insight into pathways of asthma pathogenesis and will provide novel targets for therapeutic intervention.

Yong-Jun Liu, M.D., Ph.D. — 2005 Senior Award

University of Texas M.D. Anderson Cancer Center (Now at Baylor Research Institute)

Function of TSLP-activated Dendritic Cells in the Maintenance of Human TH2 Memory T Cells

The immunopathological hallmark of allergic diseases is the infiltration and accumulation of polarized CD4 TH2 effector T cells at the sites of inflammation. Recent studies suggested that the memory-like TH2 T cells are the principle cell population responsible for the maintenance of chronic allergic inflammation and the rapid relapse of acute allergic inflammation upon re-exposure to allergens. However the nature of TH2 memory cells and mechanisms regulating their maintenance has remained elusive. Recently a subset of human CD4+ T cells that express a novel G-protein-coupled receptor for prostaglandin D2 (CRTH2) was isolated from human blood, which produced IL-4, IL-5, and IL-13, but not IFN-γ immediately upon TCR-triggering. Our microarray analyses show that human CRTH2+ T cells express the gene signatures of TH2 central memory T cells and reveal the new molecular mechanism underling the maintenance of TH2 memory T cells. We also show that DCs activated by a novel cytokine, TSLP, have the capacity to prime TH2 responses, induce more than a 10-fold expansion of the CRTH2+ T cells, and maintain their TH2 functions. Our specific aims are: 1) To determine whether CRTH2+ T cells represent TH2 memory cells; 2) to investigate the molecular mechanisms by which TSLP-activated DCs expand and maintain the CRTH2+ T cells; and 3) to compare CRTH2+ T cells from normal versus asthmatic human subjects. Isolation and characterization of TH2 memory T cells and understanding the molecular mechanisms underlying their generation, maintenance, and responses holds the future promise for developing cures for allergic diseases.

Deborah A. Meyers, Ph.D. — 2005 Senior Award

Wake Forest University Health Sciences

Gene-gene Interaction in Mouse and Man

There is evidence that multiple genes are important in determining individual susceptibility to the development of asthma. Therefore, it is important to test for gene-gene interaction in performing genetic studies of asthma. In family studies, evidence from genome wide screening and linkage analysis for interactive effects between genes on different chromosomes would facilitate gene mapping and positional cloning. In addition, previous genome wide screen linkage analyses have not shown consistent results for asthma in different family populations. Gene-by-gene interactions may contribute to this lack of reproducibility in these previous studies. Two-locus gene-gene interactions for all possible pairs of loci will be modeled across the genome in several samples of asthma families that have already been genotyped. In addition, similar analyses will be performed on two genome wide screens performed in the mouse for bronchial hyperresponsiveness. Homolog mapping will then be performed using man-mouse homology as a powerful approach to narrow chromosomal regions of interest and identify disease genes. This approach has been very productive in identifying genes for cardiovascular diseases. Evidence for these genes will be then replicated in several case-control populations. These novel approaches, utilizing data and DNA from multiple studies in both man and mouse, are needed because interactions of multiple genes influence individual risk for developing asthma.

Anne-Laure Perraud, Ph.D. — 2005 Junior Award

National Jewish Medical and Research Center

The Oxidant and ADP-ribose Gated TRPM2 Ion Channel and the Inflammatory Response in Asthma

Asthma is a disease of the immune system, resulting in a chronic inflam mation of the airways. Activated inflammatory cells recruited to the lungs release chemokines and oxidants, leading to tissue damage, and ultimately persistent airway hy per-responsiveness. Immunocytes recovered from BAL-fluid and blood samples of asthma patients have been shown to generate higher amounts of oxidants than cells from healthy subjects, further contributing to the general redox imbalance observed in the context of the disease. More recently, the concept of oxidant signaling has been intro duced, as it appears that ROS production is a common signaling event in immunology, implying that even subtle changes in the amount of generated Reactive Oxygen Species (ROS) might affect the inflammatory response. Although ROS signaling was mostly thought to be mediated by the direct oxidation of biomolecules, it has been recently shown that mitochondrial ADP-ribose production following oxidant exposure leads to the activation of the ADP-ribose gated TRPM2 ion channel. TRPM2 is the unique fusion of a Ca2+-permeable channel with an ADP-ribose hydrolase, and we have shown that TRPM2 gene expression is differentially regulated in primary mouse leukocytes, further support ing the potential role of TRPM2 and ADPR in modulating the immune response. ADP-ribose generating molecules, such as CD38, a puzzling multifunctional receptor with NAD-glycohydrolase activity, might also play a role in regulating Ca2+-entry via TRPM2. The proposed study aims at analyzing the potential involvement of the TRPM2/ADP-ribose pathway in the inflammatory response following allergen sensitization and chal lenge in a mouse model of asthma.

Christine Pham, M.D. — 2005 Junior Award

Washington University, St. Louis

Role of Cathespins in Murine Models of Asthma

Although proteases have been implicated in airway diseases, the role of cathepsins is not well studied. Cathepsin C (CtsC) is a ubiquitously expressed lysosomal protease with specific function in many effector cells, including neutrophils, mast cells, cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. In contrast, cathepsin W (CtsW) is unique among the cathepsins because of its restricted pattern of expression, namely CTL and NK cells, and its unusual endoplasmic reticulum localization. Here we showed that cathepsins are important in modulating the innate and adaptive immunity in allergen- and viral-induced airway inflammation. In a model of airway inflammation induced by ovalbumin (OVA) sensitization, we found that CtsW-/- mice developed more airway hyper-reactivity (AHR) and goblet cell hyperplasia. Similarly, CtsW-/- mice harbored significantly higher number of IFN√£-producing immune cells in their lungs following infection with the mouse parainfluenza virus type 1 (Sendai virus or SeV). Surprisingly, CtsC-/- mice display a much more attenuated response following SeV infection. These results suggest that a better understanding of the mechanisms by which these proteases control effector functions may potentially lead to novel therapeutic interventions in the treatment of airway diseases. To this end, we propose to 1) define the in vivo mechanisms by which CtsW regulates the development of allergen-induced airway inflammation and hyper-reactivity; 2) define the role of CtsW and CtsC in the development of acute and chronic airway hyper-responsiveness following SeV infection; and 3) determine the mechanisms that confer relative protection against SeV infection in the absence of CtsC.

Alexander Y. Rudensky, Ph.D. — 2005 Senior Award

University of Washington/HHMI (Now at Sloan-Kettering Institute)

Genetic Analysis of the Role of Regulatory Foxp3+ T Cells in Asthma

Chronic Th2 type responses to airborne environmental antigens or microorganisms in the lung and bronchial tissue result in allergic asthma manifested by airway inflammation and hyper-reactivity. Regulatory T cells (TR) expressing transcription factor Foxp3 serve as a critical control mechanism of autoimmunity. Recent studies have provided evidence that CD25+ TR cells likely suppress immune responses associated with the asthma pathogenesis. However, this work has been limited by the fact that CD25 is also expressed on all recently activated T cells and, unlike Foxp3, does not serve as a definitive TR marker. Furthermore, we found that the majority of TR present within the lung tissue lack or exhibit low level of CD25 expression. Therefore, TR role in asthma remains largely unknown. In this proposal we will employ genetically manipulated mice to unambiguously investigate TR dynamics and function in asthma and to dissect TR-mediated effector mechanisms. Specifically, we will investigate a role for Foxp3+ TR cells in allergic asthma using Foxp3gfp knock-in mice and mice with an inducible suicide gene "knocked" into the Foxp3 locus. We will also examine a role for TR-produced major anti-inflammatory cytokines IL-10 and TGF-β in limiting airway inflammation using mice with the TR-specific ablation of these genes. Understanding the TR mediated control of asthma will provide rationale for assessment of the immunological status of asthma patients by monitoring dynamics of Foxp3+ TR cells. In addition, our studies may facilitate development of novel therapeutic approaches to treatment of asthma by harnessing specific immunosuppressive mechanisms of TR cells.

Jenny P-Y Ting, Ph.D. — 2005 Senior Award

University of North Carolina at Chapel Hill

Innate Immune Genes and Asthma

Evolutionarily conserved pathways in host defense and immunity have been pivotal in uncovering novel bases of immunologic diseases, and in providing new drug targets. The newly discovered CATERPILLER (CLR) family share structural similarities with the NBS-LRR (nucleotide-binding sequence, leucine-rich repeat) super-family of plant disease resistance (R) proteins,,. In plants, the R proteins mediate an array of host responses to contain the spread of pathogens. Among the CLR genes, Monarch-1 (Pypaf7) is expressed by granulocytes, eosinophils, and monocytes, cells of significant relevance to asthma. Its expression is dramatically reduced by TLR2 and TLR4 agonists. Preliminary data indicate that among patients who have a history of mild asthma, the level of Monarch-1 transcript in sputum samples is reduced relative to normal controls. A reduction of Monarch-1 expression by interference RNA (RNAi) caused a profound enhancement of TLR-induced NF-kB activation, and proinflammatory cytokine/chemokine expression. These all strongly support our working hypothesis that Monarch-1 is a negative regulator of proinflammatory responses and potentially of asthma. Negative regulators of immune responses are necessary to prevent uncontrolled and overzealous responses including allergic inflammation and asthma. The goal of this proposal is to test the hypothesis that Monarch-1 is a negative regulator of allergic inflammation and asthma. The Aims are to: 1. Profile Monarch-1 expression in induced sputum samples obtained from asthmatic individuals. 2. Define the role of Monarch-1 in a mouse model of allergic inflammation. 3. Assess the binding and modulation of Monarch-1 by nucleotides. This is important because nucleotide analogs are candidates for drug therapy.