Michael Brown - US grants

The proposed research is directed towards elucidation of the role of membranes in the visual process. In the broadest of terms, our goal is to determine the molecular events responsible for membrane excitability in the vertebrate rod. At this membrane level, several puzzling and outstanding questions are evident, which may be relevant to a better understanding of vision and its disorders. The most immediate objectives of the project can be grouped into three general areas: (i) investigation of the structural and dynamic properties of the highly polyunsaturated phsopholipids which comprise the fundamental bilayer matrix of the retinal disk membranes, (ii) studies of the conformation of rhodopsin and the nature of its interaction with the native retinal disk membrane phospholipids, as well as synthetic phospholipids used for membrane reconstitution and detergents used for membrane solubilization, (iii) studies of the role of electrical and osmotic forces in determining the properties of the lipid and protein components of the rod outer segment disk membranes. The above problems will be approached primarily through the use of various biophysical techniques. A major emphasis will be to further develop and employ nuclear magnetic resonance (NMR) methods for the study of both the protein and lipid components of the photoreceptor membrane. Such NMR methods are highly novel and are capable of providing detailed information regarding the ordering and motional properties of membrane constituents, without the introduction of probe molecules, which may perturb the bilayer structure. Thus, we intend to investigate problems such as the role of membrane thickness, degree of polyunsaturation, osmotic forces, transmembrane electrical potential in determining the conformation and proper photochemical functionality of rhodopsin. Using these methods, we hope to provide during the next five years a fairly complete picture of the mutual interaction of lipid and protein and their relation to function in the vertebrate rod outer segment, a particularly promising model fo excitable membranes in general.

In broad terms, our goal is to determine the molecular nature of membrane-related events which underly visual excitation in the rod cells of the vertebrate retina. The most immediate objectives of this proposal can be specifically grouped as follows: (i) Chemical synthesis, isotopic labelling with 2H, and structural studies employing nuclear magnetic resonance (NMR) of highly polyunsaturated phospholipids. Such polyunsaturated phospholipids are found in extremely high levels in the vertebrate retinal rod outer segment (ROS) disk membranes; yet, little is known of their structural or functional properties. It is possible that such polyunsaturated phospholipids may play a specific but as yet undertermined role in the process of vision. (ii) Application and development of new solid-state NMR techniques for structural studies of phospholipid bilayers. High-resolution solid-state 13C NMR spectra will be obtained and new NMR relaxation methods will be developed to obtain information regarding the orientational ordering and molecular dynamics of phospholipids in both native and recombinant membranes containing rhodopsin. (iii) The photochemical function of rhodopsin in recombinant membranes will be studied using flash photolysis techniques as well as recently developed enzymatic assays. The role of structural variables such as the phospholipid acyl chain length and the degree and position of the acyl chain unsaturation on the function of rhodopsin will be systematically investigated. Using these methods, and following the approach outlined in this proposal, it is our intent to provide a fairly complete physical picture of rhodopsin-lipid interactions and their relationship to function during the proposed RCDA project period. Throughout this work, emphasis will be placed upon correlation of the structural properties of the rhodopsion-containing membranes with selected aspects of their vision-related function.

learning transfer; association learning; biological information processing; visual stimulus; cognition; perception; operant conditionings; behavioral habituation /sensitization; stimulus generalization;

Acquisition of a nuclear magnetic resonance (NMR) spectrometer for biochemical applications is proposed. This instrument will utilize a wide magnet bore and will operate at a field strength of 4.7 tesla. It will be capable of performing a variety of two-dimensional NMR experiments and will be equipped for studies of solids and solid-like materials. The proposed instrument is intended to provide new capabilities which are not presently available at the applicant institution and which will enhance many aspects of the NIH-funded research programs of the group of major users. The proposed NMR spectrometer will be utilized for in vivo and in vitro studies of metabolism, structural studies of lipid bilayers and biomembranes, and studies of membrane encapsulated viruses. Each of these projects is of a health-related nature.

In broad terms, the goal of this research is to obtain a better understanding of membrane structural properties and their relationship to biological function. These basic studies will contribute significantly to general medical research as well as to research in heart and muscle diseases. New experimental and theoretical methods will be developed for the study of the structural and dynamic properties of lipid bilayers and their interactions with cholesterol and integral membrane proteins. A series of different lipid bilayers and membranes containing the Ca2+-ATPase from sarcoplasmic reticulum will be investigated using nuclear magnetic resonance (NMR) techniques. Deuterium NMR will be used to derive order parameters for the individual bond segments of the lipid and protein molecules in the liquid-crystalline state to yield new structural information. The dependence of the spin- lattice relaxation rates on the segmental ordering, resonance frequency, and bilayer orientation will be studied using one- and two-dimensional (2-D) NMR methods. The results will then be used to critically evaluate different theoretical models for the molecular dynamics of lipid bilayers. The experimental studies will include detailed investigations of the influences of acyl chain length, polar head group, and degree of hydration on bilayer physical properties. Similar studies of phospholipid membranes containing cholesterol or the Ca2+-ATPase will be performed. Carbon-13 NMR studies of phospholipid bilayers and biomembranes will be conducted at natural abundance or with isotopically enriched samples using novel solid-state techniques. Dipolar tensors will be determined from 2-D NMR experiments to derive order parameters for the individual bond segments of the lipid and protein moieties. The carbon-13 NMR studies will also involve spin-lattice relaxation rate measurements in the laboratory- and rotating-coordinate frames. In addition, high-resolution proton and carbon-13 NMR studies of the Ca2+-ATPase and its 20 kD tryptic fragment in detergent micelles and membranes will be carried out. From these results, a unified picture for the structural dynamics of membranes will be developed. Finally, NMR studies of membranes containing the Ca2+-ATPase will be correlated with studies of their ATP-hydrolyzing and calcium translocating activities to relate their structural and dynamic properties to function.

The proposed research is directed at understanding the relationship between working memory and choice criteria in the context of a specific experimental situation, the rat radial-arm maze procedure. The radial maze involves rats choosing from a number of spatial locations, each of which initially contains a small amount of food. Rats quickly learn to avoid revisiting locations where they have already depleted the food. Previously visited locations are discriminated from unvisited locations using working memory. Characteristics of the working memory processes involved in this task have been extensively explored in previous investigations. The present experiments are designed to explore psychological process other than working memory that affect choice behavior in this maze. Data recently collected by my colleagues and me indicate that choices are also affected by the number of choices that have previously been made and by the distance that must be traveled between locations. We have interpreted these variables as affecting the choice criterion applied to potential choices, rather than affecting the quality of working memory. Such choice criterion effects may interact with memory in important and revealing ways, which will ultimately increase our understanding of fundamental aspects of memory performance, including that of humans. Thus, the proposed work is designed to understand the processes underlying these choice criterion effects and their relation to working memory.

The objective of the proposed research is to investigate the molecular basis for essential fatty acid deficiency in the retina, which is part of the brain and comprises a valuable and uniquely accessible model for the mammalian nervous system. Current knowledge indicates that long chain polyunsaturated fatty acids derived from essential omega3 fatty acids may play an important role in retinal and brain development. Studies of both rats and rhesus monkeys indicate a marked alteration of the electrical response functions of the eye due to dietary alteration of the content of membrane phospholipids containing docosahexaenoic acid (22:6omega3, abbreviated DHA). These studies have stimulated discussion regarding the question of whether long-chain polyunsaturated fatty acids such as DHA should be added to infant formulas. Moreover there is increased concern regarding the ratios of omega3 / omega6 essential fatty acids in the western diet. The process of vision in retinal rods is triggered by a conformational change of the visual protein rhodopsin embedded within the membranes of the rod outer segment (ROS). We will test the hypothesis that the properties of the retinal rod disk membrane lipid and protein constituents govern visual function through their influences on the MI-MII conformational transition of rhodopsin, the triggering event in visual excitation. The retinal ROS disk membranes are extraordinarily abundant in phospholipids, containing highly polyunsaturated fatty acidS, including DHA and arachidonic acid (20:4omega6). We propose that interactions of rhodopsin with polyunsaturated membrane lipids modulate the free energies of the MI and MII conformational states, thus altering key amplification steps involving the signal transducing G protein (transducin) and cGMP phosphodiesterase. These in turn affect closure of the cGMP-gated plasma membrane sodium channels, and subsequently the generation of a visual nerve impulse. Changes in the content of polyunsaturated membrane lipids due to diet or disease shifts the MI-MII equilibrium of photolyzed rhodopsin, and influence the electrical response of the retina. Flash photolysis methods will be applied and further developed to monitor the influences of polyunsaturated lipids on the MI-MII transition of rhodopsin in membrane recombinants. Particular emphasis will be placed upon investigating the role of both the polyunsaturated acyl chain composition as well as the polar head group composition of the membrane phospholipids. The influences of polyunsaturated membrane phospholipids on later amplification stages of the visual photoresponse will be investigated including the binding and activation of the G protein (transducin) to photolyzed rhodopsin, and subsequent activation of cGMP phosphodiesterase. Finally, nuclear magnetic resonance (NMR) spectroscopy will yield complementary knowledge of the physicochemical properties of polyunsaturated phospholipids and their interactions with visual proteins in membranes. A comprehensive picture of the role of polyunsaturated phospholipids in the visual process will be provided in relation to the results of dietary investigations of essential omega3 fatty acid deficiency in animals and humans.

9810244 Piccoli This award provides 29% of the funding required for the acquisition of an electron microprobe microanalyzer system to be installed and operated at the Center for Microanalysis at the University of Maryland. The instrument will be used by research faculty, as well as graduate and undergraduate students to perform a variety of microanalytical tasks applied to research in geology, geochemistry, ore genesis, industrial and environmental mineralogy, materials science, ceramics, chemistry and solid state physics. ***

9977095
Walker
This Major Research Instrumentation award to University of Maryland at College Park provides a multi-collector inductively coupled plasma-mass spectrometer for geochemical and cosmochemical research. It will be used for a wide variety of research projects involving elemental and isotopic composition, and will be integrated into a facility, the Isotope Geophysical Laboratory, which is heavily used by UMD and outside researchers. The project is supported by the Division of Ocean Sciences at NSF. University of Maryland will provide cost-share support for more than 40% of total project costs.
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The long-term objective of our laboratory is to understand how the innate immune system recognizes and responds to pathogen invasion at the molecular level, especially since such defenses may be controlled by genomic elements. In particular, this study will focus on NK cell- mediated viral immunity to murine cytomegalovirus (MCMV) as it is controlled by the NKO linked Cmy1 locus. Notably NK cells are important contributors of host innate immune defenses to a range of pathogens including, viruses, bacteria, and protozoan parasites, through cytokines released (e.g. IFN-gamma) and direct cell-mediated cytotoxicity mechanisms. Moreover, NK cells directly limit viral replication in mice during murine cytomegalovirus (MCMV) infection, this capacity directly correlates with host survival during MCMV challenge, and NK cell-mediated immunity is directly regulated by the Cmv1 locus. Cmv1 was recently mapped within the distal NKC, however Cmv1 candidate sequences were not reported. In preliminary data, we report that an additional NKC encoded NK receptor (Ly- 49H) may be involved in MCMV resistance, but this NKC gene is physically and genetically separate from the CMv1 locus. Hence, we will determine whether more than one NKC locus is required MCMV resistance by assessing MCMV resistance in novel intra-NKC recombinant mice and Cmv1 minus/minus mice (Specific Aim 1). Novel Cmv1 coding sequences will be selected form C57BL/6 mice and from MCMV susceptible strains of mice. These sequences will be compared structurally and biochemically to identify a best Cmv1 candidate sequence. Confirmation of Cmv1 candidate sequences will be performed using a transgenic approach (Specific Aim 2). Finally, should genetic data be obtained implicating Ly49h in MCMV resistance immunity using a transgenic approach. Furthermore, this gene will be thoroughly characterized in C57BL/6 and several MCMV susceptible mouse strains (Specific Aim 3). Thus, these studies should clarify our understanding of NK cell recognition of viral infection and Cmv1-regulated immunity, as these are not currently understood mechanistically.

DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is a systemic autoimmune disease process contributed by multiple genes in humans and mouse models. The usefulness of the mouse as a model organism for studies of factors that contribute to pathogenesis has been well accepted. To date, as many as 12 distinct loci have been implicated in mouse lupus, but strong genetic linkages have been assigned to chromosomes 1, 4, 7 and 17. Newly available genetic tools are transforming studies of autoimmune disease in mouse models and have the potential to support rational exploration of complex physiological pathways and genetic traits. Congenic mice are especially useful in this regard since they are amenable to the study of the effects of single genes or multiple tightly linked genes in genetically controlled experiments, thus enabling directed and rational approaches toward the study of multigenic traits. Moreover, congenic mice and recombinant congenic mice derived from them, provide the foundation for genetic mapping and positional gene cloning strategies. Ultimately, information and resources gained from investigation of mouse models may be exploited to identify and characterize human genes that contribute to autoimmune disease. In order to make these genetic tools available to the members of the SCOR, we have established the Mouse Genetics Core (MGC). The primary role of the Mouse Genetics Core is to maintain extant mouse models and to develop and maintain novel mouse models for studies of autoimmune disease in well characterized and appropriate genetic backgrounds as needed by SCOR Principal Investigators serving all four SCOR projects. Novel congenic, recombinant congenic and gene targeted mice will be generated using a "speed congenic" strategy utilizing genome-wide microsatellite screening sets that others and we have previously characterized. In particular, the MGC will serve as a breeding unit and genetic analysis facility for efficient and controlled propagation and selection of specified genetic traits or mutant genes in mice. Genetic characterization will include genotyping, microsatellite linkage, DNA sequencing, and gene expression analyses. In addition, the MGC will serve as an educational resource, providing information and training for all aspects of mouse breeding and handling and genetic characterization for SCOR Investigators and their laboratory personnel. Hence, the MGC will service the needs of the SCOR Investigators by providing an operational and centralized mouse core facility to maintain and develop the necessary mouse models for continued SLE investigation and will encourage expansion of biochemical and physiological studies from cell culture systems into whole animal models, facilitating linkage of basic research with preclinical studies.

Brown
EAR-0227553

This project, with counterparts in Brazil, investigates the relationship between high-pressure (HP; P > 1.0 GPa, T > 700EC) and ultrahigh-temperature (UHT; T > 900EC) granulites and 'common' granulites in the core of the Brasilia Fold Belt. The orogenic core of the Brasilia Fold Belt is composed of a metamorphic complex characterized by granulite facies rocks, and the recent discovery of HP and UHT granulites within the southern (Minas Gerais; kyanite-K-feldspar bearing granulites) and central (Goias; sapphirine-quartz and spinel-quartz bearing granulites) sectors of the belt provides the principal motivation for this research. We will map the distribution of HP (Minas Gerais) and UHT (Goias) granulite types within the metamorphic complex of the core of the Brasilia Fold Belt, and establish their spatial, temporal, structural and metamorphic relationship with 'common' granulites. We will investigate the role of melt loss and melt retention in the preservation and/or retrogression of mineral assemblages formed under conditions of extreme crustal metamorphism. Melt loss will be evaluated using distribution of leucosome, bulk rock chemistry, and pseudosections in multi-element model systems that allow the investigation of subsolidus and suprasolidus phase equilibria. We will characterize the microstructural relationships and mineral assemblages within HP and UHT granulites for a range of bulk compositions, particularly in samples that preserve disequilibrium reaction microstructures such as symplectites and reaction coronae. Peak and post-peak segments of P-T paths will be established using these data and detailed mineral chemistry in rocks with suitably low variance. Of general interest is the relationship between the development, preservation and mode of exhumation of HP and UHT granulites to the tectonics of convergent (subduction - collision) orogens. The Brasilia Fold Belt displays tectonic elements that developed due to ocean basin evolution and subduction that culminated with continent-continent collision in Neoproterozoic time. We will evaluate the results of our research within the framework of current plate tectonics theory, and propose a model that is consistent with the data and which accounts for the evolution of the Brasilia Fold Belt.

EAR-0549300
Walker

The new generation of thermal ionization mass spectrometer to be purchased via funding from this grant will allow scientists at the University of Maryland to measure certain isotopic ratios to better than +5 ppm, a substantial improvement relative to measurements with our current equipment that are limited to approximately +25 ppm. The improved measurement capability will allow us to pursue a host of new Earth science projects that require ultra-high precision analysis. Specific analytical tasks to be tackled include the ultra-high precision analysis of 186Os/188Os ratios of mantle-derived rocks from putative plume sources, such as Hawaii, the Canary Islands and the Azores. Our previous work has suggested that enrichments in 186Os in some putative plume sources may reflect prior incorporation of outer core materials. We will continue to pursue this concept utilizing the superior measurement capabilities of the new instrument. We will also use the new instrument to test this hypothesis via examination of isotopes of tungsten. Cosmochemical observations coupled with mass balance arguments imply that Earth's core is depleted in 182W compared to the silicate portion of the Earth. Very slight depletions in this isotope in plume derived magmas would potentially implicate incorporation of core material. The predicted effects are so small that only the highest precision measurements, made possible with the new instrument, provide sufficient resolution necessary for this project. Unequivocal identification of a core component in a plume-derived rock could potentially settle major questions in geodynamics.
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DESCRIPTION (provided by applicant): This proposal is based on recent experimental results with paired congenic and transgenic strains of mice with divergent missing-self MHC-I Dk recognition of murine CMV infected cells to examine the role of NK cells in virus infected animals. The broad long-term objective for the research project seeks to understand inhibitory receptor signaling in NK cells and the impact on the NK cell's ability to rapidly respond to and kill virus infected cells. An interesting question raised by the research proposal is whether NK inhibitory receptor recognition of virus infection simply permits activation receptor signals in NK cells or directly activates a signal cascade in NK cells needed in efficient effector responses and viral resistance. This distinction is important since NK cells have a profound effect on other immune cells, including the priming of virus-specific T cell effectors. A guiding hypothesis in the research proposal is that NK inhibitory receptor recognition of virus infection delivers a potent signal which permits or 'activates' rapid NK responses to immediately provide protection against further infection and begin to cultivate other immune cells needed to prime adaptive immune responses. Three specific aims are proposed: Aim 1. will determine the effect of inhibitory receptor signaling and missing-self MHC class I recognition of virus infection in NK cell-mediated resistance and enhancement of adaptive viral immunity. We will test the hypothesis that an inhibitory receptor is required in missing-self MHC-I Dk recognition of MCMV infected cells in Ly49G-null mice. Proposed experiments will examine the effect of Ly49G2 deficiency on innate virus resistance, priming of virus-specific T cell effectors, viral persistence and mortality. Aim 2. will assess the effect of inhibitory receptor signaling and missing-self recognition of virus infected target cells on membrane proximal signaling events in NK cells and identify key NK cell expression differences, which may be causally linked with innate virus resistance. We will investigate Ly49G2 inhibitory receptor signaling in the context of virus infection and the hypothesis that missing-self recognition of MHC-I Dk infected cells permits or activates a potent signal, marked by distinct biochemical changes in NK cells, to promote NK cell responsiveness. Aim 3. will perform refined genetic mapping, identification and characterization of non-MHC loci that enhance inhibitory receptor expression on NK cells and NK cell-mediated virus resistance. We will test the hypothesis that modifiers of Ly49G2 receptor expression and MCMV resistance determine how NK cells respond to infection, their capacity to recognize infected cells and consequently their impact on adaptive immunity. Relevance NK cells are vital to human health. They protect against malignancy and virus infection. NK cells recognize and respond to many different types of viruses. However, some viruses are able to evade NK cell detection (e.g. herpesviruses) and others (e.g. HIV) are able to outlast, outwit or defeat NK cells. Thus, it is critical to understand how NK cells recognize virus infected cells in the body. Recent advances in this field have remarkable promise since we know now that NK cells can use activation receptors that directly recognize virus infected cells and therefore target NK cell killing adequately. However, NK cells with activation receptors cannot restrain all virus infections. Despite this, NK cells use inhibitory receptors also to recognize and respond to viral infection. Unfortunately, an established model to investigate the effect of NK inhibitory receptors in viral infections was not available before. Our laboratory has succeeded in generating such an innovative model and this research proposal will use it to investigate the impact of NK cell inhibitory receptors and their contribution to antiviral NK cell effector functions.

[unreadable] DESCRIPTION (provided by applicant): Here we shall test the hypothesis that the retinal rod disk membrane lipid constituents govern visual function through their influences on signaling and amplification processes involving rhodopsin. Emphasis will be placed on the role of the membrane environment in modulating the Meta I-Meta II equilibrium, which is the signaling event in visual excitation. The retinal rod disk membranes are extraordinarily abundant in phospholipids containing highly polyunsaturated fatty acids, including docosahexaenoic acid (DHA; 22:6-omega-3) and arachidonic acid (20:4-omega-6). Alterations of visual function are found to occur in essential fatty acid deficiency. Biophysical methods will characterize the influences of membrane lipids on the Meta I}Meta II transition of rhodopsin. Specific Aims are to apply a multidisciplinary approach to (1) identify the membrane lipids that function as agonists or antagonists of rhodopsin signaling; (2) elucidate the properties of membrane lipid bilayers that influence the photochemical function of rhodopsin; (3) illuminate the role of lipid polyunsaturation in rhodopsin activation; (4) discover how electrostatic properties of the membrane govern rhodopsin activation; and (5) establish how the membrane lipid influences on rhodopsin are amplified in visual signaling. A time-resolved multi-wavelength approach based on an optical multi-channel analyzer (OMA) will be used to study the kinetics and mechanism of rhodopsin activation. In addition, Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and plasmon waveguide resonance (PWR) spectroscopy will elucidate the retinal environment, protein conformation, and oligomerization or association of rhodopsin in the dark, Meta I, and Meta II states. A new flexible surface model (FSM) will provide a framework for understanding how the signaling function of rhodopsin is driven by non-specific properties of the membrane phospholipids, including membrane lipid curvature and hydrophobic forces within the bilayer. The FSM describes the lipid-protein interactions in terms of a balance of the curvature deformation energy, due to elastic stress/strain of the bilayer, with the solvation energy of the proteolipid interface. An additional aspect entails the interplay of the bilayer electrostatics including the surface charge density and the electrical double layer with the above bilayer properties. The influences of polyunsaturated membrane phospholipids on later amplification stages of the visual photoresponse will be investigated, including the binding and activation of the G protein (transducin) to photolyzed rhodopsin, and subsequent activation of cGMP phosphodiesterase. In this manner, a truly comprehensive picture of the triggering and amplification steps of the visual process will be provided at the membrane level in relation to dietary investigations of essential ?3 fatty acid deficiency in humans. PUBLIC HEALTH RELEVANCE: The proposed research will investigate the molecular basis for essential fatty acid deficiency in the retina, which is part of the brain and comprises a uniquely accessible model for the mammalian nervous system. Current knowledge indicates that long chain polyunsaturated fatty acids derived from essential ?3 fatty acids play an important role in retinal and brain development involving human infants. Moreover, polyunsaturated lipids are involved in diseases such as Parkinson's disease, cardiovascular disease, cancer, aging, and other physiological and pathological anomalies. The proposed in vitro studies of the influence of the membrane lipid bilayer on rhodopsin activity will test a specific framework for explaining the effects of essential fatty acid) deficiency in the visual system at the membrane level. This work is pertinent to the role of polyunsaturated lipids in the function and dysfunction of central nervous system of humans with attendant insights that may be of eventual therapeutic benefit. [unreadable] [unreadable] [unreadable]

NK cells are needed in the body to kill virus infected cells. NK-mediated virus immunity has been linked with innate cytokines (e.g. type I interferons), sustained dendritic cells and accelerated acquisition of virus specific effector T cells in infected animals. However, little is known about how efficient NK-mediated virus immunity can regulate and shape maturation and/or activation profiles of other immune cells. This proposal is based on a new genetic model for NK-mediated virus immunity under MHC control in two recombinant congenic strains of mice referred to as R2 and R7. R2 and R7 only differ by ~300-kb in the MHC class I D subregion, but NK-mediated virus immunity is remarkable in R7 mice. The broad long-term objective for this research project seeks to examine how efficient NK-mediated virus immunity can impart dramatic control over immune cells, in addition to their critical function in innate immunity, through controlled genetic comparisons. A guiding hypothesis is that NK-mediated virus control differences in R2 and R7 will be linked with induction of adaptive immunity through regulation of a fine balance of cytokines before virus levels differ significantly. Three specific aims are proposed: Aim 1. To determine the effect of MHC regulated NK cell function on the induction and kinetics of the CD8+ T cell response to MCMV infection. CD8+ CTLs from R2 and R7 will be assessed in flow cytometric and cytotoxicity assays to ascertain the induction time course, response magnitude, activation state and effector activity after infection. Adoptive transfers with CD8+ TCR transgenic T-cells will further define the induction and the effector activity CDS T cells responding in the spleens of resistant and susceptible mice. Aim 2. To analyze the effect of NK cell-DC interactions on splenic NK cell function. Activation states for splenic NK cells and the frequency and subset distribution of CDllc+ DCs after infection of R2 and R7 will be analyzed using flow cytometry. Splenic cytokine production, especially type 1 interferon and IL-10, will be evaluated via multiplex cytokine assays. Aim 3. To examine a potential causal role for IL-10 underlying MHC regulated differences in NK cell-mediated virus immunity. A potential regulatory role for IL-10 on the induction of adaptive immunity will be evaluated in IL10GFP knock-in reporter mice and IL-10 deficient mice

DESCRIPTION (provided by applicant): NK cells are needed in the body to protect against malignancy and virus infection. Genetic factors in different individuals can affect how NK cells recognize and respond to target cells. However, there is still much to be learned about how genetic differences can actually influence NK cells and their role in virus immunity. This proposal is based on using several new MHC congenic and transgenic mouse strains generated in the PI's laboratory to model the effect(s) of MHC polymorphism on NK cell-mediated virus immunity. The broad long-term objective for the research project seeks to understand how MHC polymorphism imparts its effect on NK-mediated virus immunity, other immune cells and protection from disease in the given hosts. Published and preliminary data implicate multiple mechanisms. A guiding hypothesis for the proposal is that MHC polymorphism can influence membrane-bound inhibitory receptors displayed by NK cells and their capacity to recognize and respond to virus infection. Three specific aims are proposed: Aim 1. To determine the effect of Ly49G2 expression on NK cells needed in MHC-I Dk resistance to MCMV infection. The induction and kinetics of NK cells specifically responding to MCMV infection, and the effect such responses have on DCs and virus specific T cell effector cells will be examined to ascertain the underlying molecular and cellular virus control mechanisms. Aim 2. To determine the effect of inhibitory Ly49G2 receptor expression on NK cells needed in MHC-I Dk resistance to MCMV infection. Preliminary data support a critical role for Ly49G2 in virus resistance. Molecular, cellular and biochemical strategies will be used to examine the receptor and its impact on NK cells. Aim 3. To evaluate the impact of MHC-I Dk on licensing and inhibitory receptor signaling in Ly49G2+ NK cells for highly efficient MCMV resistance, protection of spleen DCs and prompt induction of virus specific CD8+ T cell immunity. Biochemical and genetic strategies will be used to probe inhibitory receptor signaling in normal and inhibitory signaling deficient NK cells, their acquisition of potent antiviral effector activity and their involvement in MHC-I Dk- dependent virus resistance. Relevance The importance and health relevance of the research described in this proposal is related to the establishment of a new mouse model to examine natural killer (NK) cells responding to and controlling virus infection. Achievement of the specific aims in the research proposal will advance our understanding of genetic, molecular and cellular mechanisms used by NK cells to provide early protection against virus infection and to help kindle or elicit virus specific adaptive immune responses needed in the eventual clearance of replicating virus. The research project therefore has the potential to unlock new targets of immune cell mediated therapy to heighten disease resistance and greater understanding of the intricate links between innate and adaptive immunity may be important for vaccine development strategies. PUBLIC HEALTH RELEVANCE: This research proposal uses several new MHC recombinant congenic and transgenic strains of mice generated in the PI's laboratory to model and investigate how MHC polymorphism can regulate NK cell- mediated virus immunity. Three aims are proposed: Aim 1 will delineate the effect of MHC polymorphism on NK and dendritic cell contributions in virus resistance;Aim 2 will examine the effect of an inhibitory Ly49 receptor expressed by NK cells needed in virus resistance;and Aim 3 will study the effect of MHC polymorphism on NK cell education of effector functions which can impact early resistance to virus infection through NK cells and consequently later virus-specific immune responses.

? DESCRIPTION (provided by applicant): MHC class I Dk mediates genetic resistance to murine cytomegalovirus (MCMV). The resistance effect requires a specific subset of NK cells marked by an inhibitory NK receptor, Ly49G2, that protects against viral spread. Preliminary data in the proposal has revealed a major genetic modifier, provisionally designated Cmv5, of Dk- dependent virus resistance. Cmv5 was genetically mapped nearby, but distal to the MHC. Preliminary results show that it also regulates protection of secondary lymphoid organ structure (SLO) and expansion of NK cells in spleen soon after MCMV infection. It is not known how Cmv5 spleen resilience to infection is regulated, how distinct alleles differently affect histopathological outcomes or if the genetic effect manifests through hematopoietic cells, non-hematopoietic cells or both. The peak genetic map position for Cmv5 coincides with the Trem/Trem-like gene cluster that encodes molecules known for regulating inflammation. Cmv5 is predicted to intensify virus clearance by protecting spleen SLO structure and by expanding competent NK cells to mediate specific virus clearance. A long-term goal is to identify Cmv5 and investigate how naturally selected alleles alter its basic biological functions and further shape immune responsiveness, inflammation and disease outcomes. A genetic basis for protection of SLO structure and regulation of necrosis and NK cell accumulation after virus infection is unknown. Cmv5-disparate mice will be used to explore the genetic relationship between lymphoid structure, NK cell accumulation and innate viral immunity. Cmv5 regulation of the biology of repair/regeneration in response to virus infection will be studied. Inasmuch as Trems are implicated by positional genetic mapping data, exome sequence analysis and for their proclivity to regulate inflammation, a possible role for Trem/Trem-like molecules in virus-induced inflammation and viral clearance will also be investigated. Two specific aims are proposed to address the long-term goal. Aim 1 investigates the cellular basis of Cmv5 locus dependent protection of secondary lymphoid organ (SLO) structure and NK cell accrual in spleen after MCMV infection. Cmv5 is predicted to protect stromal cells in the red and white pulp regions of the spleen. Aim1 therefore investigates the relationship between stromal cells and cytokines released by them, and their capability to support and/or regenerate SLO structure that may be further required to regulate leukocyte recruitment and retention. Aim 2 performs precision Cmv5 mapping and analysis of TREM expression, function and support of SLO structure after MCMV infection. A 16- Mb interval on mouse chr-17 is predicted to span the Cmv5 locus that regulates SLO structure, tissue necrosis and expansion of NK cells in spleen after virus infection. Inasmuch as the locus spans the Trem/Trem-like gene cluster that encodes myeloid-derived proteins needed to regulate inflammation, a further prediction is that one or more Trem / Trem-like proteins regulates SLO structure/function, NK cells expansion and enhancement of viral immunity.

Natural killer (NK) cells are essential immune effector cells due to their intrinsic capacity to detect and destroy virus-infected and malignant target cells. In control of this essential function is an array of highly polymorphic cell surface receptors that regulate NK cell effector functionality. Human genetics studies have shown that certain combined gene sets for killer immunoglobulin-related receptor (KIR) NK-cell receptors and their HLA class I ligands affect clinical outcomes in autoimmunity, infection, transplantation and pregnancy syndromes. However, the molecular and cellular bases of these effects are not understood. Our prior mouse genetics studies have also found that significant genetic interaction between gene sets for NK receptors and MHC class I ligands controls NK cell features, and possibly their capacity to mediate critical antiviral functions and protection from immune- or virus-mediated tissue damage and disease. A MHC-linked quantitative trait locus (QTL), Cmv5, that specifically regulates NK cells, secondary lymphoid organ (SLO) structures, and tissue necrosis in spleen after viral infection was also recently discovered. However, it is not known how Cmv5 traits are regulated, how distinct alleles affect histopathological outcomes, or if genetic control manifests through hematopoietic cells, non-hematopoietic cells, or both. We hypothesize that Cmv5 regulation of NK cells and lymphoid architecture during acute virus infection can profoundly impact the tempo/extent of viral clearance and tissue inflammation. Likewise, Cmv5 may affect priming of adaptive immunity, memory formation and ultimate disease outcomes. The broad long-term objectives of this research proposal, therefore, center on identifying and fully characterizing MHC I Dk, NKC and Cmv5 genetic modifiers to elucidate how genetic variation controls NK cells, SLO structures and tissue necrosis in infected tissues following acute viral infection. To achieve the objectives, Aim 1 investigates molecular and cellular bases of Cmv5s suppression of NK expansion and SLO structure protection during inflammation and pathogen infection. Classical genetics and genetic screens will be applied to precisely map a Cmv5 critical interval. Exome sequence variant analyses, and chromosome editing strategies will be applied to further minimize the interval by constructing Cmv5 interval/candidate sequence deletions and testing for loss of suppression mutations in experimental cross mice. Aim 2 explores the NKC effect on Cmv5 regulation of NK expansion and SLO protection in MCMV- infected spleen. We hypothesize that strain-specific regulation of Cmv5 effects is controlled at the level of NK cell effector functionality, and reactivity to viral infection. NKC variation, resulting in NK receptor diversity, will be carefully assessed for its effect on Cmv5 regulation of NK cells, SLO structural integrity and tissue necrosis during viral infection in congenic, transplanted and NK receptor gene-edited mouse strains. Our studies should ascertain how NKC and MHC genetic interaction specifically shapes NK cells, additional critical immune cells, and inflammatory responses, which may help to model combined KIR/HLA associations with clinical response. ! ! !

Project Summary. Infectious diseases are a major global threat to human life. Certain individuals, however, carry naturally selected genetic factors which provide essential protection against life-threatening infections. Genetic approaches thus are essential to unravel the basis of host resistance to viral infection and resulting disease. We have shown that host resistance to murine cytomegalovirus (MCMV) infection differs greatly in MA/My and C57L mice. Thus we combined classical genetics with immune cell phenotyping to screen large numbers of MA/My x C57L cross offspring for genetic modifiers of viral immunity. Our whole genome approach detected a quantitative trait locus (QTL) on distal chromosome 4 (dc4) which controls MCMV levels in spleen within days after infection. Two additional QTLs that control host body weight, or NK cells frequency in spleen during infection were separately mapped to the same locus (86.5 cM) on dc4, which hints that it harbors a critical gene or genes essential to NK cells and host resistance to viral infection. Intriguingly, dc4 overlaps a nest of Tumor Necrosis Factor Receptor Superfamily (TNFRSF) member genes which are known to regulate immune cell activities. TNF signaling via TNFRSF members is crucial in lymphoid organogenesis, lymphocyte survival and proliferation, autoimmune dis- ease, cancer and viral infection.!Indeed, several dc4 TNFR-family members and their conserved human coun- terparts have been shown to regulate natural killer (NK) cells in different virus infections. We predict variation in dc4 TNFR-family member gene expression or polymorphism might underlie antiviral immunity or host response differences observed in C57L and MA/My mice during MCMV infection. A broad, long-term objective of this project is to discover the genetic basis of dc4 control of MCMV resistance, weight loss and NK cells during MCMV infection. We will thus combine classical and exploratory genetics approaches to identify and characterize high priority dc4 candidates. In Aim 1, precision genetics will be applied to pinpoint a dc4 QTL in congenic and recombinant congenic mice. Assessment of immune cell responsiveness and MCMV control in key recombinant congenic mice will underpin gene expression screening and selection of high priority dc4 candidates based on positional mapping, allelic differences and expression variation in vital immune cells. Aim 2 examines antiviral NK cell responses, including NK activation, proliferation, survival and trafficking during MCMV infection in dc4- disparate mice. This effect will be further investigated by comparing mixed dc4 donor NK cells responding to MCMV in the same adoptive transfer hosts, followed by analysis of dc4-regulated NK cell-specific gene expres- sion. High priority candidates will be vetted in CRISPR-modified primary T cells developed to assess the effect of dc4/Tnfrsf expression variation or polymorphism in lymphocyte activities following antigen-specific stimulation with plate-bound antibodies, in co-culture assays with antigen presenting cells expressing key ligands, or in in vivo experiments that test OT-I T cells responding to Ova+ pathogens.!

This Interdisciplinary Training Program in Immunology (ITP) at the University of Virginia (UVA) provides comprehensive support for the development of predoctoral and postdoctoral trainees as the next generation of Immunology research scientists. It is based in a group of 32 outstanding faculty, whose research programs are centered in Immunology, but oriented towards infectious disease, cancer, autoimmunity, allergic disease, inflammation, immune system development, vascular biology, or neuroscience. They are from 7 University Departments and 6 Divisions of the Dept of Medicine, creating a rich interdisciplinary research environment where trainees are exposed to a range of ideas and cutting-edge technologies, augmented by the highly collegial and collaborative environment of UVA. Trainees and mentors are brought into contact through their involvement in activities supported by the ITP, including courses, workshops, seminars, Research-In-Progress presentations, and advisory committees. The ITP structure overcomes traditional departmental barriers by establishing cross- disciplinary interest in trainee activities and oversight of trainee performance and progress. The organization of graduate recruiting at UVA also supports this interdisciplinary approach, while at the same time increasing the visibility of Immunology as a Research Discipline to enhance our ability to attract outstanding applicants. Recognizing the inherently translational orientation of contemporary Immunology and the central importance of the immune system in many human pathologies, the structure and activities of the UVA ITP are also designed to bridge the gap between basic and clinical science. The mentors include a strong cohort of translational/clinical scientists who move work in animal models into patient settings and engage in trainee-related activities that expose PhD-based graduate students and postdoctoral fellows to clinical problems. Conversely, we support an MD postdoctoral fellow to develop the next generation of Physician-Scientists who will address questions in human Immunology at a basic level. Over the last 26 years, the UVA ITP has recruited an outstanding group of both pre- and postdoctoral trainees, including a strong cohort of trainees from disadvantaged groups, and has propelled them towards productive careers as research scientists. To continue this record of achievement, we request support for 6 predoctoral trainees, selected from a group of highly qualified first year graduate students who have already been highly scrutinized during initial recruitment into our umbrella Biomedical Sciences (BIMS) graduate program. We also request support for 2 postdoctoral trainees. They may hold a PhD, and be selected based on the excellence of their graduate student work, and their demonstrated commitment and productivity during an initial year of postdoctoral research. Alternatively, they may be Physician-Scientists, highly dedicated to a career in academic medicine, chosen from board-eligible or board-certified MD or MD-PhD fellows in UVA clinical departments. Recognizing the diversity of career paths available to both pre- and postdoctoral trainees, the ITP is strongly committed to supporting trainees who will pursue research intensive or related careers.