Jan Erikson - US grants

DESCRIPTION (Adapted from the Investigator's abstract): Systemic lupus erythematosus (SLE) is a complex disease that has a spectrum of clinical manifestations thought to be the consequence of a dysfunctional immune system. The prominent serological feature of SLE is the presence of anti-DNA antibodies. While these antibodies are useful diagnostically, neither their etiology nor their precise role in the autoimmune pathology is clear. The investigator's objectives are to define the cellular and molecular interactions that influence autoantibody expression in both healthy and autoimmune states, first, by determining how healthy animals regulate the expression of autoantibodies, and second, by defining the critical lesion(s) in these processes that lead to autoimmunity. The investigators will use immunoglobulin (Ig) transgenic (tg) mice to follow the fate of autoreactive B cells. Because anti-DNA Igs that are present in SLE are heterogeneous in terms of their apparent specificity for single-stranded (ss) versus double-stranded (ds) DNA, the investigators will use a variety of anti-DNA Ig tgs to recapitulate these different autospecificities. Specific aims are as follows: 1. Determine the relative fates of anti-ssDNA and anti-dsDNA tg B cells when they are a part of a diverse B-cell repertoire as is the case in autoimmune individuals. Evaluate how the presence of different anti-DNA Ig tgs affects the kinetics of autoantibody expression and influences the severity of renal pathology in MRL-lpr/lpr mice. 2. Examine the role of two regulators of cell death, Fas and Bcl-2, whose altered expression has been associated with the occurrence of SLE-associated autoantibodies. 3. Identify and compare cellular antigens that are recognized by autoantibodies for which the investigators have evidence of B cell tolerance and by antibodies which have been isolated from autoimmune mice. Examine the role that apoptosis plays in generating autoantigens targeted in SLE by determining if anti-nuclear antibodies from MRL-lpr/lpr mice bind to antigens exposed on apoptotic cells.

DESCRIPTION (provided by applicant): The long-term goal of this application has been to understand how autoreactive B cell responses are prevented in healthy individuals and how they are initiated and sustained in autoimmune disease. Specifically, B cells that produce anti-dsDNA antibodies (Abs) are studied. These Abs are highly significant clinically;they are one of the diagnostic criteria of the autoimmune disease Systemic Lupus Erythematosus (SLE), and they have been implicated in mediating tissue pathology. The approach has been to develop a transgenic (Tg) model using a heavy chain-only Tg (VH3H9) that can pair with endogenous light chains to generate both anti-DNA and non-DNA Abs. Here, the development of anti-dsDNA B cells can be tracked in the context of a diverse B cell repertoire in non-autoimmune and autoimmune-prone backgrounds. Taking this strategy, anti-dsDNA Abs are undetectable in the serum of BALB/c mice, yet the autoreactive B cells persist in the bone marrow and periphery with a phenotype indicative of antigen-mediated developmental arrest. In contrast, in autoimmune animals they are mature and by ten weeks of age autoAbs are detectable in the serum. Specific aims of this competitive renewal are to compare and contrast the differentiation and activation potential of anti-dsDNA B cells from non-autoimmune and autoimmune mice. The impact of signal strength on B cell differentiation pathways will be investigated using low avidity naive, TH1 and TH2 effectors. The ability of bystander T cell activation to influence anti-dsDNA B cell fate will be explored. Having demonstrated that T regulatory cells can suppress the ability of T cell help to drive autoAb production in vivo, the mechanism behind this suppression will be investigated. The extent of anti-dsDNA B cell activation and differentiation following T-independent stimulation will also be assessed both in vitro and in vivo. Specifically, the functional capabilities of B cells exposed to TLR4 and TLR9 ligands will be investigated. It is hoped that through a better understanding of autoreactive B cell activation thresholds, and the differentiation pathways that ensue as a consequence of this activation, that more rational therapeutic strategies for treating autoimmune diseases will be developed.

DESCRIPTION (provided by applicant): Anti-dsDNA antibodies (Abs) are one of the diagnostic criteria of systemic lupus erythematosus (SLE). To understand how these autoAbs are regulated in healthy individuals and to identify the mechanisms underlying their expression in autoimmunity, we have used an immunoglobulin transgenic (Ig Tg) model. The advantage of this model is that the development of anti-dsDNA B cells can be tracked in the context of a diverse B cell repertoire in non-autoimmune and autoimmune-prone (lpr/lpr) backgrounds. We present data showing that T cells play a determining role in the phenotype of anti-dsDNA B cells. Using Ab depletion to remove CD4 cells, we have demonstrated that the phenotypic changes in anti-dsDNA B cells in Fas-deficient mice - their follicular entry and maturation - are dependent on T cell help. Additionally, using a model system to provide T cell help, we can induce follicular entry, maturation, and Ab production from anti-dsDNA B cells in Fas-sufficient mice. Collectively, these results suggest that the availability of T cell help is responsible for determining whether anti-dsDNA B cells are functionally silent or become activated. Finally, we have identified unique attributes of the lpr/lpr dendritic cells (DCs) that we hypothesize play a central role in generating the (auto)reactive T cell help in vivo. Here, we propose to extend these preliminary findings to identify first, the nature of the CD4 T cell help in young Fas-deficient mice that promotes follicular entry but not terminal differentiation of anti-dsDNA B cells, and the requirements for T cell help in promoting autoAb production in 10-week-old animals. We will test the ability of distinct classes of T cells to provide T cell help to BALB/c anti-dsDNA B cells in Fas-sufficient mice and to alter the kinetics of autoAb production in lpr/lpr mice. Second, we will examine DC function in lpr/lpr mice, and determine the extent to which alterations in DC phenotype in lpr/lpr mice are involved in the initiation of autoimmunity or are secondary to the dysregulation of T and/or B cells. Defining the processes that release certain self-reactive B cells from their tolerant state is critical to the understanding and eventual treatment of autoimmune disease.

DESCRIPTION (provided by applicant): Influenza virus continues to be a major threat to human health. Current influenza virus vaccines aim to induce a strong antibody (Ab) response to the viral glycoproteins as these Abs are known to be highly protective when present at sufficient concentration in serum and respiratory tract secretions. The main problem inherent in these vaccines is that they target viral structures that are highly variable. Therefore, their effectiveness depends largely on the match between vaccine and epidemic virus strain and may be low or insignificant if the emergence of an epidemic virus strain with major alterations in its glycoproteins was not anticipated. This deficiency could be minimized by maximizing protective immune mechanisms that are broadly cross-reactive with virus strains within and between subtypes. A possible way to achieve this is through Abs directed to the ectodomain of the transmembrane protein M2 (M2e). M2e has remained highly conserved amongst human epidemic strains since 1918 to present. In the mouse model, M2e-specific Abs have been shown to be protective. Most importantly, limited evidence indicates that this response is poorly induced in humans by natural infection and current vaccines are not likely to induce it either. During the preceding granting period, we have shown that a synthetic multiple antigenic peptide (MAP) construct is effective in inducing M2e-specific Ab responses of high titer and long duration in the mouse model and for renewal, we propose to: 1) optimize the construct and immunization protocol to achieve maximum protection in the mouse model; 2) develop an ELISA for quantitative measurement of the M2e-specific Ab response in human sera; 3) investigate the propensity of influenza virus to escape anti-M2e Ab-mediated protection in vivo; and 4) study the protective activity of M2e-specific immunity in mouse models of severe pneumotropic and pantropic infections. These studies may provide better picture of the potential of M2e for human vaccination.

The Wistar Institute provides an AAALAC-accredited Animal Facility that serves faculty from the Gene Expression and Regulation, Oncogenesis, and Immunology Cancer Center programs. The facility is maintained as a modified barrier Biosafety Level 2 (BSL2) facility by highly qualified and experienced personnel. In addition to ordering and housing research animals, the facility houses the Mouse Genetics Facility. The Animal Facility supports the severe combined immunodeficiency (SCID) mouse breeding colony, quarantine housing, technical training, Intravital Imaging System (MS) 200i in vivo imaging capabilities, animal health surveillance, veterinary coverage, new employee hands-on training, and Institutional Animal Care and Use Committee (IACUC) testing. Since 2003, The Wistar Institute has invested over $1 million into the Animal Facility for structural upgrades and new equipment supporting Cancer Center programs. A new polishing chiller augments chilled water supplies for cooling, and ensures stable temperature regulation for the Facility during the hottest weather, as well as back-up chilled water supplies in the event of failure in the primary cooling system. Additional upgrades to the facility infrastructure include improved ventilation, security, and emergency capabilities. Direct Digital Control monitoring has been incorporated into all animal rooms, offering 24-hour monitoring of the temperature as well as the heating, ventilation, and air condition (HVAC) system. Research infrastructure improvements include the addition of an IVIS imager, permitting tumor growth monitoring over time at high resolution; new state-of-the-art biocontainment equipment for expanding studies involving viral vectors and other infectious agents; and a transgenic mouse suite for use by the expanding Mouse Genetics Shared Facility. Future goals reflect the needs of cancer researchers and shared facilities and include the purchase of additional bio-containment caging equipment and Class II bio-safety cabinets; an anesthesia machine; a stereomicroscope for mouse surgical procedures; and continued upgrades to security systems.

Viral infections remain a considerable cause of morbidity and mortality. Despite advances in the availability of vaccines, the mechanisms of generating and maintaining effective antiviral immunity remains incompletely understood. A major concern in this regard is influenza virus, a focal point for a number of Projects in this Program. The vaccine for influenza virus must be remade each year because it has not yet been possible to generate broadly protective immunity to this rapidly evolving RNA virus and the potential emergence of devastating pandemic influenza virus strains remains ever present. Thus, it is essential that we understand the basic principles of long-term B cell and T cell immunity to viral infections, including influenza virus, to improve existing, and develop novel, vaccination strategies. A major complication with influenza virus infection is co-infection with bacteria resulting in bacterial pneumonia. It is unclear how such a co-infection with a virus and bacterial pathogen in the respiratory tract impacts antiviral immunological memory and future protective immunity to the virus. To address these questions, the research Projects will utilize common infectious agents. The overall goal of Core B is to generate, standardize, store and provide infectious agents to the Projects. In addition, Core B will optimize and validate assays to monitor the burden of infectious agents in tissues from infected animals. Thus, the specific Aims for this core are: 1) To define a model of respiratory infection with Streptococcus pneumonia (Sp) and coinfection with Sp and influenza virus;2) To generate and characterize Streptococcus pneumoniae expressing CD4 T cell determinant(s) from influenza virus HA;and 3) To produce, standardize, maintain and store infectious agents. The activities of this Core will therefore create synergy and interaction between the different Projects by creating a common and easily accessible set of tools, and by enhancing the quality, standardization and reproducibility of the data generated by the individual Projects.