Raul M. Torres - US grants

[unreadable] DESCRIPTION (provided by the applicant): Chemokines and their receptors have emerged as important mediators of hematopoietic cell development, homeostasis, and function. However, relatively little is understood about how these molecules influence B cell development and immune response. The long-term goal of this application is to molecularly define how chemokines and their receptors participate in the selection of B cells and their response to foreign antigens as mature lymphocytes. This objective has been partially motivated by our preliminary analyses of a mouse line engineered to be deficient for a signaling molecule that regulates heptahelical receptor signaling within B lineage cells. Using these mutants in combination with wild-type, novel, and established mouse models of B cell development and function we test our central hypothesis that chemokine receptor signaling is regulated in vivo for selecting newly-generated B cells and orchestrating appropriate B cell movements during an immune response. We address this hypothesis in three specific aims the first of which examines whether chemokine receptor responsiveness is regulated during B cell development as a mechanism that aids in the selection of immature B cells. Specific Aim 2 will investigate how distinct chemokines guide mature B cells upon antigen activation during the early phase of an immune response. Successful execution of the above two Aims will illustrate how regulating chemokine responses may influence B cell biology although will provide limited insight into the molecular details of how chemokine receptor signaling is regulated within the cell. Ultimately, chemokine responses involve receptor signaling and subsequent reorganization of the actin cytoskeleton as cells migrate through a chemoattractant gradient. Specific Aim 3 investigates the molecular nature of how chemokine receptor signaling is regulated within B cells and the basis of its coupling to the actin cytoskeleton. By accomplishing the goals of this proposal, these studies will not only further our understanding of how chemokines orchestrate immune system function, but will also enhance our basic knowledge on the mechanisms leading to B cell tolerance and effective humoral responses.

[unreadable] DESCRIPTION (provided by applicant): Abstract Signals transmitted by the antigen receptor on immature or mature B lymphocytes have a direct impact on subsequent cell migration. During development in the bone marrow, antigen receptor signaling by an immature B cell determines whether the cell is rendered tolerant in the marrow microenvironment or is allowed to exit and join the peripheral lymphocyte pool. A practical requirement for either of these outcomes is that immature B cell antigen specificity dictates response to chemoattractants, adhesion, or both. However, this has not been demonstrated nor the chemoattractants identified that facilitate immature B cell exit from the marrow. Antigen receptor signaling by mature B cells in the spleen also promotes the migration of antigen-activated cells to the microenvironment best suited for the antibody response towards that antigen and progress has been made in identifying the chemoattractants participating in this movement. However, whether diverse antigens promote similar migratory responses is not clear nor do we understand how antigen receptor signaling facilitates changes in chemoattractant responsiveness. Furthermore, given that bona fide pathogens also present ligands recognized by toll-like receptors on mature B cells, how antigen receptor and toll-like receptor signaling together influence migration and antibody response has not been established. In this proposal we outline three experimental aims that explore the functional and mechanistic relationships between antigen receptor and chemoattractant receptor signaling on B lymphocytes and the consequence of this regulation on B cell selection and antibody response. To accomplish these goals we use in vitro models of antigen and chemoattractant receptor signaling and cell adhesion coupled with in vivo mouse models of B cell development, tolerance, and antibody response. In the first specific aim we ask how antigen receptor reactivity alters chemoattractant response and adhesion of immature B cells. These findings are extended in the second aim by assessing whether chemoattractants are able to influence antigen receptor signaling and which chemoattractants contribute to immature B cell exit from the bone marrow. In the last specific aim we investigate how the nature of antigen regulates the chemoattractant response of mature marginal zone B cells and whether this regulation is further influenced by toll-like receptor signaling. Throughout this study we use biochemical and genetic approaches to evaluate the molecular mechanisms by which antigen and chemoattractant receptors regulate each other and the points where these signaling pathways intersect. Together, we anticipate these experiments to define how B cell antigen receptor signaling by B lymphocytes dictates subsequent cell movement and the implications of this regulation on B cell development, selection, and humoral immunity. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The appropriate development and function of B lymphocytes is required for mounting antibody responses to foreign antigens while preventing autoimmunity. Both of these processes, development and function, rely on the ability of B lymphocytes to migrate in response to defined cues. This application studies how B lymphocytes regulate migration during their development and later during an antibody response. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The human immunodeficiency virus (HIV) continues to annually infect millions individuals with most of these infections in developing countries where current anti- retroviral therapy is not readily available or affordable. However, prospects for an effective vaccine remain viable and realistic as HIV-specific antibodies have been isolated from infected individuals that are able to confer immune protection. Moreover, our understanding about how the different mature B cell populations mount antibody responses to foreign antigen has also advanced. Thus, we argue that current vaccine strategies should be able to selectively call upon these distinct B cell subsets to elicit an appropriate anti-viral antibody response. This application, therefore, considers the mechanisms by which distinct B cell subsets mount antibody responses to propose experiments designed to enhance the production of HIV neutralizing antibodies. A major goal of this proposal is to determine whether marginal zone B cells are able to participate in the antibody response to HIV and, if so, do they participate and, if not, can they be recruited to participate. To accomplish this goal in two specific aims, we rely on in vivo and in vitro mouse models of antibody production to molecularly and functionally define the HIV envelope-specific antibodies produced by different B cell subpopulations. Specific Aim 1 characterizes the phenotype of HIV envelope-specific B cells in na[unreadable]ve and immunized mice and the antibodies they produce with respect to polyreactivity, autoreactivity and neutralization capability. In Specific Aim 2 we center our attention on the toll-like receptors expressed by B cells and how agonists specific for these receptors alter, if at all, the type of B cells recruited into the HIV antibody response and the molecular and functional properties of the antibodies produced by these cells. Human and mouse marginal zone B cells are phenotypically and functionally similar, thus we consider the use of a mouse model to study the role of marginal zone B cells in the antibody response to HIV to be appropriate. More importantly, a comparable study of human marginal zone B cells is not feasible. Thus, these experiments investigating the principles of how distinct B cell populations respond to HIV in na[unreadable]ve and immunized mice will be directly relevant to human B cell biology and will provide significant insight into HIV vaccine design strategies. The need for an effective human immunodeficiency virus (HIV) vaccine is imperative as HIV continues to infect millions of individuals annually. This proposal outlines experiments that will define the contribution of different types of B lymphocytes in the production of antibodies specific for this virus. The information gained by this study will be directly relevant to considerations in HIV vaccine design. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): B cell antigen receptor (BCR) signaling controls the development, selection and function of B lymphocytes. Work in our lab has determined that the lysophospholipid, lysophosphatidic acid (LPA), signals to the LPA5 G-protein coupled receptor expressed by B lineage cells to suppress BCR signaling and subsequent antibody response. LPA binds and signals to LPA receptors with low nanomolar affinity and as a major lysophospholipid is present in blood at high nanomolar to low micromolar concentrations. However, despite that lymphocytes express several LPA receptors; relatively little is understood about how LPA influences humoral immunity. A long-term goal of our research has been to understand how BCR-derived signals intersect with those signals transmitted via GPCRs such as chemoattractant receptors and the goal of this application is to define how LPA signaling through LPA receptors on B lineage cells regulates the development and function of B lymphocytes. LPA has also been characterized as an inflammatory lipid and whose levels are considerably elevated in a number of chronic inflammatory disorders such as cancer, autoimmunity and viral infections. We show that at these heightened LPA levels BCR signaling is further inhibited. Thus, we also investigate how pathophysiological levels of LPA alter B cell tolerance induction in the bone marrow and antibody responses by marginal zone and follicular B cell populations. To address these issues, we propose in vitro and in vivo experiments that rely on well-characterized mouse models of B cell tolerance and antibody response to elucidate the role(s) of LPA receptors on B lymphocytes during their development in the bone marrow and their function as mature B cells in the periphery. These experiments are outlined in the following Specific Aims: Aim 1: Characterize how LPA regulates immature B cell development and tolerance induction. 1A. Establish if LPA receptors guide immature B cell localization in the bone marrow during These experiments ask how LPA receptor signaling influence B lymphopoeisis and if pathological LPA levels alters central B cell tolerance. Aim 2: Characterize the molecular and cellular mechanisms that lead to LPA suppression of B cell antibody responses. Here we define the extent to which LPA regulates B cell responses and the signaling pathways used by LPA receptors to inhibit BCR signaling. In particular, we determine if antigen-specific B cell responses are suppressed by all antigens or only antigens with certain (weak) affinity. Aim 3: Characterize how local autotaxin expression influences LPA regulation of B cell responses and if inflammatory and autoimmune settings alter the regulation of its expression. These experiments are designed to define the relative contributions of locally-restricted LPA production versus global systemic LPA levels in regulating the B cell antibody response and how LPA production may be altered in inflammatory and autoimmune settings. Significance. The sphingosine-1-phosphate (S1P) lysophospholipid has emerged as a critical regulator of lymphocyte development, trafficking and localization. However, lymphocytes and most other cells of the immune system also express G-protein coupled receptors that recognize another major lysophospholipid, lysophosphatidic acid (LPA). In contrast to S1P, that serves an important role under homeostatic conditions, LPA has features of an inflammatory lipid and has been associated with a chronic inflammatory disorders. Notably, how LPA regulates immune function and, specifically, humoral immunity is largely unexplored. The successful completion of these experiments is expected to illustrate how LPA functions to regulate the development, selection and antibody response by B lymphocytes. Furthermore, as LPA-LPA receptor signaling has been associated with a number of different types of cancer and has received considerable attention for possible therapeutic intervention, these findings will also be important to inform on how such strategies might alter adaptive immunity.

PROJECT SUMMARY/ABSTRACT This application is a renewal of our T32 training grant to support the training of 6 predoctoral and 4 postdoctoral trainees in Immunology. Training is to be performed at 3 institutions, the University of Colorado, Denver (UCD) and the Barbara Davis Center for Childhood Diabetes (BDC), both located at the Anschutz Medical Campus and at National Jewish Health (NJH). All faculty on this application are primary or secondary members of the Department of Immunology & Microbiology at UCD. The number of faculty on this application have been substantially increased since our last competitive renewal (2010). The increase reflects a considerable broadening in areas in which we intend to train and now includes experts in biostatistics and analysis of large data sets as well as more faculty who are skilled in translational research. The grant will be headed by two Program Directors, Dr. Philippa Marrack and Dr. Raul Torres, whose laboratories are at NJH and UCD, respectively. This arrangement is to ensure attention is directed to faculty and trainees at both physical locations. Dr. Marrack is a recognized expert in T cell biology who has a long career in training pre-doctoral students and post-doctoral fellows and who has been PD on this training grant for the last 5 years. Dr. Torres has successfully trained a number of predoctoral and postdoctoral fellows. He has been Director of the Ph.D. training program in immunology at UCD for almost 10 years and is well known for his work on B cell biology. Responsibility for various aspects of the Training Grant will be shared by these two PDs as described in the Multiple PD Leadership Plan. Predoctoral and postdoctoral individuals will be selected for support by this Training Grant by the Program Steering Committee after application by the trainee and mentor. Selection will be based on the merit of their previous work and their proposal for future research. Trainees will be required to take a course in the Responsible Conduct in Research every 4 years, to attend and present in our weekly ?Research in Progress? forum and to complete each year an Individual Development Plan, which will be discussed with their mentor. Evaluation of progress of research and advice about career direction will be provided for predoctoral students by their Thesis Committee and for postdoctoral fellows by a mentoring committee consisting of at least 3 faculty members, chosen by the fellow. Predoctoral and postdoctoral trainees must meet their committees at least once every 6 months, and the committees must submit written reports describing the meetings. This Training Grant has a long and distinguished record, with almost all of its trainees going on to successful careers as scientists, ranging from academia to public service at NIH or the CDC to research in Biotech or with big Pharmaceutical companies. Particularly notable is its continued successful recruitment and training of a number of underrepresented minorities. The Program continues to be a major focus for immunological research in the Rocky Mountain region in particular and the USA in general.

PROJECT SUMMARY Not all autoreactive B cells are censored by central tolerance during their development. Thus, mechanisms of B cell anergy are essential for the functional silencing of autoreactive B cells that exist in the periphery in both humans and mice. However, it remains unclear why this poorly defined process of immunological tolerance allows for the temporary retention of autoreactive B cells in the periphery given that, under certain genetic and environmental settings, these cells can contribute to autoimmunity. Indeed, anergic B cells can be released from their functionally inert state but requires unique circumstances (e.g., strong TLR stimulus and highly multimerized antigen), which could presumably occur during an uncontrolled infection. As such, we propose that autoreactive anergic B cells may serve as a reserve population able to respond to pathogens not contained by an initial immune response and particularly for pathogens that aim to evade the immune response through molecular mimicry of self-antigens. Accordingly, work from our lab has recently evaluated if autoreactive B cells that are normally silenced by immune tolerance can contribute to a protective cross-reactive antibody response. To accomplish this we used both autoimmune prone B6.Sle123 mice and wild-type mice treated with pristane, a treatment characterized to impair tolerance and promote autoantibody production. These mice were immunized with HIV envelope protein (Env) and immune sera was found to neutralize tier 2 genetic subtypes of clinically relevant HIV-1, a pathogen proposed to exploit immune tolerance in order to evade the immune response. Furthermore, from these mice we isolated Env-specific neutralizing monoclonal antibodies that also recognize the H2A histone protein. Thus, the goal of this proposal is to use mouse and humanized mouse models to identify the nature and mechanisms that facilitate this antibody response by peripheral autoreactive B cells and to establish conditions that experimentally breach tolerance and promote cross-reactive autoantibody responses by anergic B cells.

PROJECT SUMMARY Cytotoxic CD8 T lymphocytes fill a crucial role in adaptive immunity by virtue of their ability to recognize and eliminate pathogen-infected cells and nascent tumors. To accomplish this important function, the T cell antigen receptor (TCR) expressed by CD8 T cells must recognize a pathogen-derived peptide in the context of the major histocompatibility complex (MHC) class I receptor (pMHC). In response to a pathogen infection this TCR-pMHC recognition event by CD8 T cells is crucial in dictating how the ensuing adaptive response manifests. Thus, TCR signaling by naïve CD8 T cells is not only important in initiating a protective response but TCR signaling by effector CD8 T cells also underlies its cytotoxic activity for the efficient elimination of infected cells. Accordingly, appropriate TCR-signaling is central for the activation, robust proliferation and function of effector and memory CD8 T cells that ultimately leads to the trafficking of pathogen-specific CD8 T cells to sites of infection and elimination of infected cells via CD8 T cell cytolytic activity. Work from our lab has revealed that an endogenous extracellular lysophospholipid, lysophosphatidic acid (LPA), signals via the LPAR5 G-protein coupled receptor expressed by mature human and mouse T cells and negatively regulates TCR signaling, proliferation and cytotoxic activity. Notably, this lipid and the secreted enzyme responsible for its synthesis are often elevated in inflammatory settings including a number of chronic pathogen infections. Yet, how LPA regulates CD8 T cell biology at these pathophysiological levels or at homeostatic endogenous levels has only been cursorily examined and is not well understood. The experiments described in this proposal are designed to provide a comprehensive understanding of the molecular mechanisms by which the LPAR5 signaling negatively impacts TCR signaling and in vivo CD8 T cell immunity. In addition, given that LPA levels are often increased in chronic infections, we also propose to determine if pathogens that establish chronic infections subvert LPA production to suppress T cell immunity and whether small molecule inhibitors are able to antagonize LPAR5 signaling to promote enhanced immunity. The successful completion of these studies is thus expected to not only extend our current understanding of how CD8 T cells are regulated to provide protective immunity against pathogen infections but also may reveal new avenues of therapeutic intervention that may enhance immunity to persistent infections of global health concern.