David Nemazee - US grants

DESCRIPTION (Adapted from the Investigator's abstract): This is an application to explore a novel approach to the in vivo analysis of antigen receptor function. Antigen receptor transgenic mice have been extraordinarily useful in the analysis of immune specificity, but they also pose a series of limitations to certain analyses. Taking advantage of these antigen receptor transgenic systems to screen the effects of various knockout mutations has been time consuming and cumbersome, primarily because of the need for introducing the transgenes onto the knockout genetic background. In this application we suggest a novel general strategy that we will apply to two particular questions in B-cell biology. The methods are however applicable to the analysis of several lymphocytic subsets and to the study of wide range of receptor-mediated processes. Specific Aim 1. To isolate single chain antibodies reactive to portions of antigen receptor chains. Specific Aim 2. To generate transgenic mice expressing these proteins as membrane antigens on the surface of selected tissue cells. Specific Aim 3. To demonstrate the feasibility of using the mice generated in Aim 2 to analyze antigen receptor-mediated processes in vivo. The long-term goal of this work is to generate a general approach to exploit the mutational analysis of receptor signalling. These studies should have broad application to question lymphocyte activation, differentiation, and survival.

DESCRIPTION (provided by applicant): This is an R01 renewal proposal to study immune tolerance using custom designed super-antigens (Ags) reactive to immunoglobulin (Ig) constant regions. Immunological tolerance regulates unwanted responses of B and T cells to self- tissue, reducing the chances of autoimmunity and focusing the immune response on foreign antigens. SuperAgs facilitate the analysis of tolerance among normal, polyclonal populations of cells. In the present proposal, we make use of two transgenic models, one ubiquitously expressing superAg to IgM (IgM-macroself) and a second expressing in the liver a superAg to immunoglobulin ? light-chain (pAlb mice). IgM-macroself mice are a model of central tolerance in which B cell development is blocked at the immature B cell stage in the bone marrow and peripheral B cells are absent. In the pAlb model, B cells carrying ? L-chain are deleted in the periphery at a later, still semi- mature CD93+ transitional stage and fail to populate lymph nodes. Previous work has suggested that tolerance mechanisms at these checkpoints are distinct. We established a transposon insertional mutagenesis system to generate and identify mutant B cells in vivo that have escaped superAg-mediated tolerance. We identified a number of potential candidate genes and verified one candidate whose mutation allowed B cell escape. We propose to validate candidate genes regulating these two tolerance checkpoints, find additional candidates, evaluate in depth the role of Caspase 2, and investigate the use of genetic reprogramming to genetically capture escaped B cells and their mutations. The long-term goal of these studies is to develop a comprehensive understanding of all of the genetic components maintaining immune tolerance at these stages. This knowledge should facilitate treatment of autoimmune disorders and impact vaccine development.

DESCRIPTION (provided by investigator): This is a renewal application of an R01 grant to study B cell tolerance in health and autoimmunity. The focus here is on peripheral B cell tolerance. Several lines of evidence, including prior work on this grant, have shown that peripheral B cell tolerance occurs and is presumably a barrier to the development of autoimmune disease. The consensus conceptual framework to explain B cell tolerance is the 2-signal model of Bretcher and Cohn, which posits that in the absence of T cell help, BCR stimulation is a negative signal for B cells. However, this model fails to explain how B cells can respond to T-independent type II (TI-2) antigens or distinguish TI-2 antigens from multimeric self determinants. In the present grant an alternative model is offered, which proposes that B cells express inhibitory coreceptors that aid in making this distinction. This "self marker" hypothesis will be tested through the following Specific Aims: 1. Determine the differences between TI-2-responsive and -non-responsive B cell populations in terms of their gene expression and ability to undergo peripheral deletion. 2. Determine if disruption of putative self-marker signals inhibits peripheral deletion. The long-term goal of these studies is to understand how the self/non-self discrimination is made, what goes wrong in the development of autoimmunity, and to identify ways that these mechanisms may be manipulated to ameliorate or prevent disease.

[unreadable] DESCRIPTION (provided by applicant): DNA vaccines have shown promise as a means to develop meaningful CTL responses in HIV and in other settings, but antibody responses are often poor. The goal of this proposal is to employ a series of improvements that are designed to overcome the poor neutralizing antibody responses to HIV envelope (Env) DNA vaccine. The key hypotheses to be explored are as follows: i) Coexpression of the extremely potent B cell stimulatory molecules BAFF or CD40L will promote enhanced antibody responses, and physical linkage between Env and these TNF family ligands will provide an even more potent stimulus, ii) HIV gp120 or gp140 variants that mask irrelevant portions of the surface with N-linked glycans will focus the antibody response to relevant epitopes, iii) Expressing HIV gp120/gp140 genes as fusion proteins with molecules that have a strong propensity to trimerize can facilitate the expression of the most relevant form of HIV Env. iv) Oral delivery of the DNA vaccine using an attenuated Salmonella typhimurium vector will promote a strong immune response in mucosal immune tissue, v) A combination of these features will yield a more effective vaccine candidate. These goals will be approached through the following Specific Aims: 1) To determine if selected HIV gp120 variants stimulate a better antibody response to DNA vaccination when introduced along with BAFF expressed either as a fusion gene or in the unlinked form. 2) To test the ability of linked or unlinked expression of BAFF or CD40L to improve the antibody responses to DNA vaccine delivered orally by attenuated Salmonella typhimurium. While the long-term goal is to generate an effective HIV vaccine, important achievable intermediate goals will be i) to assess the adjuvanticity of BAFF and other TNF family members, ii) to determine if gp120/BAFF or gp140/BAFF fusion molecules enforce envelope protein trimerization, and iii) to determine if these bioengineering steps facilitate the production of a broadly neutralizing antibody. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is an R21 application to study a novel approach to suppress IgE-mediated hypersensitivity. While IgE plays a role in certain types of host defense, notably in reactions to multicellular parasites, such as helminth worms, its undesirable side effects in reactions to harmless, but ubiquitous, environmental antigens (Ags) are of greater clinical significance. Therefore, therapies to suppress IgE mediated hypersensitivity responses are being developed. The long-term goal of this proposal is to treat human allergy and asthma with genetic therapies to suppress IgE. [unreadable] The hypothesis of this proposal is that IgE constant region-reactive superantigen ("macroself Ag") can suppress hypersensitivity reactions when expressed in vivo, and that it can function both by sequestering free IgE and by actively tolerizing membrane IgE-expressing B cells. Because free IgE levels are normally very low, it is further hypothesized that one can induce tolerance or suppression of IgE by treatment with macroself Ag even in intact individuals after sensitization, and that this can be done using a gene therapy approach. [unreadable] These hypotheses will be approached through the generation of genes encoding macroself Ags specifically reactive to mouse and human IgE, and testing the ability of these genes to suppress IgE responses either when expressed as germline transgenes or in a gene therapy setting. Testing the function of human IgE-reactive macroself Ag will be performed in mice engineered to carry human antibody gene loci. Furthermore, IgE macroself Ag-mediated suppression by gene transfer will be assessed both during and after antigenic sensitization. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Sjogren's Syndrome (SS) is a systemic autoimmune disease associated with chronic inflammation of salivary and lacrimal glands that is characterized by autoantibody formation and B cell hyperactivity. Elevated levels of the B cell survival cytokine BAFF are associated with lupus and SS in both humans and experimental animals. The absence of BAFF, by contrast, leads to a severe deficiency in B cells. The applicant's laboratory recently discovered an inhibitory BAFF splice isoform, deltaBAFF, that has not been taken into account in most studies, but which may be a key regulator modulating the potential extreme effects of too much or too little BAFF activity. Studies from autoantibody transgenic mice have indicated that autoreactive B cells are more dependent on BAFF for survival than are non-autoreactive B cells. However, this differential dependence can only be revealed in situations in which autoreactive B cells make up a large fraction of all B cells, i.e., in the absence of cell: cell competition. Therefore, it is not clear how excess BAFF promotes autoimmunity in a polyclonal immune system. As BAFF has effects on T cells as well as B cells, it is not excluded that excess BAFF breaks B cell tolerance indirectly, through T cell dysregulation. Alternatively, BAFF may rescue only those autoantibodies of relatively low affinity. These hypotheses will be tested in the following Specific Aims: 1) To determine in a polyclonal immune system whether or not BAFF over-expression selectively rescues low affinity self-reactive clones. 2) To determine if reduction in BAFF levels by deltaBAFF over-expression leads to subnormal levels of basal autoantibody activity and more stringent self-tolerance. 3) To assess autoantibody formation and B cell tolerance in BAFF over-expressing mice lacking T cells. The long-term goal of these studies is to understand how BAFF promotes Sjogren's Syndrome and to determine the basic parameters regulating immune tolerance.

DESCRIPTION (provided by applicant): The design of a component to elicit broadly neutralizing antibodies is thought to be crucial for a successful HIV vaccine but is proving very difficult. In particular, immunogens designed from study of broadly neutralizing human monoclonal antibodies have generally failed to elicit antibodies with the appropriate specificities. A number of hypotheses have been advanced for these failures including restricted access to broadly neutralizing epitopes in recessed sites on the virus surface, proximity of the epitopes to viral membranes and epitope mimicry of self-antigens. In this new R01 proposal, we propose to generate transgenic mice carrying genes encoding two of the broadly neutralizing HIV antibodies 4E10 and b12 to investigate these hypotheses and to provide tools to study and rank potential HIV vaccine candidates. In the first instance, we propose to use mice carrying HIV antibody genes targeted to the physiological Ig loci to test if 4E10 and b12 have biologically relevant self-reactivity. If self-reactive, the B cells generated in 4E10 and b12 transgenic mice will be regulated by the immune tolerance mechanisms of receptor editing, anergy induction, or deletion and these mechanisms will be dissected. This may allow the design of immunogens in the future that do not evoke self-responses. If non-tolerant, transgenic mice carrying the 4E10 or b12 specificities should carry a greatly increased precursor frequency of B cells that are broadly neutralizing for HIV. We will then use an array of immunogens to explore the origins of any restrictions on eliciting 4E10 and b12 antibody specificities, to evaluate the most promising immunogens and ultimately to guide the design of modified and new immunogens to elicit broadly neutralizing antibodies. Relevance: To be successful, it is likely that an HIV vaccine must stimulate neutralizing antibodies. This proposal provides a mouse model to understand some of the problems in stimulating such antibodies by various vaccine candidates and to thereby suggest how better candidates can be designed.

DESCRIPTION (provided by applicant): This a renewal T32 application for graduate student training in immunolgy, microbial sciences, and related topics at The Scripps Research Institute (TSRI). This program is currently in its 10th year. The present proposal includes a larger faculty base than in the past (42 laboratories drawn from a total of 289 faculty members), reflecting the merger of the former Immunology Department with other faculty into the "Department of Immunology and Microbial Science", and outreach to all departments at TSRI, including Genetics, Molecular Biology, Cell Biology, Chemical Biology and Chemistry. The wider scope reflects both the growth of our institution and the multidisciplinary nature of science at TSRI. The proposed support shall be available to students at TSRI regardless of department affiliation, provided that their research is related to immunology, microbiology, or host defense. This training program is part of a highly successful "Doctoral Programs in Chemical and Biological Sciences". The TSRI graduate program attracts top-notch applicants and has been successful in training high quality scientists who have continued research careers. During the previous award period, all positions have been filled with talented individuals. TSRI is the largest non-profit research organization in the country, offering world-class research facilities. The faculty mentors in this training program are at the forefront of research into the mechanisms involved in generating and regulating immune responses, including responses of both the adaptive and innate arms of the immune system. In partial recognition of these achievements, this year two of our participating faculty members, Bruce Beutler and Michael Oldstone, were elected to the National Academy of Sciences. The unusually wide diversity of areas of investigation within the Training Program, from basic science to clinical applications as well as bioterrorism-related research, affords excellent opportunities for graduate student research and training in Immunology. Participation in scientific meetings, research-in-progress seminars, journal clubs, and lecture series gives students exposure to a multitude of different disciplines and experience in presenting their research to others.

DESCRIPTION (provided by applicant): This is an R21 proposal to investigate the biochemistry and genetics of Phospholipase D4, a novel gene of unknown function with homology to classical phospholipase D genes. Related family member proteins, such as PLD1 and PLD2 convert phosphatidyl choline (PC) to phosphatidic acid, releasing choline, a reaction that has effects on both cell signaling and membrane dynamics. PLD4 carries key active site amino acids of PLD1 and 2, but lacks classical phospholipase D activity. More distantly related family members have nuclease or DNA-tyrosine phosphodiesterase activity. Particularly close homologues to PLD4 are found in pox viruses. PLD4 is expressed in a variety of antigen presenting cells, including B cells, with highest levels in endosomal compartments of myeloid cells, especially plasmacytoid dendritic cells. Very low, but detectable, levels of expression are also seen in T cells. We have recently generated a conditional knockout allele of Pld4 and have begun the analysis of Pld4 germline null mice. At three months of age these mice already exhibit evidence of multiorgan inflammation, splenomegaly, possible defects in B cell development and thymic selection, and excess numbers of activated and memory B and T cells. We hypothesize that PLD4 plays a role in antigen presentation through the regulation of endosomal function. To understand how Pld4 deficiency causes the immunological phenotype we propose to characterize mice lacking Pld4 in selected tissues and cell types. A second goal is to determine the enzymatic activity of PLD4 using a variety of biochemical approaches, including "lipidomics" screens. PUBLIC HEALTH RELEVANCE: This is a R21 exploratory grant proposal to elucidate the function of a novel gene called Pld4. We have mutated this gene in mice and found that the mutants develop severe inflammation in many organs, including lung, salivary gland, liver and kidney, suggesting dysregulation of the immune system. However, the key cell types in which Pld4 must function to avoid disease are not known, nor is it clear what biochemical role the Pld4 gene product plays, though it is implicated in the regulation or formation of small vesicles within cells. This project will attempt to shed light on these issues. Because human beings carry a nearly identical gene to that of mouse, our studies have direct relevance to the understanding of the human gene and possible diseases caused by Pld4 deficiency or dysregulation.

DESCRIPTION (provided by applicant): This is a revised R01 proposal to address the biochemistry and function of two novel, leukocyte restricted signaling molecules. Fgd2 and Fgd3 are guanine nucleotide exchange factors (GEF) for the Ras homology (Rho) GTPases. They have significant sequence identity with known Cdc42 GEFs and are members of the Dbl homology family of GEFs. The Fgd subfamily includes 6 members and is distinguished by inclusion of FYVE domains that in other systems promote phospholipid binding and vesicular targeting. RhoGEFs play a number of crucial roles in receptor signaling, vesicle trafficking, cytoskeletal regulation, migration and the regulation of cell growth. Prior to our work, Fgd2 had not been characterized as a protein. Fgd2 is expressed at high levels in B cells and in macrophages. In B cells, Fgd2 expression is regulated by antigen receptor signaling and is expressed in memory B cells. In some contexts Fgd2 appears to be regulated reciprocally with a distinct family member, Fgd3. We have recently generated a conditional knockout of Fgd2. In this proposal the roles of Fgd2 and Fgd3 are addressed through the following three Specific Aims. 1) To characterize the biological functions of Fgd2 in the contexts of genetic knockouts and overexpression. 2) To characterize biochemical properties and specificities of Fgd2 and the related molecule Fgd3. A structure/function analysis will be carried out to determine the roles of individual motifs, to generate mutant forms, and to assess Fgd2 posttranslational modifications and interacting proteins. 3) To generate and analyze Fgd3 deficient mice and to address the issue of possible compensation of Fgd2 function by Fgd3. The long term goal of these studies is to understand how B cell development and antibody responses are regulated at the molecular level.

DESCRIPTION (provided by applicant): This is a R21 proposal to investigate Phospholipase D4 (Pld4) a novel gene of unknown function with homology to classical PLD enzymes. Pld4 was chosen for study initially because its expression pattern appeared to be restricted to B cells and its mRNA levels changed in immature bone marrow B cells upon BCR stimulation. We now know that it is expressed most in certain myeloid lineage cells including plasmacytoid dendritic cells. We have recently generated a conditional knockout allele of Pld4. We find that Pld4 null mice have several subtle phenotypes, with provocative alterations in the immune system, including massive up-regulation of MHCII expression on a subset of macrophages, an increase in marginal zone B cell numbers, and a decrease in peritoneal B-1 cells. Antibody responses are subtly altered. The most striking functional phenotype we have identified is that Pld4 null mice are completely resistant to induction of experimental autoimmune encephalomyelitis (EAE). The exciting implication is that inhibition of the action of this enzyme may facilitate treatment of multiple sclerosis. Pld4 carries a conserved phospholipase D catalytic domain with active site histidine, lysine and aspartic acid (HKD motif), but otherwise differs significantly frm well-known family members such as PLD1 and 2. No catalytic activity has been established for Pld4. We propose a two-pronged approach to determine if the putative active site is important for biological function and to assess in which cell types Pld4 bioactivity is required for its multple effects. The long-term goal of this study is to understand the biology and biochemistry of this novel protein.

DESCRIPTION: This is a renewal R01 application to exploit previously generated mouse models of germline-reverted HIV Env broadly neutralizing antibodies (gl-bNAbs) and to generate new ones. Developing an effective HIV vaccine is an important goal, as it would facilitate the major public health objective of HIV prevention. The recent identification of bNAbs to HIV Env has shown that such responses are possible. Moreover, animal challenge studies after passive immunization have shown that these bNAbs can be protective. Structural studies have identified the key epitopes at the molecular level, aiding vaccine design. Small animal models potentially can aid HIV vaccine development by allowing one to rapidly assess vaccine candidates and to determine the roadblocks to appropriate gl-bNAb B cell activation and maturation. The hypothesis of this proposal is that mice and B cell lines expressing gl-bNAbs can be useful tools in defining how best to initially stimulate, and then later to train, B cells to generate the desird bNAb responses. Because the problem of poor precursor affinity to antigen is a limitation to all immune responses, knowledge obtained here should be applicable to a variety of vaccine targets. We address these goals through the following Specific Aims: 1) To elicit bNAbs from exisiting gl-bNAb targeted (knock-in) B cells using designer immunogens. Antigens that target naive gl-b12 and gl-4E10 B cells will be used in immunization studies to characterize how best to generate high affinity bNAbs. These studies will be facilitated by the availability of our recently generated germline b12 and 4E10 knock-in mice and specially designed ligands directed to these gl-bNAbs. 2) To generate new gl-bNAb knock-ins for VRC01, 10E8, and either PGT121 or PG9. bNAbs VRC01, 10E8, PG9 and PGT121 see independent, conserved Env epitopes. These potent bNAbs currently appear to be better models than b12 and 4E10 for the development of a good HIV vaccine. Genes for VRC01, 10E8, and either PGT121 or PG9 will be used in the generation of gl-bNAb knock-in mice and in the testing of epitope-scaffold immunogens. These knock-in gl-bNAb mice will be ideal for working out the best ligands and methods for testing in human immunization.

Abstract This is an R21 proposal to investigate Phospholipase D4 (Pld4) a novel gene of unknown function with homology to classical PLD enzymes. Pld4 has been linked to inflammatory and autoimmune diseases. We generated a conditional knockout allele of Pld4, Pld4 monoclonal antibodies, and Pld4-mutated cell lines using CRISPR to study Pld4 expression and function. Pld4-/- plasmacytoid dendritic cells were found to have defects in type I interferon and other cytokine responses to endocytosed nucleic acid ligands. The present proposal explores several hypotheses related to how Pld4 might function in pDCs and other leukocytes to facilitate these responses. The immediate goal is to understand how Pld4 functions in pDCs and other leukocytes. Long-term goals are to understand all of the roles of Pld4 in health and disease and to explore whether Pld4 might be a good target for therapeutic intervention, particularly in autoimmune disease or vaccinology.

Abstract Influenza virus is a major public health burden that is currently controlled by yearly vaccination. Protection is largely conferred by antibodies. However, public health officials fear that such immunizations might miss newly emerging viruses or those with novel subtypes of hemagglutinin or neuraminidase proteins, with the potential to lead to a pandemic. Moreover, the costs of the current approach represent a major burden to society. Recently, hope has arisen that it may be possible to formulate a universal vaccine, capable of conferring resistance to numerous subtypes. Rare monoclonal antibodies targeting the stem region of influenza virus can be broadly neutralizing, illustrating the possibility of raising a universal influenza virus vaccine, but also indicating that the natural response is limited or natural influenza antigens do not stimulate the immune system in an appropriate way. Broadly neutralizing human antibodies reactive to the stem of influenza usually use VH1-69 with moderate numbers of mutations, but have few other features in common. The VH1-6+D3-3 broadly neutralizing antibody class targets the stem in a distinct way and has been seen in independent patients. In this RO1 project, we propose to study existing and new knock- in mice carrying germline human VH1-69 or VH1-6+D3-3 targeted to the physiological loci for their responses to influenza vaccine candidates. We shall isolate new rationally designed immunogens by evolving HA variants or scaffolds with affinity for germline- reverted broadly neutralizing VH1-69 and VH1-6+D3-3 class antibodies. These novel immunogens will then be tested for the ability to stimulate B cells using germline VH1- 69 or VH1-6+D3-3 in vivo, using novel mouse strains generated for the purpose. Finally, we will assess the ability of these immunogens alone or together with boosting immunogens for the ability to confer broad neutralizing protection. Our long-term goals are to understand the limitations of the natural influenza antibody response, to learn how to optimize immunogens, and to identify candidate universal vaccine immunogens suitable for human trials. 

Abstract A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). While many HIV bnAbs have been isolated from chronically infected patients, raising bnAbs by immunization has been difficult. The principal problems appear to be (1) that bnAb epitopes are sub-dominant and so the response must be primed and boosted in such a way as to avoid off-target responses and favor the desired specificities, (2) that most forms of Env bind poorly to unmutated precursors of bnAbs so that bnAb responses are not typically primed appropriately and (3) we do not understand how best to boost responses once primed to yield bnAbs that typically have an unusual amount of somatic mutation. In this proposal, we address these problems by study of engineered immunogens in bnAb knockin mice. We focus on two classes of bnAbs, VRC01 and PGT130, which we predict will be readily raised by vaccination in humans using appropriately engineered immunogens. VRC01 is the prototype bnAb of a class that recognizes the CD4 binding site (CD4bs) and PGT130 is a representative of a class of glycan-dependent bnAbs that bind the V3 loop and mannose patch on Env. Both antibody classes have been described in many HIV infected individuals and are particularly potent and broad suggesting that a vaccine able to elicit such Abs would provide protection at relatively low Ab concentrations. We have previously generated mice engineered to carry B cells with germline reverted (gl)-VRC01 immunoglobulin heavy (H) and light (L)-chains, and here we propose to generated new knock-in mice carrying gl-PGT130 H- and L-chain genes. The mice express the B cell receptors that should be triggered and matured to generate VRC01 or PGT130 class bnAbs and will be used to investigate immunogens and immunization strategies. For priming, we will use existing germline-targeting immunogens reactive to gl-VRC01 and we will generate novel engineered immunogens targeting gl- PGT130 and. For boosting we will test a range of engineered and natural immunogens to focus and enhance intermediate and final steps in bnAb maturation. This study will test the generality of our germline-targeting approach, discover new vaccine candidates, and teach us lessons on how to maximize subdominant anti-viral responses that should inform human vaccination strategies in general.

Project Summary/Abstract This is a R01 project to study the role of Phospholipase D4 (PLD4) in nucleic acid sensing. Leukocytes carry conserved sensors for endocytosed DNA and RNA that trigger the production of proinflammatory cytokines, including type I interferon. The ensuing inflammation can be beneficial for host defense to microorganisms, viruses, and tumors, but can also be pathogenic, and may thwart the use of potentially therapeutic oligonucleotides. PLD4 is a lysosomal protein whose function is not understood. Genome-wide association studies have linked PLD4 to human rheumatoid arthritis and systemic sclerosis. We find that PLD4-deficient mice have a phenotype similar to the human disease macrophage activation syndrome, which is mimicked in mice by repeated injections with Toll-like receptor 9 agonists. Here we test the hypotheses that PLD4 breaks down ingested DNA and RNA and regulates host defense and inflammation by promoting the destruction nucleic acids and regulating recognition by TLR7, TLR8 (in humans) and TLR9. We will characterize how PLD4 deficiency promotes a MAS-like phenotype and how deficiencies in PLD4 or PLD3+PLD4 affect TLR7 and TLR9 responses to specific ligands, leading to altered cytokine secretion. The possible role of PLD4 in self/nonself discrimination will also be assessed. The long-term goal of these studies is to understand the basic biology of PLD4, its functions in host defense and inflammation, and to develop a basis for potential future translational research.

The long-standing Immunology Training Program at The Scripps Research Institute currently provides advanced research training for 5 postdoctoral fellows in diverse areas of immunology. One additional position is requested to support a total of 6 postdoctoral trainees each year. The 24 designated Mentors, remaining faculty of Scripps and the interactions with the La Jolla immunology community provide a rich, challenging, multidisciplinary, and highly collaborative environment for trainees. The goals of this Immunology Training grant remain unchanged: to recruit and train the best candidates from a diverse applicant pool to become independent scientists with promising careers in basic and applied immunology. Underrepresented minority candidates and both Ph.D. and M.D. candidates are encouraged to apply. An individualized training and mentoring plan is designed for each trainee including training in research ethics, biostatistics, communication skills, grant preparation, career options, and proper use of human subjects and animals in research. The 24 designated Mentors have a distinguished record of training postdoctoral fellows and have research interests that include autoimmunity, innate immunity and inflammation, lymphocyte biology, signal transduction, structural immunology, and viral immunology. The Immunology Training Program will continue the tradition of training highly productive, independent, creative scientists who will make substantial contributions to the field of immunology and related disciplines.

Project Summary Autoreactive B cells play critical roles in many autoimmune diseases. Multiple immune tolerance checkpoints exist to remove autoreactive B cells or keep them under control. Defects in these checkpoints constitute the basis for the development of autoimmune diseases. Despite intensive study, our understanding of these checkpoints remains incomplete and fragmentary. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate a large diversity of biological processes. Hundreds of miRNAs are expressed in the immune system. While some miRNAs have been shown to play important roles in lymphocyte development and function, the roles of miRNAs in controlling immune tolerance remain poorly understood. We performed in vivo functional analysis of hundreds of miRNAs in the recently established IgMb-macroself mouse model and identified miR-148a as an important regulator of B cell central tolerance and autoimmunity (Nature Immunology 17:433-40, 2016). Further molecular analysis identified 119 target genes regulated by miR-148a in immature B cells. We examined 4 of these target genes and demonstrated that 3 of them, Gadd45a, Bim and Pten, regulate B cell central tolerance. In this proposal, we will 1) further investigate the cellular and molecular mechanisms underlying miR-148a regulation of immune tolerance and autoimmunity, focusing on its role in controlling various B cell tolerance checkpoints and plasma cell differentiation; 2) evaluate the possibility of treating systemic autoimmunity through miR-148a ablation and inhibition by genetic and chemical approaches, respectively, and 3) perform an in vitro functional screen of the other 115 miR-148a target genes to identify novel regulators of B cell tolerance. Our pilot screen has identified B4galt5 as a positive hit. As B4galt5 is a major enzyme in the glycolipid biosynthesis pathway, we speculate that this pathway plays important roles in immune tolerance. Therefore, we will elucidate the function and mechanism of B4galt5 and the glycolipid biosynthesis pathway in controlling B cell tolerance and autoimmunity.

This is a R01 proposal to study the function of Phospholipase D3 (PLD3) and its contribution to neurodegeneration in mouse models. PLD3 missense mutations and under expression have been implicated in Alzheimer's disease (AD) and in Spinocerebellar Ataxia, but the mechanisms by which these mutations alter function are unclear. PLD3 protein is associated with neuritic AD plaques and has been implicated in Ab and Tau processing. We have recently discovered that PLD3 and a related protein PLD4 are not phospholipases, as was thought, but are in fact single- stranded DNA and RNA exonucleases localized in endolysosomes. PLD3 and PLD4 can strongly influence nucleic acid recognition by toll-like receptors (TLR) 7,8 and 9. Mice lacking both PLD3 and PLD4 enzymes die of massive autoinflammation, whereas the phenotype of Pld3?/? mice is relatively subtle and has not been investigated in older mice. Here we propose to study the roles of PLD3 in more detail by evaluating functional alterations of disease associated PLD3 alleles and by generating tools to assess the effects of deficiency of PLD3 and PLD4 in microglia and other tissues of the brain.