Deepta Bhattacharya, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Hematopoietic stem cells (HSCs) are the only cells within the bone marrow that possess the ability to both differentiate to all blood lineages and to self-renew for life. These two properties allow for the proper maintenance of hematopoietic homeostasis, but genetic abnormalities within HSCs can lead to profound negative consequences such as immunodeficiency, anemia, or leukemia. Because the replacement of genetically abnormal HSCs with normal HSCs can correct many of these diseases, it is of critical importance to understand how best to achieve such an exchange while minimizing risk to the patient. One of the most important parameters for the success of bone marrow transplantations is the availability of sufficient numbers of appropriate bone marrow microenvironments for donor HSCs to engraft. The research proposal outlined below describes a series of both classical and technologically novel approaches designed to better understand the functional interactions of HSCs with the highly specific bone marrow microenvironments in which they reside and the potential for therapeutic intervention by capitalizing upon these dynamic interactions. It is anticipated that significant contributions to this field will allow me to transition to an independent faculty position within the next 3 years. [unreadable] Specific Aims: [unreadable] Aim 1: To characterize the kinetics, magnitude, and therapeutic potential of HSC niche emptying and refilling under steady state conditions. [unreadable] Aim 2: To isolate and characterize HSC-supportive niche cells. [unreadable] Aim 3: To localize and visualize HSC homing and engraftment in vivo. Relevance: The hematopoietic stem cell is a remarkable cell type that exists within the bone marrow and is responsible for the production of all red and white blood cells for life. In order for them to function appropriately, they must reside within specific areas, or niches, within the bone marrow. The goal of this proposal is to better characterize the properties of these niches and to determine how best to utilize these niches for bone marrow transplantation. [unreadable] [unreadable]

Self-reactivity in antibodies isolated from patients with autoimmune disorders is often imparted by somatic mutations. Indeed, reversion of immunoglobulin genes to germ line sequences typically eliminates reactivity against self-antigens. These data suggest that many autoreactive B cells are derived from the germinal center reaction, in which somatic hypermutation of immunoglobulin variable genes occurs. The mechanisms by which self-reactive B cells are eliminated during early development are well-understood. In contrast, very little is known about how somatic mutation-induced self-reactivity is normally prevented in healthy individuals, and which of these pathways fail in patients with autoimmune disorders. In this proposal, we will identify the survival pathways downstream of B cell receptor (BCR) signals and CD4+ T cell help that allow autoreactive B cells to survive.

DESCRIPTION (provided by applicant): Effective vaccines exist for many pathogens. But rational design methods have failed to generate vaccines against many important pathogens. Importantly, our understanding of the molecular pathways involved in generating long-lived and broadly protective immunity is incomplete. Using a newly discovered molecule, this application is directed at understanding pathways that regulate the ability of B cells to provide durable and cross-protective antibody responses. We have discovered that the transcription factor Zbtb20 is required for maintenance of long lived plasma cells, with only high affinity clones persisting afte immunization. These findings provide us with the unique opportunity to: 1) Identify the signaling and survival pathways that regulate plasma cell diversity and lifespan through genetic complementation experiments; 2) Determine the function of low and moderate affinity antibodies in cross-protection against viruses and in the pathogenesis of autoimmunity.

The usage of metabolic pathways is tailored to meet the specific functions and demands of a given cell type. Of particular interest is how metabolism supports the survival and antibody secretion of long-lived cells, the primary cell type that is responsible for humoral immunity. Using plasma cells as a model system, we will define how glucose and amino acid uptake supports long-term persistence of these antibody-secreting cells. In preliminary data, we demonstrate that long-lived plasma cells import far more glucose than do their short-lived counterparts. Glucose is primarily used to glycosylate antibodies, but a portion of this glucose can be used for glycolysis, and the resultant pyruvate imported into the mitochondria. Short-lived plasma cells do not have this capacity. We propose to define the metabolic fates of pyruvate and explain why this pathway is important in long-lived plasma cells. We also propose to define how long-lived plasma cells balance enhanced antibody production with nutrient uptake and ER stress. Finally, we seek to define the physiological signals that promote plasma cell metabolic programs following vaccination. The application heavily emphasizes new genetic and in vivo approaches to define, report, and functionally test critical metabolic pathways in a cell type critical for humoral immunity.

Abstract: Vaccines against devastating infectious diseases represent some of mankind's greatest medical breakthroughs. The underlying principle of vaccines is the establishment of immunity using attenuated or inactivated vectors that mimic the natural pathogen without causing the systemic damage a live infection can trigger. Thus the prerequisite for all successful vaccines to date is that the human immune system must be inherently capable of eventually generating adaptive immunity when faced with the natural live infection. In cases like polio, the damage achieved before adaptive immunity can control the natural virus is unacceptable; nevertheless, lasting immunity is ultimately achieved and protects against subsequent infections. Currently, however, many of the most problematic infectious diseases worldwide cannot be effectively controlled by the human immune system and do not elicit lasting immunity against re-infection. For example, by virtue of its mutability, HIV establishes chronic infections that cannot be cleared and are ineffectively controlled without the assistance of antiviral drugs. Dengue virus infection leads to lasting immunity against re- infection by the same serotype, but in fact causes far more severe disease if the individual is re-infected with a different Dengue serotype than if he/she were completely naïve. Similarly, influenza strains continuously alter themselves genetically; thus immunity to a given strain rarely affords complete protection against all subsequent influenza viruses that circulate in time and space. Natural infection with malaria does not necessarily lead to lasting immunity, as the same individual can be re-infected many times over the course of a lifetime. Pathogens like these thus pose a conundrum: how can vaccines designed to mimic the natural pathogen elicit immunity when the immune system is intrinsically incapable of generating broad and effective immunity when faced with the actual infection? Innovative alternate strategies, such as structure-guided sequential immunizations, gene therapy, and cell-based therapy have all been proposed, though the latter approach is the least developed. In this application, we thus focus on cell-based therapies and propose to 1) develop strategies to differentiate human pluripotent stem cells into transplantable pathogen-specific plasma cells, and 2) Use CRISPR/Cas9 genome editing to eliminate major determinants of immunogenicity from human pluripotent stem cells to allow scalable off-the-shelf therapies for infectious disease.    

Abstract: Imaging flow cytometry merges the capabilities of modern flow cytometry with fluorescence microscopy. Flow cytometry provides single-cell resolution of complex mixtures of cells through size, expression of surface proteins, and intracellular levels of other markers. With the advent of new fluorophores, detection systems, and analysis algorithms, dozens of distinct parameters can be analyzed on cells simultaneously. Moreover, thousands of cells can be analyzed per second, providing substantial statistical power to identify even very rare populations. A limitation of traditional flow cytometry, however, lies in its inability to resolve subcellular features or morphological changes between cells aside from overall size. Thus, processes such as autophagy or NF-?B activation that operate through the cellular re-localization of proteins to autophagosomes or the nucleus, respectively, are difficult to quantify by flow cytometry. Such features can be monitored by fluorescence microscopy, but low throughput, especially for rare cell subsets, limits its utility. Moreover, most microscopy configurations have a limited number of fluorescence parameters that can be assessed simultaneously. Imaging flow cytometry addresses these limitations by merging the advantages of flow cytometry and microscopy, taking high resolution pictures as cells pass through at low pressure but great speed. Up to 11 distinct parameters can be assessed at once, allowing for a robust separation of even rare cells with distinct combinations of surface and intracellular markers, morphology, and subcellular features. No such instrument exists at the University of Arizona or within 100 miles of Tucson. Acquisition of such an instrument would transform the capabilities of the community of local scientists studying basic cell biology, immunology, and cancer.