Steven L. Reiner - US grants

The reciprocal expansion of Th1 and Th2 subsets is now recognized as a central feature of the immunopathogenesis of several human and experimental diseases. Infection of inbred strains of mice with Leishmania major is the model system for studying the in vivo regulation of protective and non-protective CD4+ subsets and a paradigm for disease that have a genetic basis for susceptibility. Dr Reiner is uniquely positioned to dissect the exact mechanism(s) responsible for the Th1 and Th2 switch in a well- characterized infectious disease model. To facilitate this, he has spent the past year developing an assay that provides a precise method for analysis of in vivo cytokine regulation and successfully generating transgenic mice that express a T cell receptor recognizing a dominant leishmanial epitope. There are three specific aims proposed: 1. To characterize the early immune response to murine leishmaniasis by in vivo analysis of gene expression for various cytokines and effector molecules. 2. To determining the necessary and sufficient conditions governing the preferential expansion of a Th1 and Th2 response to experimental leishmaniasis by in vivo administration and neutralization of critically implicated cytokines in TcR-transgenic mice. 3. To identify the mechanism(s) of genetic susceptibility by in vitro modeling using antigen-presenting and accessory cell populations from resistant and susceptible mice plus transgenic Leishmania-specific T cells.

Lyme disease is a major source of morbidity due to the high incidence of rheumatic, cardiovascular and neurologic complications that follow infection with the etiologic agent, Borrelia burgdorferi. In the U.S., close to 10,000 cases are reported annually and it was recently named a "High Priority Research Topic" by the NIAID. Among humans infected but not treated with antibiotics, 80% develop complications but 20% remain disease-free after resolution of a self-limited skin rash. An undetermined number of patients never develop any overt sign of infection and, despite adequate antibiotic therapy, a significant subpopulation still develops debilitating complications. Although described almost twenty years ago, the pathogenesis as well as the immunologic and genetic basis for differing host responses to Lyme disease are poorly understood. Experimental infection of lymphocyte-deficient mice indicates that the innate immune system has sufficient capacity to mediate the most extreme pathologic outcome in this model. Infection of immunocompetent inbred strains of mice recapitulates the spectrum of responses seen in humans. The genetic model of Lyme arthritis using inbred mice is a powerful system to delineate the mechanisms by which adaptive immunity can regulate innate immunity and pathology. Our preliminary evidence indicates that a major immunologic difference between susceptible and resistant hosts is the magnitude of pro-inflammatory response directed by T helper type I cells. Pathology-prone hosts also uniquely exhibit increased activation of NK cells following infection as well as an MHC association with the H-2k haplotype. This proposal seeks to define the immunoregulatory requirements, host polymorphisms and genetic control that lead to differing phenotypic outcomes in experimental Lyme arthritis. Specific aims encompass the following areas: Cytokine gene expression in the lymphoid organs and sites of pathology of infected mice, in vitro analysis of inter-strain differences in cellular immunoresponse, infection of immunologically perturbed mice with reconstitution of immune cell populations, and genetic analysis of select inter-strain crosses of susceptible and resistant mice. Because this model deals with the microbiologic control of a foreign pathogen, the pathogenesis of inflammation and differential host response to a fixed stimulus, the proposed studies should provide broad insight into microbial pathogenesis, inflammatory conditions of multiple organ systems and the genetic basis for susceptibility to infectious and rheumatic diseases.

Leishmaniasis and many other parasitic diseases remain a major cause of world-wide morbidity and mortality. A central regulatory event in the defense against parasitic diseases is the post-thymic maturational fate of CD4+ (helper) T cells. Murine infection with Leishmania major has a well-defined sequence of cellular events. These features make it a versatile model to study the molecular regulation of CD4+ T cells in vivo. First, the parasite invades mammalian macrophages. Class II- restricted helper T cells become activated and expand after the presentation of parasite antigens. The maturing helper T cells assume one of two developmental fates. The Th1 fate results in control of infection while the Th2 fate results in disseminated disease. Resolution of infection involves elimination of the parasite as well as the host's own formerly-useful immune cells. This proposal will systematically define the molecular events regulating the activated life and death of CD4+ T cells during murine infection with L. major. The proposal has three specific aims. The experimental methods rely on in vivo infection of normal and mutant mice and are complemented by in vitro cellular and molecular analyses of macrophages and T cells. The first aim will provide a detailed assessment of MHC class II-restricted antigen presentation in macrophages. The influence which antigen presentation has on T cell function during infection will be examined. Additionally, the interactive functions of two MHC class II cofactors, invariant chain and DM, will be assessed by infection of mice deficient in these molecules. The second aim examines the regulation of helper T cell lineage commitment by defining the cytokine and non-cytokine factors which influence maturational fate and, thus, disease outcome. In addition, a novel hypothesis regarding the molecular basis for T helper subset commitment will be tested using murine leishmaniasis as model system. The third aim will analyze the homeostatic elimination of parasite-specific T cells which is necessary to avert the potentially pathological consequences of an immune reaction. Cell death molecules which are responsible for killing CD4+ T cells during leishmaniasis will be identified by analysis of loss-of-function mutations. The results of these studies should provide important regulatory information about the control of helper T cells during an immune response. Since many infectious and autoimmune conditions are mediated by the heterogeneous life and death of CD4+ T cells, this should ultimately result in improved treatment for diseases.

DESCRIPTION (provided by the applicant): Parasitic diseases are major causes of morbidity and mortality throughout the world. The outcome of many parasitic infections depends on whether or not an infected host mounts the correct type of helper T cell response against the invading microbe. T helper-1 (Thl) cells are critical for protection against intracellular protozoa, and T helper-2 (Th2) cells are critical for expulsion of intestinal helminthes. Mature Thl and Th2 cell subsets are thought to arise from a common naive progenitor. How states of gene activity are acquired and transmitted to daughter cells are critical issues that currently remain unresolved. This proposal investigates the mechanisms controlling how highly polarized immune responses against parasitic pathogens develop. Evidence is offered in support of a model that the molecular signatures of Thl and Th2 cells, the specific effector cytokines that mediate control of the parasites, are regulated by gene silencing. In naive progenitor cells, effector cytokine genes appear to exist in a non-permissive structure, which is determined by methylation of cytosines in DNA and specific modifications of histone tails. The nonpermissive structures appear to be plastic, giving way to more active structures in some daughter cells. This proposal uses genetic and biochemical approaches to try to define how a model gene, interleukin-4 (IL-4), becomes re-assembled into an active structure as a naive cell becomes a Th2 cell. It also examines how gene silencing of IL-4 is maintained in Thl cells. The proposal also tests the consequences of defective gene silencing when hosts are confronted with parasitic infections in vivo. Successful execution of the 3 specific aims of this proposal should provide novel insight into the way that helper T cells mature, and novel strategies to defend humans against parasitic invaders.

DESCRIPTION (provided by applicant): Cell-mediated immunity is critical for host defense against all classes of pathogens and cells that have undergone cancerous transformation. Strategies to vaccinate or potentiate T lymphocyte-mediated cellular immunity have been remarkably ineffective, probably owing to our limited understanding of the mechanisms for establishing and maintaining T cell effector function and memory. This proposal investigates how two T- box family transcription factors contribute to the formation of cellular immunity. Eomesodermin and T-bet redundantly ensure CD8+ T cells become cytotoxic effector cells but they also seem to oppose each other's functions in normal and abnormal CD8+ T cell differentiation. This allows the two transcription factors to form an adjustable balance between the opposing demands of terminal differentiation and self-renewal. The specific aims of this project will address how and when Eomes functions in memory T cell programming. The aims will also resolve whether the predominant actions of Eomes in memory cells are on a unique set of genes or whether Eomes is controlling CD8+ T cell memory through interplay with T-bet at loci they regulate in common. Successful execution of the 3 specific aims of this proposal should provide new insight into the mechanisms of gene induction and cellular differentiation in the immune response. It is also anticipated that these studies will yield new strategies for defending us against a variety of infectious diseases that are the focus of our CD8+ T cell responses. PUBLIC HEALTH RELEVANCE: Specialized white blood cells, called lymphocytes, increase in number to help protect us against infections. This project will provide important information about how these cells are programmed for immediate elimination of infections, how they are trained to provide immunity against re-infection for our entire life, and how they can be re-programmed if they become ineffective.

[unreadable] DESCRIPTION (provided by applicant): CD8+ T cells play a critical role in host defense against microbes pertinent to biodefense. A hallmark of adaptive immunity against agents such as Listeria monocytogenes, LCMV virus, and Toxoplasma gondii is heterogeneity of cell fate among antigen-experienced CD8+ T cells. Substantial preliminary evidence outlined in this proposal indicates that the first division of a CD8+ T cell responding to a pathogen, in vivo, is characterized by unequal partitioning of proteins with established roles in signaling, cell fate specification, and asymmetric cell division. In addition, the first daughter T cells of the immune response appear to be differentially fated as precursors of the effector and memory lineages. This project will test whether asymmetric cell division is a general feature of the CD8+ T cell response against pathogens, whether ancestral regulators of cell polarity are responsible for establishing cytoskeletal features necessary for asymmetric division, and how asymmetrically inherited signaling proteins could mediate fate disparity in daughter T cells. These studies should provide a framework for rational engineering of immune responses and vaccines against agents of biodefense, and address fundamental uncertainties regarding the principle of clonal selection of lymphocytes in response to infectious diseases. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Specialized white blood cells, called lymphocytes, increase in number to help protect us against infections. This project will provide fundamental insight into how immunity against re-infection is maintained for one's entire life. This proposal is a response to a continuing initiative in Biodefense research sponsored by the NIAID. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): B lymphocytes and the antibodies they produce play a critical role in host defense. Cell fate diversification of activated lymphocytes is essential to achieve the regenerative state required for long-lived immunity. The mechanisms responsible for generating B cell fate diversity, however, are poorly understood. Substantial preliminary data suggest B cells can diversify the fates and functions of their daughter cells using an evolutionarily conserved strategy to allocate unequal amounts of key components. This project will test whether asymmetric cell division is a cardinal feature of the B cell- mediated immune response. The proposal develops novel methodologies to image the characteristics of dividing B cells during the course of an immune response. The aims of the project are to test the importance of unequal cellular inheritance of three different transcriptional regulators of B cells Bcl-6, Pax5, and T-bet, each of which is known to regulate critical cell fate decisions. The experiments will test the hypotheses that asymmetric division is iteratively used to meet the opposing demands of terminal differentiation and self-renewal, as well to refine and diversify the functional properties of antibodies by regulating germinal center entry and class switch choice. This project will also examine whether ancestral regulators of cell polarity are responsible for establishing asymmetric B cell division, and how asymmetrically inherited proteins could mediate fate disparity in daughter B cells. These studies should provide a framework for rational engineering of immune responses and vaccines against microbial agents. This project should also address fundamental uncertainties regarding the principle of clonal selection of lymphocytes in response to infectious diseases or during situations when our immune cells attack our own selves.

DESCRIPTION (provided by applicant): T lymphocytes play a critical role in host defense against microbes and cancer. A remarkable feature of the signals that trigger T cell participation in the immune response is that every cell appears to have a mandate that assures its daughters will not uniformly adopt the same fate. This proposal develops novel methodologies to image the characteristics of dividing T cells during the course of an infectious disease. The aims of the project are to test the importance of unequal cellular inheritance in the earliest stages of the immune response, where an abundance of cell fate decisions are being made (Aim 1), in the re-challenge response wherein memory T cells may be mimicking the regenerative behavior of adult stem cells (Aim 2), and in chronic infection because this scenario may tax the regenerative limits of normal memory T cells (Aim 3). The studies proposed herein should offer novel approaches for maintaining lifelong immunity against chronic infections and for eliminating many life-threatening infectious diseases.

DESCRIPTION (provided by applicant): The proposed goal of the Medical Scientist Training Program grant is to produce the next generation of physician scientists who have the skills and experience to move medical science in a positive and significant direction. We plan to continue training students in both clinical medicine and basic science with a very strong foundation in both areas. Students will fulfill an entire medical school educational curriculum including caring for and treating patients in the hospital and clinic. In a full PhD program, they will also learn t plan and carry out research that will delve deeply into the scientific basis of disease and find new, innovative therapies. Our graduates will learn from outstanding mentors in medicine and science to play a key role in the translation of scientific findings to clinical research. They wil be uniquely qualified to be the leaders of medical science.

Project Summary/Abstract When a T lymphocyte is engaged in an immune response, it must divide and produce functional daughter cells, often repeatedly. To maintain continued, clonal production of fresh effector T cells requires that some daughter cells self-renew instead of differentiating. Our laboratory identified the activating signals that induce progenitor T cells to undergo irreversible commitment to differentiation. Preservation of self-renewal, however, requires a dampening mechanism to oppose full activation. Among the most critical signals that dampen T cell activation are the inhibitory receptors, which are now key targets of a revolutionary approach to unleash T cell attack against tumors. While blockade of inhibitory receptors offers clinical benefit in some cases, many treated patients do not experience durable anti-tumor immunity. This project addresses a novel and clinically important question that may represent a major barrier for improving the efficacy of inhibitory receptor blockade: Are inhibitory signals an essential part of a regenerative mechanism allowing some T cells to self-renew as their kindred cells undergo differentiation? Using preclinical models of cancer and chronic-active infectious diseases, 3 specific aims will be addressed: (1) Determine if inhibitory receptor blockade impacts the balance of T cell differentiation and self-renewal in vivo, (2) Define the cell biological mechanisms that support T cell self-renewal under in vivo conditions of repetitive, high-level antigen activation and response intensification by inhibitory blockade, and (3) Test whether the efficacy of inhibitory receptor blockade will be improved by addition of agents that promote T cell self-renewal. The results of these studies could offer novel immune response biomarkers, new strategies for vaccination, and novel or repurposed compounds to augment the efficacy of immunotherapy.