Christopher Hunter - US grants

DESCRIPTION (provided by applicant): Infection with the parasite Toxoplasma is of public health importance in the context of maternal-fetal transmission, and is a leading cause of food borne mortality in the US. Approximately 60 million people in this country are chronically infected and reactivation of the latent cyst form of the parasite in patients with defects in T cel function remains a significant cause of morbidity and mortality. As primary infection of immune competent individuals can result in severe disease and T. gondii is easily transmitted, this parasite is a Class B Biodefense pathogen and an NIAID Emerging/Re-emerging Pathogen of concern to human health. Resistance to this parasite in the brain is mediated by CD4+ and CD8+ T cells that produce IFN-?, which activates local cells to control parasite replication. Astrocytes are the most numerous cell populations in the CNS and have been implicated in direct anti-microbial activities as well as coordinating the entry and migration of T cells that respond to infections that affect the brain. The proposed studies focus on the role of the cytokines IFN-? and the type I IFNs in activating astrocytes to control Toxoplasma. Experiments are proposed to identify the pathways downstream of these cytokines that allow human and murine astrocytes to control parasite replication and which coordinate innate and adaptive responses in the CNS. These studies will be complemented by work to understand how a novel nuclear hormone receptor TLX impacts on astrocyte and immune cell function. Together, the approaches proposed will provide new insights into how astrocytes respond to T. gondii, control this pathogen and regulate local effector T cells with the ultimate goal of being able to develop strategies to augment immunity to T. gondii (and other pathogens) within the CNS.

Project Summary Understanding the transcriptional regulation of polarized T cell responses and their link to inflammatory conditions and resistance to infection has been a major theme in immunology for the last 20 years and has led to the identification of T-bet, GATA3 and RoR?t as ?master regulators? of the development of Th1, Th2 and Th17 type responses, respectively. As such, the ability of T-bet to promote IFN-? production has dominated our appreciation of how T-bet contributes to T cell responses in the setting of infection. This is reinforced by the increased susceptibility of T-bet-/- mice to various intracellular infections, including the opportunistic parasite Toxoplasma gondii. In CD4+ and CD8+ T cells there is good evidence that T cell activation is accompanied by two waves of T-bet expression (the T-bet two step) and that the initial TCR- mediated induction of T-bet sensitizes T cells for polarizing signals provided by cytokines such as IL-12 that reinforce further T-bet expression and commitment to differentiation. However, our recent published and preliminary studies indicate that in CD8+ T cells T-bet may have an unanticipated role in controlling a myriad of early T cell activation induced events including upregulation of adhesion molecules and chemoattractants and entry of CD8+ T cells into cell cycle. These results have led to the novel hypothesis that the early induction of T-bet optimizes the interactions between recently activated T and DC populations to promote entry into the cell cycle, optimize expansion and the acquisition of effector functions. To test this hypothesis we will combine transcriptional profiling to identify early targets of T-bet with state of the art cellular immunology and imaging approaches to understand the role of T-bet and in hand candidates in the initial events that are essential for the generation of effector responses required for resistance to an intracellular pathogen.

Project Summary The goal of this program is to provide pre-doctoral students with strong research training in specific basic science disciplines in combination with broad training in parasitology. Parasitic diseases remain an important cause of morbidity and mortality in humans and have an important impact on food safety and production. Most of these diseases are associated with tropical and subtropical areas and characterized as Neglected Tropical Diseases (NTDs), but they are not limited to developing countries and many have recently emerged or re-emerged in temperate regions. Penn has a vibrant program in Parasitology and members of this T32 program have diverse interests and study at least 16 different parasitic infections. This program has evolved from 8 faculty in 1998 with a strong emphasis on immune-parasitology to a group of 14 faculty who are involved in basic research that includes immune- parasitology, cell and molecular biology of these organism, as well as their population biology. These form the underlying core of our program. The faculty participants in this proposal have primary appointments in the Schools of Medicine, Veterinary Medicine and Arts and Sciences and in the last 20 years this T32 has provided support for 35 graduate students. The majority of these trainees have gone on to successful careers in Parasitology or related disciplines. Each faculty member offers strong research training in a basic science discipline as it relates to Parasitology. As a group, we also offer didactic training in parasitology and provide an environment in which students will gain an appreciation for broader aspects of parasitic disease research.

Project Summary The development of vaccines that can specifically generate cell mediated immunity has remained a major challenge in the field of vaccinology for both microbial pathogens and cancer. Prior work has shown that the use of replication deficient live microbes is one way to generate a potent pathogen-specific CD8+ T cell response. For example, even a single low dose of the non-replicating vaccine CPS strain of Toxoplasma gondii elicits a robust parasite specific CD8+ T cell response that provides long-lived protective immunity. However, many questions remain about how the immune system recognizes and processes the antigens from these attenuated organisms to prime and expand parasite-specific T cells. Prior work has shown that the CD8+ T cell response is dependent on the Batf3-dependent dendritic cells (DC1), which are commonly involved in antigen cross-presentation to CD8+ T cells as well as IL-12 production. Paradoxically, preliminary data indicates that DC1 are not required for early CD8+ T cell priming, but are required to generate a protective CD8+ T cell response. Importantly, the loss of the autophagy pathway in DC also results in a failure to generate protective CD8+ T cells. These studies emphasize two important questions: 1) what priming-independent role do DC1 play in generating a CD8+ T cell response, 2) what autophagy- dependent pathway in DC1 is required for the expansion and differentiation of CD8+ T cells? The autophagy pathway has been implicated as being required for leukocyte survival in inflammatory setting as well as an alternative pathway for the processing and presentation of parasite antigen. The studies proposed here will use a variety of unique microbial tools (fluorescent reporters, parasite auxotrophs, Cre- exressing parasites) and mouse strains (Batf3-/-, unique reporters, and mice which do not express MHCI H- 2Kb on DC1) combined with our imaging expertise to address I. Do DC1 interact directly with parasite- specific CD8+ T cells in a peptide-MHCI dependent fashion to provide the signals required for T cell expansion and differentiation and II. What autophagy-dependent processes in DC1 are required for the generation of protective effector CD8+ T cells.

Project Summary There are over 10 trillion endothelial cells (EC) that line the vasculature of the human body and these are an important replicative niche for a subset of viral, bacterial and parasitic pathogens. Consequently, it makes sense that the immune system has mechanisms to monitor this extensive network for signs of infection. While it has been proposed that activated EC have an important role in mediating resistance to these organisms this is an idea that has been difficult to test in vivo because of: 1) lack of tractable in vivo systems to modify EC immune functions 2) the size of and dynamic flow within the vascular system that hinders the detection of rare immune processes and 3) a lack of understanding of whether there are T cells specialized to operate in this environment.

The apicomplexan parasite Cryptosporidium is a leading cause of death due to diarrheal disease in young children, particularly in the context of malnutrition, milder infections often result in growth faltering. The incidence of cryptosporidiosis drops off sharply after the age of two in areas of high transmission, yet in regions with low transmission adults remain susceptible, as demonstrated by frequent outbreaks in the U.S. More than 50% of waterborne disease in the U.S. is caused by this parasite which is resistant to water chlorination and considered a category B potential bioterrorism agent. The epidemiology in children suggests that initial infection results in long lived protective immunity, a phenomenon also observed in the context of veterinary cryptosporidiosis. Harnessing this immunity into a vaccine could prevent the disease and dramatically impact child mortality and development. However, there are significant gaps in our knowledge of the mechanisms that underlie immunity to Cryptosporidium. We developed a new natural model of Cryptosporidium infection in immunocompetent C57BL/6 mice that mirrors important aspects of human cryptosporidiosis and in which host and parasite are genetically tractable. This proposal assembles an interdisciplinary team of experienced parasite molecular biologist and immunologist to define how the infected enterocyte senses infection, how it responds to and restricts infection, and how it interacts with dendritic cells and T cells to trigger and execute a protective immune response. This research will impact our fundamental understanding of immunity in the gut and drive translation towards prevention of an important contributor of child mortality. !

Project Summary One of the major challenges in vaccine development is access to adjuvants that promote long-lived pathogen specific CD4+ and CD8+ T cell responses and provide protective cell mediated immunity. Our previous studies identified a molecular adjuvant that uses CD40 and TLR agonists (CD40/TLR) that induces protective long- lived T cell populations in mice and primates but why this approach is so effective is unclear. We believe that if we can understand why this adjuvant is different from other adjuvants or infection induced responses then it will help in the rational design of vaccines to generate protective cell mediated immunity. We have documented that vaccine-elicited T cells (Tvax) are distinct from infection-elicited T cells (Tinf) and this includes a unique metabolic program and unanticipated requirement for the cytokine IL-27 for Tvax generation. In addition, the ability of a specialized subset of dendritic cells (cDC1), to produce IL-27 predicts the magnitude of vaccine- elicited CD8+ T cell expansion and memory formation. Our recent studies have identified an IL-27-dependent c-Myc transcriptional signature within Tvax that is associated with T cell proliferation and survival. We developed a computational model which mathematically recapitulates T cell expansion data derived from antigen challenge studies in vivo. This unsupervised analysis indicates that the ability of this adjuvant to rapidly promote T cell interactions with APC at the initiation of T cell priming is the major predictor of the magnitude and quality of the T cell response. Based on these data sets will use intravital imaging of T?DC interactions and the manipulation of IL-27, DC functions and c-Myc pathways to understand the mechanistic determinants of the combined CD40L/TLR adjuvant. These data sets will be integrated into a stochastic agent-based mathematical model to predict and validate the key events involved in Tvax formation. The proposed studies bring together the combined efforts of three productive laboratories and their respective expertise in adjuvant discovery, CD8+ T cell biology, cytokine and transcriptional networks, multi photon imaging, and computational modeling in order to understand the molecular basis for adjuvant-elicited cellular immunity.

Project Summary Neurons are poorly recognized by the immune system which contributes to the ability of neurotropic pathogens to persist in the CNS but there is evidence that T cells can promote clearance of these organisms. For the parasite Toxoplasma gondii, T cell production of the cytokine IFN-? is important for resistance in the CNS because it activates hematopoietic and non-hematopoietic cells to control the tachyzoite (lytic) stage of the infection. Conversely, in response to cellular stress T. gondii transforms to the latent bradyzoite stage and forms long lived cysts in neurons. The lack of therapies that target the latent stage of T. gondii is a significant impediment to the management of this infection. Current dogma holds that because this stage is in neurons it evades immune surveillance and ensures chronicity. However, there is accumulating evidence of a more active battle between the host and parasite in the CNS. These observations indicate that T cell production of IFN-? activates neurons to control T. gondii but the ability of this parasite to persist may be because bradyzoites evade recognition and/or modulate cyst specific responses. In support of this idea, comparisons between tachyzoite and bradyzoite specific responses suggest that cyst-specific CD8+ T cells have reduced effector functions. To understand how T. gondii is recognized in neurons and how the parasite can evade surveillance, novel transgenic reporter systems for parasites will be combined with host reporters to track the fate of infected neurons in vivo. Additional studies will determine the impact of IFN-? on neurons and live imaging studies will visualize interactions between T cells and infected neurons and if this results in parasite clearance or evasion of T cell activities. The findings that emerge from these studies will have a significant impact on understanding how CD8+ T cell-neuron interactions lead to pathogen control and will be relevant to other neurotropic infections and neuroinflammatory conditions.

Project Summary The ability of recently activated T cells to express the cell surface molecule CD40L allows them to communicate with other immune and non-immune populations. This molecule is of particular importance in the gut to help control the parasitic infection caused by Cryptosporidium. Here we leverage a novel, natural mouse model of Cryptosporidium to dissect the impact of the CD40-CD40L interaction in T cell-mediated resistance to infection in the gut. In this model, WT mice (like humans) develop sterile immunity mediated by T cell production of IFN-?, but mice that lack CD40L mice (like humans) do not resolve infection. In addition, treatment of chronically infected CD40L-deficient mice with soluble (s)CD40L results in rapid parasite clearance. We will test if protective effect of CD40L may be explained by either I. its ability to promote T cell responses essential for resistance and/or II. because CD40L directly activates EC to limit parasite growth. We are uniquely equipped to utilize parasite transgenesis, combined with sophisticated genetic approaches to define the key cellular interactions that allows CD40L to determine the outcome of an enteric infection.