PhD, University of Cincinnati, 2024, Medicine: Immunology

Natural killer (NK) cells are a subset of innate lymphocytes characterized by their ability to eliminate infected or non-self cells. In addition, NK cells play a noncanonical role in regulation of adaptive immune responses. After certain viral infections and vaccinations, NK cells kill a subset of activated T cells in a perforin-dependent manner. The clearance of these T cells early in the immune response negatively affects downstream antiviral immunity. Ergo, NK cell suppression of adaptive immunity dictates the outcome of viral infections and efficacy of vaccinations. However, the mechanisms driving immunoregulatory activity of NK cells remain incompletely defined. The established requirement for perforin in NK cell immunoregulation suggests the necessity for cell-to-cell contact between NK cells and their targets. At homeostasis, NK cells and T cells localize in different tissue compartments. Chapter II explores re-localization of NK cells to T-cell rich sites after infection, a phenomenon shown to be dependent on the dose of virus given. This is shown to be associated with reduced induction of type I interferons during the lower doses of virus, which limits upregulation of the necessary chemokine ligands involved in NK cell enrichment within T cell rich regions of lymphoid tissues. Localization of NK cells and subsequent regulation of T cells is dictated by the pattern of type I interferon expression. However, pleiotropic effects of interferon on NK cells, T cells, and other immune cells precluded clear dissection of the precise dose-dependent governance of NK-cell immunoregulatory capacity. Cell transfer experiments complement current strategies to study NK cell immunoregulation but are predominantly used in anti-tumor models or in mice with incomplete immune systems. The conventional need for proliferative expansion of NK cells in a less competitive, immunodeficient environment when performing adoptive transfer studies precludes use of such models to e (open full item for complete abstract)

Committee: Stephen Waggoner Ph.D. (Committee Chair); David Hildeman Ph.D. (Committee Member); Grant Schulert M.D. Ph.D. (Committee Member); Leah Claire Kottyan Ph.D. (Committee Member); Koichi Araki D.V.M. Ph.D. (Committee Member) Subjects: Immunology

PhD, University of Cincinnati, 2023, Medicine: Pathobiology and Molecular Medicine

Head and neck cancer (HNC) is the 6th most common cancer worldwide, the majority of which are head and neck squamous cell carcinomas (HNSCC). Recurrent or metastatic HNSCC are often treated with immune checkpoint inhibitors (ICIs), which have dramatically increased survival, but unfortunately, only 20% respond to treatment necessitating improved therapies. The diabetes drug, metformin, was shown previously to have both anti-cancer and immune stimulating (primarily T cells) activity. In a phase I clinical trial (NCT02325401), metformin was administered to HNSCC patients prior to standard of care treatment. Metformin specifically increased NK cell numbers as well as activation more robustly than T cells in HNSCC patients. Therefore, we studied the direct effects of metformin on NK cells. Pre- and post-metformin- treated tumor tissue from a clinical trial (NCT02083692), was analyzed by immunofluorescence. Post-metformin tissue exhibited an increase in infiltrating NK cells. Ex vivo metformin treatment of HNSCC NK cells resulted in increased perforin production correlating with higher cytotoxicity of tumor cells. To determine pathways in which metformin may be regulating in order to promote cytotoxicity in NK cells, we utilized bulk RNA-sequencing (RNA-seq) on ex vivo pre- and post-metformin treated HNSCC patient NK cells. The chemokine, CXCL1, expression was significantly reduced with metformin treatment. Exogenous CXCL1 prevented metformin-mediated NK cell perforin release, but this could be reversed by an inhibitor of the CXCL1 receptor, CXCR2. We next investigated the mechanism in which metformin mediated CXCL1 inhibition may be preventing perforin release and subsequent cytotoxicity. Given metformin is known to inhibit mTOR and pSTAT3, we investigated the roles of these pathways in perforin release. Perforin was decreased by pSTAT1 inhibition and increased by mTOR inhibition suggesting that both pathways may be important. In order to understand if the CXCR2/CXCL1 p (open full item for complete abstract)

Committee: Trisha Wise-Draper M.D. Ph.D. (Committee Chair); Michael Borchers Ph.D. (Committee Member); Stephen Waggoner Ph.D. (Committee Member); Vinita Takiar (Committee Member); Scott Langevin Ph.D. (Committee Member) Subjects: Oncology

DNP, Otterbein University, 2024, Nursing

In the United States, the rate at which cancer is diagnosed is on the rise. In women, breast cancer is the second most common cancer diagnosis, following skin cancer, with approximately one in eight women developing breast cancer in their lifetime. When a patient receives a breast cancer diagnosis, the oncologic team will discuss the most suitable treatment plan for the patient, this often includes chemotherapy, radiation therapy, and surgery. Within the last few decades, research has shown that there may be a correlation between anesthetic medications and breast cancer metastasis. This project aims to create an evidence-based anesthesia guideline for patients undergoing breast cancer surgery. Interventions will include a paravertebral block, total intravenous anesthesia (TIVA), and non-opioid analgesics; evidence has shown that volatile agents and opioids can decrease natural killer cell activity and increase postoperative metastasis. The Johns Hopkins Evidence-Based Practice Guideline Model for Nurses and Healthcare Professionals will be used to guide this process.

Committee: Brian Garrett (Advisor); Kerrie Rodgers (Committee Member); Joy Shoemaker (Committee Member) Subjects: Health Sciences; Medicine; Nursing

PhD, University of Cincinnati, 2021, Medicine: Immunology

Natural killer (NK) cells are innate lymphocytes classically defined by their ability to eliminate infected or transformed cells. Within the past decade, a novel and unconventional role of NK cells in directly regulating adaptive immunity has been described. Following viral infection, NK cells eliminate a subset of activated T cells in a perforin-dependent manner. This early T cell suppression negatively impacts downstream antiviral cellular and humoral immunity and may lead to viral persistence while preventing fatal immunopathology. Hence, NK cell adaptive immune regulation dictates the outcome of viral infection. Importantly, the mechanisms involved in facilitating NK cell immunoregulatory activity remain incompletely understood. The requirement of perforin for NK cell suppression of T cells suggests that immunoregulation is cell-contact dependent. Yet NK cells and T cells reside in different tissue compartments at homeostasis. In chapter II, we find a transient accumulation of NK cells within T-cell rich follicles during viral infection. The chemokine receptor CXCR3 was essential for this redistribution as well as for NK cell suppression of antiviral T cells. Type 1 interferons mediated NK cell migration by promoting CXCR3 ligand expression. Adenoviral vectors that weakly induced type 1 interferon and NK cell follicle accumulation also do not stimulate NK cell suppression of T cells. Exogenous supplementation of type 1 interferon sufficiently rescued NK cell migration during adenoviral vector immunization. Thus, type 1 interferons mediate crucial CXCR3-directed redistribution of NK cells proximal to T cells to exert suppression. Therapeutic targeting of CXCR3 represents a novel strategy to bolster vaccine-elicited protective immunity. Whether NK cells exert additional immunoregulatory functions during viral infection remains unknown. Notably, chronically infected individuals exhibit worsened morbidity and mortality to bacterial pneumonias. Whether NK cell (open full item for complete abstract)

Committee: Stephen Waggoner Ph.D. (Committee Chair); George Deepe M.D. (Committee Member); Chandrashekhar Pasare DVM PhD (Committee Member); Joseph Qualls Ph.D. (Committee Member); Sing Sing Way Ph.D. (Committee Member); William Zacharias M.D. Ph.D. (Committee Member) Subjects: Immunology

MS, University of Cincinnati, 2020, Medicine: Molecular, Cellular and Biochemical Pharmacology

Influenza is a global pathogen of major public health impact. Despite the availability of vaccines for seasonal influenza, there are still 3-5 million severe cases of influenza globally each year. Therefore, understanding the mechanisms that the host utilizes to defend and kill the influenza virus is essential. An important host mechanism that has been associated with protection in animal models is Antibody-Dependent Cellular Cytotoxicity (ADCC). ADCC is mediated via innate immune cells that lyse the infected target cells, clearing the infection. There are several in-vitro assays that have been developed to measure influenza-specific ADCC. Most of these assays employ an endpoint that measures a surrogate marker of cell lysis. The major goal of this project was to develop and optimize an ADCC assay for Influenza A virus (IAV) whose endpoint quantification is cellular death due to cytotoxicity, and which can also distinguish responses to the Haemagglutinin (HA) and Neuraminidase (NA) components of 2019-2020 circulating strains. Codon-optimized HA and NA genes were designed, synthesized, and cloned into expression systems allowing stable, inducible expression in a mammalian cell line. An additional reporter construct expressing firefly luciferase and GFP was also stably introduced into the target cells. Robust total and cell surface expression of HA and NA along with luciferase expression was documented upon induction with doxycycline. An in-vitro assay for ADCC was then developed using an immortalized human NK cell line that is mixed with HA or NA-expressing target cells at given effector: target ratios, with lysis measured as the loss of luciferase signal compared to control cells. We next evaluated sera from influenza vaccine recipients in this assay. H1 and H3-specific ADCC responses were readily observed with these sera. N1-specific responses were detectable but appeared weaker in general than either H1 or H3-specific response. Evaluation with additional sera gat (open full item for complete abstract)

Committee: Paul Spearman M.D. (Committee Chair); Andrew Norman Ph.D. (Committee Member); Karnail Singh Ph.D. (Committee Member); Tongli Zhang Ph.D. (Committee Member) Subjects: Immunology

PhD, University of Cincinnati, 2020, Medicine: Pathobiology and Molecular Medicine

While cytomegalovirus (CMV) infection poses a considerable burden to public health, an efficacious vaccine is not yet available. Classical vaccine efforts focusing on the induction of Band T-cell memory have proven ineffective and, therefore, CMV remains a significant public health threat. Notably, CMV infections in both mice and humans trigger accumulation of unique adaptive natural killer (NK) cell subsets that putatively possess enhanced effector functions against CMV. This suggests that a pioneering approach to creating an efficacious CMV vaccine would include stimulation of the accumulation and long-term maintenance of these CMVreactive NK cells. It remains unknown, however, whether current CMV vaccines can stimulate the expansion of these NK-cell subsets. Here, we interrogated this question using a unique set of samples collected during a CMV vaccine trial. Vaccine study participants did not show an expansion of the CMV-dependent NKG2C+ NK cell subset, but marked oscillations of the frequency of FcR?neg NK cells uniformly lacking FcR?, EAT-2 and SYK adaptor molecules. Interestingly, the FcR?neg NK cells in this healthy cohort were phenotypically and functionally distinct from the ones reported in CMV-infected individuals. This unique longitudinal data in healthy CMV-seronegative individuals indicates that NK-cell subsets may react to unknown environmental or inflammatory cues and demonstrate greater flux than appreciated from past cross-sectional studies. We further evaluated the functional capabilities of CMV-associated NK subsets present in healthy individuals by using peripheral blood mononuclear cells (PBMC) from healthy blood donors. Since NKG2C+ and FcR?neg NK subsets are present in minute quantities in the blood of healthy individuals, we used a K562 feeder cell line with membrane bound IL-21 in order to expand those ex vivo for subsequent functional interrogation. Within the limit of our small sample size we report no significant d (open full item for complete abstract)

Committee: Stephen Waggoner Ph.D. (Committee Chair); David Bernstein M.D. (Committee Member); Jason Blackard Ph.D. (Committee Member); Laura Conforti Ph.D. (Committee Member); Rajat Madan M.D. Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Immune cells of myeloid origin have a unique role in the body's response to non-self entities. The cells, including monocytes and macrophages, carry out a diverse array of functions including phagocytosis, the uptake and presentation of foreign antigens, environmental debris, and damaged cells; the release of cytokines that coordinate acute inflammatory responses; and cytotoxic effector functions that result in the destruction of targets. In acute myeloid leukemia (AML), a differentiation block in the myeloid cell lineage prevents proper maturation of monocytes and macrophages. Instead, leukemic blasts rapidly accumulate and proliferate in the bone marrow, blood, and organs preventing proper hemocytic development. Patient death is caused mainly by infection, followed by hemorrhage and organ failure. The most common form of adult leukemia, AML has a low five year survival rate of 26.6% and a high rate of patient relapse. Taken with the high average age of diagnosis and the fact that certain elderly patients are unable to participate in the standard treatment of high-intensity chemotherapy, it is clear that there is a need for innovative, less toxic therapeutic approaches to the disease. One such approach is the re-invigoration of the patient's own immune system, typically suppressed in a myriad of ways due to the disease. This is explored in two novel studies presented here. The first, detailed in Chapter 2, takes advantage of the effector function that myeloid cells naturally possess; expression of Fcγ receptors on the cell surface allow for interaction with antibody opsonized targets. By eliciting expression of the antigen for the α-CD38 antibody daratumumab on the surface of AML blasts with all-trans retinoic acid (ATRA), we demonstrated it was possible to induce antibody-dependent blast-to-blast killing amongst the cancer itself, with blasts functioning as both targets and effectors, a phenomenon we termed fratricide. This antibody-induced fratricide (open full item for complete abstract)

Committee: Susheela Tridandapani PhD (Advisor); James Blachly MD (Committee Member); John Byrd MD (Committee Member); Amanda Toland PhD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2019, Biomedical Sciences

Studies of human lymphopoiesis are important towards gaining a thorough understanding of normal immune cells and can provide insights into the pathophysiology of immune disorders and deficiencies. Innate lymphoid cells (ILCs) are a newly discovered family of immune cells with emerging roles in the defense against cancer, viral and bacterial infections, allergies, and autoimmune diseases. These immune cells are unique in that they share features of both innate and adaptive immunity. While ILCs lack the ability to respond in an antigen-specific manner, they closely resemble adaptive lymphocytes in terms of transcriptional signatures and effector function. There are four types of ILCs: cytotoxic natural killer (NK) cells and as many as three subsets of non-cytotoxic, cytokine-producing “helper” ILCs termed ILC1s, ILC2s, and ILC3s (the existence of ILC1s in states of normal human physiology remains controversial). Currently, little is known about the development and regulation of helper ILCs. Previously, our laboratory discovered a comprehensive pathway of human NK cell development in secondary lymphoid tissues (SLTs) such as tonsils and lymph nodes exclusive of other ILCs, and have begun to explore human ILC development in general. In particular, we and others have described and characterized multipotent CD34+CD117- progenitor cells as well as CD34+CD117+ common ILC progenitors in human tonsils. Based on these data, we hypothesized that in humans all ILCs share a common developmental pathway in tonsils and that at some point, each ILC subset terminally differentiates along its own developmental trajectory. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular, transcriptional, and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originate from a common ILC precursor cell identified as CD34-CD117+, final steps of ILC2 development deviate (open full item for complete abstract)

Committee: Michael Caligiuri M.D. (Advisor); Aharon Freud M.D., Ph.D. (Advisor); Don Benson M.D., Ph.D. (Committee Member); Susheela Tridandapani Ph.D. (Committee Member) Subjects: Biomedical Research; Immunology

Doctor of Philosophy, Case Western Reserve University, 0, Pathology

Cancer is the second-leading cause of death in the United States and worldwide leading to 8.8 million deaths in 2015. Despite major advancements in science and health, patients at advanced stages of particular cancer types experience significant mortality. Cancer patients often relapse following treatment with conventional therapeutic modalities such as chemotherapy and radiation therapy. Conventional treatment approaches do not successfully address the heterogeneity present in most solid tumors, result in severe off-target effects resulting in severe side effects, and fail to kill dormant cancer cells. Immunotherapy is the use of immune cells as tools to combat diseases. Natural killer (NK) cells are lymphocytes that lyse tumor cells and virally-infected cells through diverse mechanisms including release of cytotoxic granules. NK cell therapy has shown much promise in preclinical studies however it fails to demonstrate efficacy in various cancer populations. This body of work tackles two crucial barriers to NK cell therapy in patients with solid tumors. NK cell numbers in the tumor has been correlated with good prognosis in cancer patients. Obtaining clinically efficacious numbers of NK cells is limited by the relatively small proportion of NK cells in peripheral blood. We, therefore, designed and engineered a novel IL-21-based NK cell expansion platform consisting of OCI-AML3 cells transduced with membrane-bound IL-21 (NKF cells). We demonstrated that NKF cells are effective at expanding NK cells. The NK cells expanded using NKF cells were able to lyse a wide array of tumor types and demonstrated a favorable metabolic signature as compared to non-expanded NK cells. Additionally, NKF-expanded NK cells significantly reduced the tumor burden in sarcoma cell-infected mice. Cancer cells have developed mechanisms to evade anti-tumor activities of NK cells, dampening the efficacy of NK cell-mediated lysis of cancer cells. Transforming growth factor-ß (TGF-ß) is an i (open full item for complete abstract)

Committee: David Wald MD/PhD (Advisor); Alan Levine PhD (Committee Chair); John Wang PhD (Committee Member); Mark Jackson PhD (Committee Member); Sanford Markowitz MD/PhD (Committee Member); Clive Hamlin M.S./PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

In 1997 rituximab became the first monoclonal antibody (mAb) approved by the Federal Drug Administration (FDA) specifically for the treatment of cancer. A major mechanism underlying the effectiveness of rituximab is its ability to activate the innate immune system through stimulation of Fc receptors (FcR) expressed by innate immune cells such as natural killer (NK) cells. FcR stimulation of NK cells results in the direct destruction of tumor cells through antibody dependent cellular cytotoxicity (ADCC) and production of cytokines with anti tumor function. In the 20 years since rituximab's approval there has been a vast expansion of mAbs in cancer therapeutics with over a dozen such molecules receiving FDA approval. In addition, a new class of mAbs which target regulatory elements of the immune system known as immune checkpoints have begun to be successfully utilized in the clinic. Regardless of the mechanism by which a mAb works for these agents to be clinically effective the effector functions of the immune cell populations they target must be intact. Unfortunately, it has been observed that in the setting of cancer the function of both NK cells and T cells is significantly impaired, and it is thought that this dysfunction limits the response to mAb therapy. While some of the mechanisms that cause impaired NK cell and T cell function are linked to processes inherent to tumor cells such as cytokine production and metabolic alterations it is also clear that cancer is linked to the expansion of multiple immune suppressive cell populations such as regulatory T cells (Treg), tumor associated macrophages (TAMs), and myeloid derived suppressor cells (MDSC). MDSC are a heterogeneous population of immature myeloid cells that expand in the setting of cancer and accumulate in tumors. MDSC have been shown to exert potent suppression of anti tumor immune responses by contact dependent and contact independent mechanisms. Contact dependent mechanisms include expression (open full item for complete abstract)

Committee: William Carson III (Advisor) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2017, Pathology

HCV-infection affects approximately 170 million worldwide, with 60-85 percent of acute-infections resulting in chronic-infection, and a significant proportion are co-infected with HIV. HCV-infection causes increased morbidity and mortality in HCV-HIV co-infection. HIV-infection alters HCV-disease pathogenesis, accelerating progression to cirrhosis and liver failure. While HCV-therapy has greatly improved, the cost remains a barrier and is not expected to completely remove HCV from the population. We need a better understanding of the immune-response to HCV to develop successful vaccine strategies. HCV-containment and -clearance is dependent on efforts of the innate and adaptive immune-response. CD4 memory T-cells are critical in viral-clearance, by producing IFN¿ and mediating CD8 T-cell responses. NK cells have been shown to exert an important role in control of HCV-infection by lysis of infected-cells and cytokine release. Monocytes may partly shape this HCV-directed response, via direct-contact with CD4 T-cells and NK cells and in production of soluble factors. IL-7 has been demonstrated to enhance NK cell IFN¿ production, and the IL-7 receptor a chain (CD127) is expressed on NK cells. We measured CD127 expression on NK cell subsets of uninfected donors, chronic HCV-infected treatment-naive, HIV-infected on ART and HCV-HIV co-infected subjects on ART. We demonstrate that CD56bright NK cell CD127 expression negatively correlated with HCV plasma levels in HCV mono-infection and HCV-HIV co-infection. We observed IL-7-induced NK cell activation, cell-cycling, IFN¿ release and cytolytic function, with impairments in HCV- and HIV-infections. These findings offer a role for IL-7-dependent NK cell function in control of chronic viral infections. Within chronic HCV- and HIV-infection, there have been observations of elevated levels of IL-6 and sCD14, produced in part by monocytes, which contribute to immune-activation. We extended these studies to evaluate th (open full item for complete abstract)

Committee: Donald Anthony MD, PhD (Advisor); Dave Canaday MD (Committee Chair); Scott Sieg PhD (Committee Member); Daniel Popkin MD, PhD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2016, Integrated Biomedical Science Graduate Program

Natural killer (NK) cells are innate immune cells that play different roles depending on their stage of development. NK cells develop through five stages that can be identified in secondary lymphoid tissue. Stage 3 immature NK (iNK cells) cells play important roles in homeostasis and defense against infection. They produce the cytokines interleukin (IL)-17 and IL-22 and express the transcription factor ROR¿t. Because IL-22 is critical to the anti-microbial immune response, we sought to identify factors that can promote proliferation and induce or maintain IL-22 production by iNK cells and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an iNK cells cell survival factor, IL-15, to induce proliferation of iNK cells, as well as induce and maintain IL-22 production. We found that this effect was mediated through the NF¿B pathway. Furthermore, we observed that IL-18-producing CD11c+ dendritic cells are found proximal to iNK cells in human tonsil. Stage 4 and 5 mature NK cells are distinguished from iNK cells in several ways. Mature NK cells play roles in antiviral and antitumor responses. They express CD94, produce the cytokine IFN-¿, and display cytotoxic activity. Stage 5 NK cells are distinguished from stage 4 NK cells by expression of CD16. Because very little is known about how CD16 is regulated, we also sought to identify pre-translational mechanisms governing this process. First, we used chromatin immunoprecipitation (ChIP) to test for the association of STAT1, GATA3, SRF, and SP1 with the CD16 promoter. While each of these transcription factors has a consensus binding sequence in the CD16 promoter, we did not find enrichment of any of these factors. We also investigated DNA methylation in the CD16 promoter and found that it was hypermethylated in stage 4 NK cells and hypomethylated in stage 5 NK cells. These data identify DNA methylation as a possible regulatory mechanism for CD16 expression in NK cells. Fi (open full item for complete abstract)

Committee: Michael Caligiuri MD (Advisor); Mark Wewers MD (Committee Member); Natarajan Muthusamy DVM/PhD (Committee Member); Jianhua Yu PhD (Committee Member) Subjects: Biomedical Research

MS, University of Cincinnati, 2016, Medicine: Immunology

Modern medicine has advanced tremendously over the past century to develop vaccines that have successfully reduced the spread of, or eradicated, many infectious diseases including measles, smallpox and polio. While this progression has dramatically improved global health, the development of vaccination strategies for protection against deadly pathogens, such as human immunodeficiency virus (HIV) and hepatitis C virus, have been elusive. Further investigation of immune-regulatory mechanisms that are influential in the development of protective humoral immunity must be pursued for the generation of robust vaccinations to continue. Here, we present our findings on a regulatory role for natural killer (NK) cells in the generation of the humoral immune response to viral infection. Specifically, we have determined that in mice infected with the acute strain of lymphocytic choriomeningitis virus (LCMV), NK cells impair the development of T follicular helper cells and the germinal center response. We have determined that this suppressive function is carried out in a perforin-dependent manner at an early stage of infection. Importantly, during this early time frame, an anatomic redistribution of NK cells occurs in the spleen to areas involved in generation of the germinal center responses, including the B cell follicle and T/B border of the white pulp. Mechanistically, we have identified a subset of these NK cells expressing the B-cell follicle homing receptor CXCR5 that are present in the spleen at these same early time points of infection. Taken together, these data support out hypothesis that this subset of white pulp localizing NK cells traffics to this region early in infection to facilitate direct contact with cells involved in the generation of T follicular helper cell and germinal center responses. While in contact with these precursors, we suspect NK cells carry out perforin-dependent cytolysis of target cells to ultimately suppress the generation of humoral immune (open full item for complete abstract)

Committee: Stephen Noel Waggoner Ph.D. (Committee Chair); Leah Claire Kottyan Ph.D. (Committee Member); J, Matthew Kofron Ph.D. (Committee Member); Jonathan Katz Ph.D. (Committee Member) Subjects: Immunology

PhD, University of Cincinnati, 2015, Medicine: Immunology

Aging is a complex process that negatively affects a wide variety of physiological functions, including the development and maintenance of robust immune responses. Developing successful strategies that promote improved immune responses in the elderly against infectious pathogens and cancers that cause high mortality represents a growing public health priority. Natural killer (NK) cells play a critical role in eliminating tumor cells and viral infections, both of which occur at a high incidence in the elderly population. Aging has a considerable negative impact on the function and development of NK cells. These defects attenuate NK cell cytotoxicity to virus infected cells and tumors. Thus, studies to determine the causes of aged-related NK cell dysfunctions are critically required to address the health needs of a growing elderly population. The precise mechanism(s) underlying the impaired maturation and function of NK cells in aging remain unidentified. This dissertation aims to fill this gap in knowledge by evaluating whether key factors known to be important in NK cell biology could be dysregulated in aging, thus impairing their functional maturation. Importantly it highlights novel avenues for research in the field geared at discovering the specific factor(s) that impair the function and terminal maturation of NK cells in aging. Herein, we provide evidence that the aged environment and in particular, the aged BM microenvironment contributes to the impaired maturation and function of NK cells. NK cells derived from both young and aged BM cells adopted an impaired maturation phenotype in an aged host i.e. were hyporesponsive to stimuli in vitro, while adopting an augmented maturation phenotype following transfer in young hosts. In our pursuit to identify the environmental factor(s) that may contribute to this phenotype, we observed a differential pattern of expression of key transcription factors including a decrease in T-bet and Eomes which are known to regulat (open full item for complete abstract)

Committee: Claire Chougnet Ph.D. (Committee Chair); Fred Finkelman M.D. (Committee Member); David Hildeman Ph.D. (Committee Member); Edith Janssen Ph.D. (Committee Member); James Mulloy Ph.D. (Committee Member) Subjects: Immunology

Master of Science in Biological Sciences, Youngstown State University, 1999, Department of Biological Sciences and Chemistry

Natural Killer (NK) cells are white blood cells that participate in the direct cellular elimination of tumor and virus-infected cells. NK cell function is determined through the use of cytotoxicity assays. Our study investigates the use of an LDH assay (Promega Cytotox 96 LDH assay) to characterize murine NK cells. Four target cell lines were tested for their effectiveness in this assay.

Committee: Diana Fagan (Advisor) Subjects: Biology, Cell

Doctor of Philosophy in Medicinal Chemistry, University of Toledo, 2010, Medicinal Chemistry

Myasthenia gravis (MG) is characterized by autoantibody-mediated reduction of nicotinic acetylcholine receptors (AChR). In myasthenia gravis, anti-acetylcholine receptor (AChR) antibody is thought to cause damage through the activation of complement, via the classical complement pathway, in addition to causing internalization of AChRs. Interleukin-12 (IL-12), a major inducer of interferon-gamma (IFN-gamma) production, has been shown to enhance active and clinical passive experimental autoimmune myasthenia gravis (EAMG). IFN-gamma is known to be produced by various cell types, including CD4+ and CD8+ T cells, natural killer (NK) cells, and NK1.1+ T (NKT) cells. Our current research was designed to investigate the effects of IL-12 on skeletal muscle tissue. We performed passive transfer experiments with mAb D6, a mouse anti-AChR monoclonal antibody recognizing the immunodominant mouse AChR epitope, using both B6 and IFN-gamma knockout mice. The role of complement in disease induction in these mice was examined using fluorescent microscopy. When pre-treated with IL-12, B6 and IFN-gamma knockout mice had similar amounts of antibody and complement bound at the neuromuscular junction, although IFN-gamma knockout mice were resistant to EAMG development. This suggests that IL-12, through IFN-gamma, may increase susceptibility to disease through mechanisms downstream of antibody and complement binding. In order to do investigate the roles of known IFN-gamma producing cell types in passive disease enhancement, NK and NKT cells were depleted using the anti-NK1.1+ monoclonal antibody PK136 prior to administration of IL-12 and passive transfer of mAb D6 in C57BL/6 (B6) mice. Comparatively, IL-12 treatment and passive transfer of mAb D6 was performed using TCRbeta/TCRdelta dual knock-out mice (TCRbdKO mice), which possess B cells and NK cells but completely lack functional T cells and NKT cells. Interleukin-12 treatment failed to enhance passive disease in mAb PK136-treated B6 mi (open full item for complete abstract)

Committee: Katherine Wall Dr. (Committee Chair); Marcia McInerney Dr. (Committee Member); Surya Nauli Dr. (Committee Member); Francis Pizza Dr. (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Treatment with a monoclonal antibody (mAb) to HER2 (trastuzumab) is now the standard of care for patients with HER2-overexpressing breast cancer, however, only 25-30% of patients will respond to this form of therapy. The anti-tumor actions of trastuzumab have generally been attributed to the inhibition of tumor cell proliferation or the induction of apoptosis. However, recent studies have shown that the anti-tumor effects of trastuzumab are dependent on immune cells that express receptors for the constant (Fc) region of immunoglobulin G. Natural killer (NK) cells are innate immune cells that express an activating Fc receptor (FcgammaRIIIa), which allows them to recognize and interact with Ab coated targets. We proposed that the immune response to anti-tumor mAbs could be enhanced via the co-administration of NK cell activating adjuvants. NK cells co stimulated with trastuzumab-coated breast cancer cells and interleukin-12 (IL-12) secreted >10-fold higher amounts of interferon-gamma (IFN-gamma) as compared to NK cells stimulated with either agent alone. Co-stimulated NK cells also secreted abundant quantities of a number of chemokines that could induce the chemotaxis of naive and activated T cells. We detected elevated levels of IFN-gamma and T cell-recruiting chemokines within the sera of human cancer patients receiving trastuzumab and IL-12. These factors were functional for T cell chemotaxis and their presence correlated with the infiltration of tumor tissue by cytolytic CD8+ T cells. Furthermore, administration of IL 12 enhanced the actions of an anti-HER2 mAb in a murine model of HER2-positive cancer, an effect that was dependent on NK cell production of IFN-gamma and on the presence of CD8+ T cells. These results suggest that the anti-tumor effects of trastuzumab are mediated through the secretion of IFN-gamma and T cell-recruiting chemokines by activated NK cells, leading to the invasion and destruction of tumor tissue by cytolytic CD8+ T cells. Furthermore, t (open full item for complete abstract)

Committee: William Carson (Advisor) Subjects: Health Sciences, Immunology

Doctor of Philosophy, The Ohio State University, 2006, Oral Biology

Activation of neuroendocrine responses by restraint stress (RST) suppresses natural killer cell (NK) activity during an experimental influenza A/PR8 viral infection. RST-induced activation of the HPA axis upregulates glucocorticoids (GC) and endogenous opioids which may be responsible for the observed suppression of NK activity. GC receptor antagonism modulates trafficking and splenic cellularity. However, the specific effects on NK activity remain unclear. Opioid receptor antagonism with naltrexone (NTX) abrogates RST-induced suppression of splenic NK cytotoxicity suggesting endogenous opioids modulate NK cytotoxicity. However, the specific subtype of opioid receptors involved remains unclear. These studies examined the effects of GC and specific opioid receptor subtype antagonism on splenic NK cellularity and cytotoxicity. Additionally, these studies examined whether the opioid signaling mechanism involved a direct effect on NK cells or a possible modulation in NK-stimulatory cytokine mRNA expression. C57BL/6 mice were treated daily with the GC receptor antagonist RU486, the opioid receptor antagonist NTX, or i-, a-, or e-opioid receptor specific antagonists. Mice were infected intra-nasally with A/PR8 virus and underwent daily RST. Splenocytes were obtained three days post infection. For in vitro studies, splenocytes were incubated with NTX or morphine (MOR) and tested for cytolytic activity at 24 and 48 hours. NK cytotoxicity was assessed using a standard chromium release assay. FACS analysis was used to detect the number of CD3-DX5+ NK cells. Cytokine mRNA expression was assessed using realtime PCR. RST-induced suppression of NK activity was a result of decreases in number of splenic NK cells and cytotoxicity. GC Receptor antagonism restored cellularity and i-opioid receptor antagonism restored NK cytolytic activity. Splenic NK cells incubated with morphine or NTX for up to 48 hours showed no change in NK cytotoxicity. Preliminary data suggested that antagonism (open full item for complete abstract)

Committee: John Sheridan (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Medical Microbiology and Immunology

Cells from normal breast tissue have on their surface a specific protein known as HER2. Breast cancer cells over-express this protein around twenty thousand times that of normal tissue. Targeting of HER2 over-expressing tumor cells can be achieved using an agent called a monoclonal antibody (mAb) that specifically binds to over expressed HER2 on the surface of the breast cancer cells. Binding of this antibody to HER2 can potentially inhibit tumor cell growth or lead to tumor cell destruction by the immune system. Unfortunately, only 20-40% of patients with HER2 over-expressing breast cancers currently respond to this treatment. We show here that natural killer (NK) cells secrete a distinct profile of potent immune stimulatory cytokines and T cell-recruiting chemokines in response to dual stimulation with mAb-coated tumor cells and interleukin (IL)-12. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (Fc-gamma-RIIIa) and the IL-12 receptor expressed on NK cells. The importance of this NK cell cytokine profile was confirmed in a phase I trial of breast cancer patients receiving IL-12 in combination with the anti-HER2 mAb, trastuzumab. Upon examination of the intracellular signaling pathways within primary human NK cells responsible for the elevated cytokine production, we found that levels of activated Syk and extracellular signal-related kinase (Erk) were synergistically elevated in Fc-gamma-RIIIa and IL-12 co-stimuated NK cells as compared to either stimulation alone. Moreover, inhibitor studies suggested that these molecules were critical for synergistic produciton of IFN-gamma. Interestingly, inhibition of lipid rafts within NK cell membranes abroaged the enhanced Erk activation and subsequent IFN-gamma production, suggesting the importance of lipid raft signaling in mediating cytokine production. Confocal microscopy of NK cells confirmed co-localization of both Fc-gamma-RIIIa and the IL-12R w (open full item for complete abstract)

Committee: William Carson (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Medical Microbiology and Immunology

The immune system can be viewed as a diffuse sensory organ that is responsible for detecting and eliminating infiltrating pathogens. However, the immune system must strike a balance between limiting microbial replication and immune-induced pathology. The immune system has various regulatory mechanisms that can do this; one such immunoregulatory pathway is the bi-directional communication among the nervous, endocrine and immune system. Extrinsic factors, e.g. infection, and intrinsic factors, e.g. psychogenic stress, can activate the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS). Activation of these systems by either stress or infection leads to the secretion of glucocorticoids (GCs), catecholamines and opioids. Chronic elevation of GCs by restraint (RST) stress modulated the immune response to an experimental influenza A/PR8 viral infection. As demonstrated by experiments contained herein, RST altered cytokine gene expression, suppressed NK cell activity, and attenuated lymphocyte trafficking. Finally, RST enhanced viral replication (probably as a consequence of the RST mediated immunomodulation). Thus, alteration of normal immunoregulatory mechanisms by RST modulated the inflammatory, innate and adaptive response to an influenza A/PR8 viral infection. Previous studies from our laboratory using a social stress paradigm, social disruption (SDR), showed that SDR induced a state of functional GC resistance in cultured splenocytes. SDR, depending on the timing of the infection relative to the SDR cycle, differentially affected immune response to an influenza viral infection. When SDR occurred concurrently with the influenza infection, cytokine gene expression in the lung was suppressed at early time points during the infection. However, cytokine gene expression returned to control levels after SDR was stopped. In contrast, SDR prior to the influenza challenge attenuated the infection-induced weight loss and corticosterone secretion. Furt (open full item for complete abstract)

Committee: John Sheridan (Advisor) Subjects: Health Sciences, Immunology