BD BIOSCIENCES RESEARCH GRANTS

Spring 2012 Research Grant Recipients Talk About Their Research


Marie Andrée Forget, PhD
Post-Doctoral Fellow

Abstract Title:
Genetically Modified Artificial Antigen-Presenting Cells (aAPCs) to Improve Tumor Infiltrating Lymphocyte Expansion Product for Adoptive Cell Therapy


BD: Tell us about your educational background.

Marie-Andrée Forget: I hold an accreditation in biomedical lab technology from Saint-Jean-sur-Richelieu College, which allowed me to pursue an undergraduate degree while working as a hematology technician in my home town hospital. I earned my BS in biomedical sciences from the University of Québec Trois-Rivière. I was then awarded a master's and PhD in biomedical sciences from the University of Montréal, where I studied breast and lung cancer immunotherapy.

BD: How did you become interested in science?

Marie-Andrée Forget: My interest in immunology began while I was working as a technician in the hematology lab. My interest further grew during my undergraduate studies through attending lectures. But specifically, my passion for cancer immunology and immunotherapy arose through contact with my thesis director, Dr. Rejean Lapointe.

BD: Describe the project for which you were awarded the BD grant.

Marie-Andrée Forget: Adoptive cell therapy (ACT) is a type of immunotherapy that consists of expanding tumor-infiltrating T lymphocytes (TILs) from the patient's tumor to between 20 and 150 billion, and re-infusing them into the patient. These expanded TILs can recognize and eliminate tumor cells. Recently, TIL ACT has resulted in response rates up to 50% in metastatic melanoma patients. Currently, TIL adoptive cell therapy for MM is performed only at three centers in the USA (including MD Anderson Cancer Center (MDACC)) and one overseas. A major obstacle to the adoption of this approach is overcoming the technical challenges associated with the production of a large-scale, autologous TIL ACT product. A critical step during TIL expansion is the two-week rapid cell expansion protocol. The current protocol involves TIL activation in the presence of a large excess (200:1) of irradiated allogeneic normal donor peripheral blood mononuclear cells (PMBCs) added as "feeder cells." The procurement of such large numbers of feeder cells (1010 per patient treated) is difficult, expensive, and the cells' quality varies. The heterogeneity of these PBMC feeder cells also makes it difficult to define the key signaling pathways (especially T cell co-stimulatory pathways) needed to optimize TIL expansion and generate T cells with the improved anti-tumor and persistence properties.

We hypothesize that feeders can be replaced by the K562 cell line transformed into artificial antigen-presenting cells by the expression of co-stimulatory molecules. We also hypothesize that we can generate TILs with improved anti-tumor and persistence properties for ACT by targeting stimulation of other key co-stimulatory signaling pathways during TIL expansion by further engineering our antigen-presenting cell using technology developed in Dr. Laurence Cooper's lab at the MD Anderson.

We are currently successfully expanding TILs while assaying their quantity and quality compared with cells generated using conventional protocols. We are also building new antigen-presenting cells expressing different surface molecules and cytokines that serve as boosting factors during TIL expansion. We will soon be testing this second-generation antigen-presenting cell for its capacity to expand TILs and be able to tease apart the effectiveness of various co-stimulatory molecules expressed by these aAPCs.

BD: What are your long- and short-term scientific goals?

Marie-Andrée Forget: Our goal is to use K562 antigen-presenting cells as a platform to develop a novel approach to expand melanoma TILs with enhanced functional properties. We hope to replace the feeder-cell protocol with these new cells in a clinical trial at MD Anderson Cancer Center. We also hope to use this new platform to facilitate implantation of TIL-adoptive cell therapy programs in other centers.

BD: What are the implications of your project for human health?

Marie-Andrée Forget: TIL ACT has demonstrated a clinical response rate of about 50% in late-stage melanoma, as reported by three different centers. Patients enrolled in these clinical trials were suffering from metastatic melanoma that was refractory to available treatments. Facilitating access to TIL ACT in other centers across the country and beyond could extend this success to a broader patient population. We hope that this new generation of antigen-presenting cells will provide us with tools to manipulate TILs, to amplify or extend their anti-tumor responses and the persistence of these cells in patients, leading to better protection against cancer recurrence.

BD: Which BD reagents do you plan to use, and for what purposes?

Marie-Andrée Forget: Multiparameter flow cytometry is vital for this project, so BD's antibodies for flow cytometry will be the main reagents we will employ. The use of antibodies is essential to address the different molecules expressed at the surface of the T cells after expansion. This will enable us to define the cells' phenotypes following different types of co-stimulation, and to define the activation and/or differentiation status of the expanded T cells. Intracellular staining with BD's antibodies directed against different human cytokines or cytotoxic enzymes will also help in further defining the T cells' potential for anti-tumor response and allow assessment of T cell polyfunctionality.


Elizabeth Fritz, PhD
Assistant Professor

Abstract Title:
Innate Immune Response to Ebolavirus Infection


BD: Tell us about your educational background.

Elizabeth Fritz: I received my BS from the University of Illinois-Urbana/Champaign, with a major in microbiology and a minor in chemistry. I obtained my PhD in microbiology and immunology from Rush University Medical Center. I completed my post-doctoral fellowship through the National Research Council (NRC), National Academies of Science at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) in immunology and virology.

BD: How did you become interested in science?

Elizabeth Fritz: Science and math were my favorite subjects, and I had amazing high school science and math teachers who supported and encouraged my strengths. I became interested in immunology and virology after my undergraduate studies and laboratory rotation at the University of Illinois-Urbana/Champaign. My NRC post-doctoral fellowship at USAMRIID was a unique and rewarding opportunity for specialized training and experience in infectious diseases that ultimately prepared me for my current position.

BD: Describe the project for which you were awarded the BD grant.

Elizabeth Fritz: Ebolavirus (EBOV) and Marburgvirus (MARV) are single-stranded, negative-sense RNA viruses of the Filoviridae family recognized for their impressive lethality. Filoviruses are causative agents of viral hemorrhagic fever (VHF), which is characterized by hypotension, inflammation, lymphopenia, thrombocytopenia, coagulation disorders, hemorrhage, and a rampant cytokine-storm response resulting in multi-organ failure and death. There are no FDA-approved vaccines or therapeutics to combat EBOV or MARV infections, and we still lack an understanding of the host's innate immune response to these category A priority pathogens.

We will study the DC and NK cell response to EBOV infection in vitro with the objective of elucidating the innate immune response to EBOV. We hypothesize that filoviruses immediately target DC and NK cells, allowing rampant viral replication/spread resulting in an immunosuppressive state incapable of battling infection. We expect to find that ZEBOV, but not REBOV, inhibits DC maturation/activation and inhibits NK cell activation/function, thereby leading to a severely compromised innate immune response in the host.

We previously identified changes in DCs (HLA-DRlo and DC-SIGNlo) and depletion of NK cells following filovirus infection. To determine the effect EBOV has on cellular maturation, activation, and function, DC and NK cells will be purified from whole blood specimens. DCs will be infected with ZEBOV, a highly pathogenic virus in humans, or REBOV, a nonpathogenic virus in humans, and stained for cellular markers by using BD fluorochrome-conjugated antibodies (CD123, CD11c, HLA-DR, CD209, CD86, CD80, CD40) to determine changes in cellular activation and/or maturation. In parallel, DC cytokines (IL-2, IL-10, IL-12, IL-6, IL-18, IL-15, TNF-alpha, IFN-alpha/beta, IFN-gamma) will be measured as a measure of cellular activation and/or maturation. EBOV-infected DCs will be co-cultured with homologous donor NK cells to determine altered NK cell activation and function.

Additionally, EBOV and MARV are known to interfere with host cell signal transduction pathways, especially that of STATs phosphorylation leading to an inhibited interferon response. We will examine the effect of ZEBOV and REBOV on DC and NK cell signal transduction pathways and whether these viruses induce apoptosis in these cell populations.

BD: What are your long- and short-term scientific goals?

Elizabeth Fritz: Tremendous milestones have been made in filovirus vaccine development and testing. Dendritic cells and natural killer cells are innate immune cell populations affected during EBOV infection. However, a comprehensive analysis of how EBOV affects the activation, maturation, function, and/or survival of these individual cell populations is currently lacking. These cell populations are integral components of the innate immune response to viral infection. The primary goal of these studies is to provide an understanding of the dendritic and natural killer cell response to EBOV infection that will aid in unraveling the mechanisms by which these viruses affect the host's primary antiviral defense network. Eventually these studies will provide beneficial knowledge for filovirus vaccine and therapeutic development and testing to combat EBOV infection and potentially other viral hemorrhagic fever diseases

BD: What are the implications of your project for human health?

Elizabeth Fritz: The innate immune response to EBOV infection appears to be minimal to nonexistent during infection, ultimately leading to death. These studies will aid in our understanding of how EBOV affects and/or alters two primary cell populations intricately involved in the innate immune response to viral infection. This new understanding will aid in the development and testing of novel therapeutics and/or vaccines to combat EBOV infection and possibly other closely related viral hemorrhagic fever viruses.

BD: Which BD reagents do you plan to use, and for what purposes?

Elizabeth Fritz: We plan to use BD fluorochrome-conjugated antibodies in flow cytometry studies for measuring and monitoring cell activation, maturation, and function. We will work with many other BD products, including BD Pharmingen™ stain and BD Cytofix™ buffers as flow cytometry support reagents, BD GolgiPlug™ protein transport inhibitor, and BD Cytofix/Cytoperm™ buffer to help measure intracellular cytokine levels through flow cytometry, BD Phosflow™ reagents for analyzing signal transduction pathways, and the BD Pharmingen™ Annexin V and APO-BRDU™ apoptosis detection kits for apoptosis studies.


Gianluca Matteoli, DVM, PhD
Post-Doctoral Fellow

Abstract Title:
The Vagus Nerve as Modulator of Intestinal Homeostasis


BD: Tell us about your educational background.

Gianluca Matteoli: I received my veterinary medical degree from the University of Naples "Federico II," Naples, Italy, and my PhD in immunology jointly from the University of Naples and the Institute of Medical Microbiology, Tübingen, Germany. I completed a post-doctoral fellowship at the European Institute of Oncology, Milan, Italy, where I studied the immunobiology of dendritic cells in a group led by Dr. Maria Rescigno.

BD: How did you become interested in science?

Gianluca Matteoli: Like many children I was amazed by nature and the different forms of life on our planet. During my veterinary studies, I became interested in the relationship between pathogens and our immune system. I realized that besides fighting pathogens, the main challenge of our immune system is to control unnecessary response against microorganisms or foreign antigens present on our skin and in our intestines. In other words, the immune response must be tightly controlled to avoid undesirable immune reactions against microbiota and food antigens.

BD: Describe the project for which you were awarded the BD grant.

Gianluca Matteoli: A complex and independent nervous system called the enteric nervous system (ENS)—also known as the "little brain"—lies within the gastrointestinal tract. The ENS consists of a network of hundreds of millions of neurons embedded in the intestinal wall and comprising the intrinsic innervations of the gut. The ENS, together with the assistance of extrinsic innervation, enables us to "feel" the inner world of our intestine and its contents and to regulate motility and digestion of nutrients. However, the intercommunication between the ENS and immune system cells is poorly understood. Functionally, the ENS is quite similar to the central nervous system (CNS). And growing evidences suggests that neurotransmitters can convey signals to immune cells and modulate their function. In line with these findings, a primary alteration of enteric neurons is often found in patients afflicted by inflammatory bowel disease. Therefore, in my research proposal I aim to study the crosstalk between the ENS and the mucosal immune system, to define neuromediators that regulate immune tolerance and avoid chronic intestinal inflammation.

We will focus our attention mainly on the immunological changes that occur in the intestine after activation or inactivation of the parasympathetic nervous system. We will use two approaches: electrical stimulation of the vagus nerve to induce the release of neurotransmitters in the intestine, and resection of the vagus nerve which leaves the enteric nervous system without vagal control.

BD: What are your long- and short-term scientific goals?

Gianluca Matteoli: Communication with the autonomic nervous system is believed to affect the immune system. Crosstalk between nerves and immune cells may be of great importance for maintaining immune homeostasis, thereby controlling locally inflammatory responses and preventing collateral damage or disseminated disease. Although the mechanisms involved are becoming better known, the anatomy and molecular pathways involved remains a matter of debate. Therefore, we will attempt to unravel the neural circuit and the molecular mechanism involved in this process. Defining the cells targeted by the vagus nerve in the intestine will be the ultimate goal in order to develop new drugs or therapeutic approaches.

BD: What are the implications of your project for human health?

Gianluca Matteoli: Interest in the cholinergic anti-inflammatory pathway is growing exponentially as two new clinical trials using electrical stimulation of the vagus nerve are already ongoing in patients affected by rheumatoid arthritis and Crohn's disease. These studies may provide proof of the therapeutic potential of activating the cholinergic anti-inflammatory pathway. So the concept of an "inflammatory reflex," and the discovery of the anti-inflammatory potential of the vagus nerve, could be major breakthroughs with enormous therapeutic potential. A better understanding of the molecular and cellular mechanisms underlying this phenomenon will be of utmost importance to develop new therapeutic strategies for disease such as inflammatory bowel disease, celiac disease, or food allergies.

BD: Which BD reagents do you plan to use, and for what purposes?

Gianluca Matteoli: We will mainly use flow cytometry and cell sorting to characterize the immunological changes mediated by the vagus nerve in the intestine in the steady state and after an inflammatory insult. Towards this end we will rely on several BD antibodies. We will study the lamina propria T and antigen-presenting cells using fluorescently conjugated BD antibodies and cytometry reagents through multiparametric flow cytometry. Data will be collected and analysed, respectively, with a BD FACSCanto™ II flow cytometer and BD FACSDiva™ software. In addition, we will examine secretion of inflammatory molecules using BD™ Cytometric Bead Array.


Abinav Singh, PhD
Post-Doctoral Fellow

Abstract Title:
Understanding Narcolepsy


BD: Tell us about your educational background.

Abinav Singh: After earning my master's degree in microbiology from Gurukul University in India, I joined the Institute of Genomics and Integrative Biology, a CSIR institute, also in India, for my PhD. During this time I studied the immunology of various novel proteins introduced in transgenic plants with an emphasis on the proteins' allergenic potential. After earning my doctorate I joined Dr. Omid Akbari's lab at the University of Southern California as a post-doctoral fellow, where I studied the role of programmed cell death markers in influenza pathogenesis and the role these viral infections in initiation of asthma. I am now doing a second post-doc, in the lab of Dr. Emmanuel Mignot at Stanford University, where I study the involvement of influenza viruses in recent-onset narcolepsy. Our group is working towards developing a mouse model to understand this disease.

BD: How did you become interested in science?

Abinav Singh: My passion for science began in high school. Immunology has always been my favorite topic, learning for example how the chemistries of B cells and T cells influence the initiation and outcome of diseases. With the pandemic of influenza making news every year, especially during the last decade, I became interested in researching influenza pathogenesis. Influenza pandemics can lead to initiation of many other diseases, such as asthma and—as has been recently reported—narcolepsy.

BD: Describe the project for which you were awarded the BD grant.

Abinav Singh: To date we know that the selective loss of hypocretin neurons, and a complex genetic susceptibility with a strong human leukocyte antigen association, strongly support an autoimmune mechanism for narcolepsy. Hypocretin neurons, present in the hypothalamus, produce the hypocretin peptides hypocretin-1 and hypocretin-2, which are involved in sleeping and alertness. However, the major obstacle for understanding narcolepsy disease is the absence of a functional mouse model. Recent findings have correlated H1N1 influenza infection and vaccination containing a squalene-based adjuvant with increased incidence of narcolepsy. These results, we believe, will lead finally to the development of a narcolepsy model in mice. We will study in detail the immunology of influenza infection, especially in the brain and peripheral nervous system, and will try to elucidate factors that might cause a loss of hypocretin-producing cells, which subsequently leads to onset of narcolepsy.

BD: What are your long- and short-term scientific goals?

Abinav Singh: Influenza pandemics have a major impact on our society. These infections modulate our immune systems and lead to many other disease conditions. In the long-term I wish to understand, how the influenza infection leads to changes in the human immune-regulatory mechanism that subsequently triggers other immune-related diseases. My short-term goals are to understand the immune-specific mechanisms associated with hypocretin loss, and to understand the role of influenza infection as a triggering factor in narcolepsy.

BD: What are the implications of your project for human health?

Abinav Singh: Narcolepsy has a prevalence of 1 in 3,000 in America. There is a great need to understand why the disease develops in order to prevent it. The experimental results from this project will increase our knowledge of the immune-specific mechanisms associated with hypocretin loss and lay the foundation for additional research that will aim at understanding the triggering factors in narcolepsy.

BD: Which BD reagents do you plan to use, and for what purposes?

Abinav Singh: We will characterize various dendritic cells, T-cell and regulatory T cell subsets, chemokines, and cytokines with multicolor flow cytometry (BD FACSCanto™ II) techniques using BD Pharmingen™ antibodies and BD™ Cytometric Bead Array kits.


Stephanie Smith
Graduate Student

Abstract Title:
A Novel Method Using Multicolor Flow Cytometry and FACS to Enable Multiparametric Examination of the CNS Cellular Response to Intermittent Hypoxia


BD: Tell us about your educational background.

Stephanie Smith: I hold a BS in microbiology from the University of Texas, Arlington. At the University of Texas, I conducted research on coral reef biology under the direction of Professor Laura Mydlarz. I'm now enrolled at the Comparative Biomedical Sciences Program at Wisconsin, where I study neuroimmunology in the lab of Professor Jyoti Waters. Specifically, I am investigating microglia, which are the resident macrophages or immune cells in the brain and spinal cord.

BD: How did you become interested in science?

Stephanie Smith: I originally wanted to go into medicine. After taking courses and working in the laboratory I decided that research in immunology and cell biology was more to my liking. I performed an internship at a contract research organization, and during that time became familiar with the University of Wisconsin. It was during my work with Dr. Mydlarz that I realized how much I enjoyed research, which to me seemed much like putting together a puzzle. I particularly enjoy neuroimmunology because it bridges the gap between two different fields, thus facilitating and promoting collaboration. My collaborations with neurophysiologists often take me out of my comfort zone and push me to grow as a scientist.

BD: Describe the project for which you were awarded the BD grant.

Stephanie Smith: The main question that led to this work is how does chronic intermittent hypoxia—repeated episodes of low oxygen followed by re-oxygenation to normal levels—affect microglia, the resident immune cells in the CNS. We expose mice or rats to intermittent hypoxia in custom-built atmospheric chambers. This model simulates the changes in oxygen levels experienced by individuals with sleep apnea. A good deal of research suggests that intermittent hypoxia results in neuronal loss and cognitive and behavioral impairment, which is believed to be linked, at least in part, to inflammation in the brain. We are investigating the role of microglia in this process, how their activities affect other cell populations, and how to regulate their activities in order to promote CNS repair.

To achieve these goals we need to simultaneously analyze multiple cell types in the same sample—principally microglia, neurons, and astrocytes—as well as levels of inflammatory and signaling molecules. I believe flow cytometry is under-utilized in CNS as it is considered more of an immunology technique. However, flow cytometry has enabled us to start tackling questions that we previously were not able to address.

In addition to looking at protein levels, we are interested in understanding how intermittent hypoxia is influencing these cells and the DNA and RNA levels. To do this, we must sort out pure populations of cells of interest using fluorescently-activated cell sorting, and analyze them further. The principal cell markers for neurons are intracellular, so the cells must be fixed to preserve their structure, and permeabilized so antibodies can enter and bind to the proteins of interest. Typically, this technique makes RNA and DNA from these cells inaccessible. We've employed a non-traditional fixation technique that allows us to fix the cells, identify them, collect protein data, sort them based on intracellular markers, and then perform downstream RNA and DNA analyses.

BD: What are your long- and short-term scientific goals?

Stephanie Smith: Our short-term goal is to expose the animals to intermittent hypoxia and use the method we've developed to analyze microglial responses. We hypothesize that over time, with repeated exposure, the microglia will switch from a predominantly pro-inflammatory phenotype to a more trophic and supportive one. We will determine this through multicolor flow studies on proteins, and subsequent DNA and RNA analysis. We expect the protein studies will give us insight into the microglial phenotypes and signaling pathways induced by intermittent hypoxia. Studying DNA and RNA will shed light on the effect of intermittent hypoxia on transcription and translation.

BD: What are the implications of your project for human health?

Stephanie Smith: The most obvious application is to human sleep apnea, which is linked to other neurologic disorders including such as Alzheimer disease and Parkinson's. Very few studies have examined the long-term effects of sleep apnea in these patients, and none to my knowledge have examined the effects of intermittent hypoxia on microglia in these conditions. Understanding the role of hypoxia on glial cells could lead to the ability to intervene therapeutically and perhaps prevent the most serious consequences of sleep apnea.

BD: Which BD reagents do you plan to use, and for what purposes?

Stephanie Smith: The most important category for us is the antibodies. BD has a tremendous portfolio of antibodies conjugated to fluorochromes, which makes multicolor flow cytometry possible. Another reagent is the BD GolgiPlug™ protein transport inhibitor, which causes proteins of interest to accumulate in the golgi apparatus. We are also interested in cytokine performing arrays. In addition, all of our samples are run on flow cytometers.


Xiaoxian Zhao, PhD
Project Staff

Abstract Title:
Multicolor Flow Cytometry-Based Detection of Phosphoproteins in Lymphoma


BD: Tell us about your educational background.

Xiaoxian Zhao: I received my bachelor's and master's degrees in microbiology from Shandong University of China, and my PhD in applied biochemistry at University of Tsukuba, Japan. I completed my post-doctoral training at Lerner Research Institute of the Cleveland Clinic, where I studied signal transduction. I am currently on the project staff at the Cleveland Clinic.

BD: How did you become interested in science?

Xiaoxian Zhao: Biology was my favorite subject in high school as the many puzzles of cells and life attracted me deeply. I selected microbiology as my major in college because of the excellent introduction to the subject that I received from my professor at the time. The PhD program prompted me to study basic questions of how normal cells become malignancies. Thereafter I developed a strong desire to detect disease-related molecules using advanced technologies.

BD: Describe the project for which you were awarded the BD grant.

Xiaoxian Zhao: Numerous studies have demonstrated that dysregulated kinase activation and phosphorylation events are common abnormalities related to the pathogenesis of many diseases, including leukemia and lymphoma. More and more selective inhibitors targeting dysregulated kinases are under development in both preclinical studies and clinical trials. For that reason the reliable detection and measurement of phosphoproteins in human tissues is becoming more important. However, accurate and applicable methods of tissue handling quality control are required for a wide range of both basic and clinical laboratory testing. Furthermore, these tests must be applicable to specific cell types.

Flow cytometry has many advantages, among them the ability to test different targets simultaneously in mixed cell populations, including many rare cell populations. Based on results of our preliminary experiments, we propose studies to apply multicolor flow cytometry–based assays for accurately and reproducibly measuring targeted phosphoproteins in selected fresh primary lymphoma tissue samples. We expect to establish standard protocols for analyzing specific phosphoproteins over a wide range of concentrations using a quantitative flow assay.

BD: What are your long- and short-term scientific goals?

Xiaoxian Zhao: My short-term goal is to finish the correlative study of flow cytometry–based detection of selected phosphoproteins with immunoblotting, the gold standard assay for this type of analysis. We will use several cell lines that constitutively express selected phosphoprotein targets such as STAT3, STAT5, and pERK1/2. Among these is a well-established mouse xenograft model of Raji cells. In the long-term, we would like to translate these laboratory studies into tools useful for clinical testing of specific phosphoproteins for diagnosis and disease monitoring of lymphoma.

BD: What are the implications of your project for human health?

Xiaoxian Zhao: Rapid detection of abnormal phosphoproteins via a flow cytometry–based assay in lymphoma samples could provide a useful tool for studying the molecular mechanisms of the pathogenesis, as well as hold potential for clinical testing and diagnosis, and providing prognosis in patients already diagnosed with lymphoma.

BD: Which BD reagents do you plan to use, and for what purposes?

Xiaoxian Zhao: We will use BD antibodies against selected phosphoproteins, B-cell markers, apoptotic assay kits, fixation and permeabilization buffers, and cell culture reagents to develop our assays. High-quality BD Biosciences reagents are critical for us to collect reliable and repeatable data.


Cindy Zuleger, PhD
Post-Doctoral Fellow

Abstract Title:
Isolation of Viable Antigen-Specific T Cells Ex Vivo Using Density Centrifugation, Cell Cycle Position, and Flow Cytometry


BD: Tell us about your educational background.

Cindy Zuleger: I received my BS degree in microbiology from the University of Wisconsin Oshkosh, and my master's degree in medical microbiology and immunology from the University of Wisconsin, Madison. I remained at Madison, where I earned my PhD degree studying immune responses in human melanoma. I'm currently a National Institutes of Health post-doctoral trainee, also at Madison Bachelor of Science, where I continue to study immunology and melanoma.

BD: How did you become interested in science?

Cindy Zuleger: When I was growing up I watched nature shows on television with my Dad. I was always fascinated by the diversity of animals, insects, and environments depicted in these programs. This initial fascination translated to an interest in general biology. Then in high school, I took a wet-lab "biotechnology" course in which we planned simple experiments and I realized I enjoyed the logical steps in the scientific process. In college, majoring in microbiology, I took a course titled "Immunology and Serology." Through this course I found the human immune response more interesting than the mode of action of the invading pathogen. At that point I realized I was hooked on immunology and switched my major to medical microbiology and immunology.

BD: Describe the project for which you were awarded the BD grant.

Cindy Zuleger: Metastatic melanoma is a refractory disease. Few available therapies have shown any significant impact on survival. For example, adoptive cell therapy, where patients receive autologous tumor-reactive T cells, provides response rates of between 50% and 70% in select patients, and the immunotherapeutic Ipilimumab that potently induces T cell activation benefits about 20% of patients. So clinical data suggest that T cells play a critical role in the immune response to melanoma in some patients. Thus, it is imperative to develop methods to isolate viable antigen-specific T cells for further study. To address limitations in the current methodologies to isolate such cells, we propose to utilize cell cycle position to isolate viable activated T cells ex vivo without relying on a preconceived relevant antigen. As T cells progress through the cell cycle, their density decreases and quiescent T cells, which are more dense, can be separated from less-dense activated T cells by density centrifugation. We are therefore using density centrifugation as a means to isolate in vivo activated T cells from melanoma patients. We expect the activated and cycling fraction to demonstrate reactivity to melanoma antigens.

BD: What are your long- and short-term scientific goals?

Cindy Zuleger: The goal of the proposed research is to determine if cell cycle position of T cells can be utilized to isolate viable melanoma-reactive T cells from melanoma patients. We anticipate that an improved understanding of in vivo anti-melanoma T cell responses will improve clinical outcomes for melanoma patients.

BD: What are the implications of your project for human health?

Cindy Zuleger: Only a subset of patients with advanced melanoma benefit from current systemic therapies. Our T cell selection method does not rely on a preconceived relevant antigen, dependence on MHC allele, reliance on T cell lineage, or in vitro stimulation as do current methods, and thus may identify novel melanoma-reactive T cells and/or tumor antigens that may be amenable to therapeutic intervention.

BD: Which BD reagents do you plan to use, and for what purposes?

Cindy Zuleger: Specific BD Biosciences reagents for this project include BD Pharmingen™ Hoechst 33342 Solution, BD Cytofix/Cytoperm™ Kit, BD Cytofix/CytoPerm™ Plus, BD fluorchrome-conjugated antibodies, BD™ CompBead reagents, and tissue culture disposables.