Winner Interviews

Fall 2014 Research Grant Recipients Talk About Their Research


Dean Franckaert
Graduate Student
Catholic University of Leuven, Belgium

Abstract Title:
Development and Validation of Follicular Regulatory T Cells as a Diagnostic Marker for Autoimmunity


BD: What is your educational background?

Dean Franckaert: I earned my bachelor's and master's degree at the Catholic University of Leuven, Belgium. During this period, I worked on the mechanisms driving immune senescence, further investigating the process of thymic involution. After that, I began my doctoral studies in the Autoimmune Genetics Laboratory, headed by Dr. Adrian Liston. We concentrate on the basic biology of one of the most key components of immune homeostasis, regulatory T cells.

BD: How and when did you become interested in science?

Dean Franckaert: It wasn't until the first university biology courses, where I was fascinated by the beauty of biological systems, that I considered a career in academic science. Thanks to helpful, passionate instructors I was able to pursue a master's degree in biomedical sciences.

BD: How did you become interested in your broad field of study?

Dean Franckaert: During my undergraduate days I worked as a student researcher in an immunology laboratory doing basic laboratory tasks such as mouse husbandry, PCR genotyping, and buffer preparation. Through that same position, I was exposed to high-level immunology research in mice. That was when my interest really spiked. I went on to pursue a master's degree in biomedical sciences, specializing in immunology. After that, I focused on the process of thymic involution, investigating why the thymus shrinks over time, thereby reducing T-cell output in the elderly. My interest in immunology has led to my current position, where I work on the basic biology of regulatory T cells in the context of autoimmune disease.

BD: Describe the project for which you received the BD grant.

Dean Franckaert: To avoid converting to autoimmune disease after an effective immune response, the body needs to be able to also stop an immune response adequately. Regulatory T cells are key components in maintaining immune homeostasis by patrolling unwanted immune activation. However, a break in self-tolerance can occur, leading to autoimmune disease as a consequence. Rheumatoid arthritis (RA) is an autoantibody-mediated autoimmune disease that is hallmarked by failure of self-tolerance. In RA, a T-cell-driven response fuels a disease process that is directly mediated by the destructive properties of autoantibodies. Follicular T helper cells (Tfh), a subset of CD4+ T cells that home to the B-cell follicles and aid in generating high- affinity antibody generation, have been directly implicated in RA development. Interestingly, the recently identified follicular regulatory T cells (Tfr), a subset of normal regulatory T cells, modulate Tfh numbers and activity. Loss of Tfr leads to unwanted B-cell help and subsequent production of autoantibodies. Therefore, measuring the number of Tfr cells can allow detection of the earliest failures in immune tolerance and proactive monitoring of disease pathology, since current biomarkers have very limited predictive potential. In order to realize the use of Tfr cells as a diagnostic marker in RA, first, the predicted role of Tfr cells needs to be evaluated and second, a robust method for quantifying relative Tfr numbers needs to be generated. The aim of this project is to address both of these unmet needs, in order to develop a validated Tfr-based diagnostic flow cytometry panel for autoimmune diseases such as RA.

BD: What are the long- and short-term scientific goals of this project?

Dean Franckaert: In this BD grant application, we hypothesize that Tfr frequencies and numbers can be used as a diagnostic tool to expose the earliest failures in self-tolerance. The role of Tfr in RA has been suggested, but still needs formal validation in mouse models of specific Tfr deficiency. Secondly, characterization of Tfr is mainly based on markers not restricted to the Tfr lineage. To identify surface marker signatures specific for Tfr, we will generate mouse models in which bona fide Tfr cells are specifically labeled so they can be isolated and evaluated for additional markers.

Ultimately, a diagnostic panel capable of quantifying Tfr numbers throughout a disease progress needs to be translated from the murine to the human context. As part of a human immunophenotyping project running in the laboratory, we will collect flow cytometry-based peripheral blood mononuclear cell immune profiles from a large cohort of healthy individuals and patients with autoimmune disorders, thus establishing a large human dataset against which to test the identified murine markers.

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

Dean Franckaert: RA is a chronic disease in which the pathological features wax and wane, requiring differential therapeutic modulation. Currently, physicians rely on late-stage prognostic markers to guide therapy—either fluctuations in the level of autoantibodies or changes in disease severity. This approach is reactionary, requiring higher drug dosages for intervention, and causes patients greater levels of pain and tissue damage. An inexpensive, rapid diagnostic could detect early alterations in RA by monitoring Tfr, allowing treatment adjustment to be prognostic rather than reactive.

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

Dean Franckaert: We will employ a large part of the BD immunology portfolio, including BD IMag™ magnetic separation reagents, BD Lyoplate™ screening systems, and different immunoassays. Most importantly, we will rely on the vast range of BD monoclonal antibodies for flow cytometry.


Carole Guillonneau, PhD
Research Scientist
INSERM Center of Research in Transplantation and Immunology

Abstract Title:
CD8+ Treg in Allotransplantation: From an Animal Model to a Clinical Application


BD: What is your educational background?

Carole Guillonneau: I received my PhD from the University of Paris 7, France, in 2005. During my PhD studies on tolerance in transplantation, I identified a unique population of CD8+CD45RClow Tregs in an animal model. I then spent three years as a Marie-Curie Postdoctoral Fellow in Nobel Laureate Peter Doherty's laboratory in Melbourne, Australia. In 2009, I received a permanent position in France as a junior tenured researcher from the National Center for Scientific Research (CNRS), at which point I joined Ignacio Anegon's group at the INSERM UMR1064 Center for Research in Transplantation and Immunology in Nantes, France. My current work focuses on CD8+CD45RClow Tregs. I am particularly interested in understanding the cells' mechanisms of action and in developing new therapeutic strategies of tolerance induction.

BD: How and when did you become interested in science?

Carole Guillonneau: I have always been interested in science, and always knew I would be a researcher. As a child, I spent hours reading science magazines. I was very curious of the mystery and complexity of the human body.

BD: How did you become interested in your broad field of study?

Carole Guillonneau: My interests in immunology and transplantation began while I was a master's degree student. I was lucky to have a Dr. Ignacio Anegon as a mentor, and to receive a fellowship from the French Ministry of Science to complete my PhD studies in this field. During those years, I made several exciting discoveries and confirmed my interests in immune tolerance.

BD: Describe the project for which you received the BD grant.

Carole Guillonneau: Transplantation is a necessary procedure for many patients with severe illnesses. Unfortunately, transplantation is always associated with immunosuppressive drugs which, while inhibiting acute rejection, cannot prevent late graft failure associated with secondary effects such as cancer. New therapeutics are needed with more specific and long-term potential on the anti-donor immune response and less side effects. I have discovered, in a rat model of tolerance in transplantation, a new population of CD8+CD45RClow Tregs with a dominant suppressive effect capable of inhibiting long-term anti-donor immune response. In the animal model, we have described their mechanisms of action and identified new markers and the antigenic target stimulating their activity. To date, there is no description of the regulatory potential of this cell population in humans. The project for which I received the BD grant focuses on the identification and characterization of CD8+CD45RClow Tregs in humans. We will study the proportion of, and suppressive capacity and markers of CD8+CD45RClow Tregs in healthy individuals as well as in a cohort of transplanted patients to determine the predictive capacity of our Tregs on transplant outcome. In parallel, we will develop a protocol of expansion and acquisition of specificity of CD8+CD45RClow Tregs and evaluate their potential in a preclinical protocol using humanized rodents.

BD: What are the long- and short-term scientific goals of this project?

Carole Guillonneau: The short term goals of this project are to characterize the CD8+CD45RClow Tregs in healthy individuals and transplanted patients. Long-term, we hope to identify their potential as a cellular therapy and their markers/properties as diagnostic/prognostic for transplanted patients. The definition of the role of new markers on CD8+ Tregs will help us better define how this subset acts to induce allograft survival. In addition, identification and characterization of molecules with immunosuppressive properties is of great interest for the induction of donor-specific tolerance. In the very long term, we expect these findings will lead to innovative therapies.

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

Carole Guillonneau: Preclinical attempts to use regulatory cells therapeutically, to induce transplant tolerance, have demonstrated success, safety and feasibility. Phase I studies are ongoing. With this project, we expect to use CD8+CD45RClow Tregs in therapeutic strategies, but also as biomarkers of tolerance. We expect to develop and provide patients and physicians with more reliable tools regarding patient care. This includes enhanced prognosis for organ transplantation patients with improved monitoring and prediction of transplants, development of more targeted treatments, and quality of life improvements. This will in turn help in optimizing the allocation of the available grafts, minimizing the return to dialysis (for kidney transplant patients), and lower mortality.

In addition, we expect to establish innovative strategies of tolerance induction that will be applicable to a vast array of diseases such as transplant rejection, autoimmune diseases, gene therapy, and immunization against therapeutic proteins.

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

Carole Guillonneau: We will be using essential BD Biosciences equipment and reagents, for example: BD FACS Aria™ II cell sorter, BD FACSCanto™ II flow cytometer, BD FACSVerse flow cytometer, BD LSRFortessa™ II cell analyzer, BD FACSDiva™ software, FlowJo™ Software, FCAP Array™ Software. For the characterization of our CD8+CD45RClow Tregs, we will need fluorochrome-conjugated antibodies, BD™ CompBead reagents, 7-AAD staining solution, BD Cytofix/Cytoperm™ Kit, BD™ Cytometric Bead Array (CBA) Flex Set System, BD OptEIA™ TMB substrate reagent set; BD OptEIA™ ELISA Set and tissue culture disposables.

FlowJo is a trademark of Tree Star, Inc.

FCAP Array is a trademark of Soft Flow Hungary Ltd.


Alexandra Kadl, MD
Assistant Professor
University of Virginia School of Medicine

Abstract Title:
Mass Cytometry Profiling of Immune Cells in Lung Transplant Recipients Samples with the Goal to Develop Prognostic Markers


BD: What is your educational background?

Alexandra Kadl: I received my medical training at the University of Vienna. At the time, Austrian schools combined undergraduate and medical school, which is not the case today. For my doctoral thesis I studied gamma radiation-induced oxidative modification of vascular wall matrix proteins. I did my internship and residency in the Department of Medicine at the University of Virginia, where I also did a fellowship in the Division of Pulmonary and Critical Care. I am board-certified in internal medicine, pulmonary medicine, and critical care medicine.

BD: How and when did you become interested in science?

Alexandra Kadl: I was always a curious person. As a child I had a microscope and chemistry box. I began working in a lab in 1998, during medical school, and never left.

BD: How did you become interested in your broad field of study?

Alexandra Kadl: I would say immunology is the best description of what I do now, with a bit of lipid biochemistry. I began working in atherosclerosis—that's where the lipids came in, particularly oxidized phospholipids—then with macrophages, then disease models, and finally the lung. My medical specialty, pulmonary critical care, has a natural tie-in with immunology. The role of immune cell trafficking, particularly of macrophages and their role in lung transplant rejection remains poorly understood. I felt that with my background in macrophage biology I would be in a good position to tackle this question.

BD: Describe the project for which you received the BD grant.

Alexandra Kadl: Lung transplant survival is quite low. Acute rejection is a significant risk factor for chronic rejection and transplant failure. However, at this point we cannot predict which patient is at risk for acute rejection. Essentially, all patients are immunosuppressed, and often when the patient complains of respiratory symptoms, we have found that the patient is in acute rejection. At this point, we have to immunosuppress the patient even more, knowing all the possible side effects. So, finding patients at risk for rejection, and treating them before clinical signs develop, may improve overall survival. It is also possible that rejection markers we identify could be used in other transplant populations.

We will take advantage of normal screening bronchoscopies on transplant patients in our program. Normally, they will come in several times a year during the first year, and once yearly thereafter. We will take bronchoalveolar lavage samples as well as peripheral blood leukocytes and perform high-level phenotyping of cells using mass cytometry, a combination of flow cytometry and mass spectrometry. Mass cytometry, which allows identification of several dozens of cell markers simultaneously, is interesting in that it does not use fluorochromes. Instead, antibodies are labeled with isotopes. After primary cell separation through sorting instrumentation, individual cells are injected into a time-of-flight mass spectrometer, which positively identifies the isotope. The high bandwidth of the isotope approach arises from the fact that there is no overlap of fluorescence signals, and molecular weight signals are unequivocal. We expect that macrophage markers and specific immune cell combinations will be informative indicators of transplant condition.

BD: What are the long- and short-term scientific goals of this project?

Alexandra Kadl: Short term, we hope to establish a robust screening method for cell populations that emerge before clinical symptoms develop, and that are predictive for acute rejection. We expect that from tens of cell populations we will be able to reduce the number of target cells to a more manageable number. Long term, we hope to make this screening protocol available for transplant teams at other institutions.

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

Alexandra Kadl: Five-year survival for lung transplant recipients is only about fifty percent, which is the lowest for solid organ transplants. Identifying recipients at risk for rejection over, say, the following three months could significantly improve outcomes because the fewer the number of acute rejection episodes, the lower the risk of chronic rejection.

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

Alexandra Kadl: We will mainly use primary antibodies to human cell surface markers and signaling molecules, plus BD's cytometric bead assays.


Damian Maseda, PhD
Postdoctoral Fellow
Vanderbilt University

Abstract Title:
Control of Regulatory and Pathogenic T-Cell Expansion and Function in Murine IBD by PGE2


BD: What is your educational background?

Damian Maseda: I earned my BSc degree in molecular biology at the University of Malaga, Spain, my MSc in biological sciences at the Technical University of Munich, Germany, and my PhD in immunology at the University of Erlangen-Nuremberg, Germany. I spent four years as a postdoctoral associate at Duke University, where I received support through a grant from the Juvenile Diabetes Research Foundation. I am currently in a post-doc at Vanderbilt University, where I work on autoimmunity, more precisely in how inflammation affects rheumatoid arthritis and inflammatory bowel disease. My current mentor is Dr. Leslie Crofford in the Division of Rheumatology and Immunology.

BD: How and when did you become interested in science?

Damian Maseda: As a child I was fascinated with Carl Sagan's "Cosmos" series. But I really discovered I wanted to be a scientist as an undergraduate student, during my first semester in genetics and physiology classes. That was when I realized the potential of scientific experimentation to understand natural processes.

BD: How did you become interested in your broad field of study?

Damian Maseda: I clearly remember learning immunology as an undergraduate and thinking that I appreciated it as a mixture of both basic and applied science, and it had a direct impact in people's health. And, I also especially liked the holistic approach toward understanding the complex immune system and how immunology combined different disciplines within the biosciences.

BD: Describe the project for which you received the BD grant.

Damian Maseda: Mediators of inflammation are critical for controlling the pathogenic potential of immune cells during inflammatory bowel disease (IBD). We will study how the levels of the immunomodulatory lipid prostaglandin E2 (PGE2) alter the cell fate and pathogenic capacities of T cells, using mouse models of IBD. The effect of PGE2 seems to be especially relevant in regulatory T cells, which suppress undesired T-cell responses, and Th17 cells, which under certain instances can be highly damaging. Our hypothesis is that fine regulation of local PGE2 concentrations within a certain range will promote Treg function while suppressing Th17 pathogenicity.

BD: What are the long- and short-term scientific goals of this project?

Damian Maseda: The long-term goal is to define how the pathogenicity of Tregs/Th17 cells is altered due to inflammation, and to discover how prostaglandins can be used to modulate the function of these T cells to promote a healthy resolution of an inflammatory process. Short-term, we hope to define how endogenous and exogenous prostaglandins regulate T-cell fate in vitro and the plasticity among these subsets. Also, we will investigate how PGE2 levels from both autocrine (secreted by T cells) and paracrine (from neighboring cells like dendritic cells or macrophages) sources affect intestinal tissues and their function.

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

Damian Maseda: Many current therapeutic approaches for controlling inflammatory events, for example nonsteroidal anti-inflammatory drugs that block production of all prostaglandins, have undesired effects that include gastrointestinal tissue damage. It is unclear how inflammatory events affect the pathogenicity of other cells. While very strong or chronic inflammation is very dangerous for one's health, a certain degree of inflammation is absolutely necessary to mount a proper immune response. Better understanding of how prostaglandins, which are critical mediators of inflammation, control pathogenic T-cell responses will lead to therapies that control pathogenicity in a more selective manner while avoiding off-target effects. For example, by targeting an enzyme (mPGES1) that is more terminal and controls more precisely local PGE2 levels, we will be better positioned to decrease inflammation without causing tissue damage or suppressing the activity of other immune cells that need to stay active.

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

Damian Maseda: I plan to use many different antibodies and kits to prepare and detect T-cell populations both as surface markers (eg, CD4 and CD25) and transcription factors (eg, FoxP3), and also for intracellular staining (eg, IL-10) of diverse cytokines. To induce selective T-cell differentiation, we will use a combination of BD cytokines (recombinant mouse IL-2, IL-6, and IL-12) and BD stimulating and neutralizing antibodies like the anti-mouse CD3 and CD28 to induce proliferation and neutralizing anti-mouse IL-4 and IFNgamma for Th1/Th17 conditions. For detection of differentiated cells, we will use the BD Cytofix/Cytoperm™ Plus Kit (with BD GolgiPlug™) for intracellular stainings, and the BD mouse Th17/Treg phenotyping kit, with slight modifications for simultaneous detection of Tregs, Th1 and Th17 cells. We will also evaluate surface expression patterns of receptors involved in intestinal T-cell migration like CCR7 and CCR9 with the corresponding BD anti-mouse antibodies.


Paolo Monti, PhD
Project Leader
San Raffaele Scientific Institute

Abstract Title:
Autoreactive Memory Stem T Cells in Type 1 Diabetes


BD: What is your educational background?

Paolo Monti: I earned my undergraduate degree from the University of Milan, where I studied the role of dendritic cells in transplant rejection. I then enrolled in the doctoral program at the San Raffaele Vita-Salute University to study autoreactive T-cell expansion after islet transplantation in patients with type 1 diabetes. After a training period on the use of major histocompatibility complex tetramers in Seattle, and proliferation assays in Melbourne, Australia, I completed my PhD project under the supervision of Profs. Ezio Bonifacio and Maria Grazia Roncarolo, who are well-known researchers in type 1 diabetes. After receiving my PhD in immunology in 2007, I moved to Prof. E. Bonifacio's lab in Dresden, Germany, where I did postdoctoral training on homeostatic proliferation of autoreactive T cells. I then moved back to Milan where I established my own research group at the San Raffaele Scientific Institute. My research focuses on understanding mechanisms of expansion of autoreactive T cells in type 1 diabetes and islet cell transplantation.

BD: How and when did you become interested in science?

Paolo Monti: My fascination with science began in primary school. I don't remember a specific event or class that helped me decide –I believe that science was just one of my natural tendencies. My father, a physician, strongly supported my scientific interests.

BD: How did you become interested in your broad field of study?

Paolo Monti: In the late 1990s, when I was at university, immunology began to gain in prominence as the key to understanding and curing major illnesses such as cancer, cardiovascular diseases, and diabetes. At the same time, new technologies were emerging that allowed in-depth study of the immune system and its components.

BD: Describe the project for which you received the BD grant.

Paolo Monti: Type 1 diabetes is an autoimmune disease in which insulin-producing beta cells are selectively destroyed by autoreactive T cells. Consequently, therapeutic approaches are based on the inhibition or deletion of autoreactive effector T cells. But this treatment strategy has enjoyed only modest success. A novel strategy for controlling autoimmunity is to target memory stem T cells, a novel T-cell population responsible for maintaining long-term immunologic memory. However, medical researchers do not know if memory stem T cells are capable of recognizing self-antigens that exist in patients with type 1 diabetes. That is the aim of this BD-supported project.

To address this point, we designed a 12-parameter flow cytometry panel, which we will employ on a four-laser equipped BD LSRFortessa™ cell analyzer. The panel will allow us to determine the relative frequency of GAD65-specific clones within the pathogenic memory stem T-cell subset. As samples, we plan to use sixty-eight subjects whose tissues are already stored in our repository. These include thirty-five patients with recent onset type 1 diabetes, thirteen autoantibody-positive at-risk subjects, and twenty healthy control subjects. We expect to find GAD65-specific memory stem T cells in subject at risk and patients with type 1 diabetes, but not in healthy control subjects.

BD: What are the long- and short-term scientific goals of this project?

Paolo Monti: The overall goal of my research is to identify cellular mechanisms and cell populations that are potential targets for novel therapies for controlling autoimmunity and preventing the onset of diabetes. With respect to this project, our short-term goal is to detect and measure the frequency of GAD65-specific T cells in healthy donors, subjects at risk for type 1 diabetes, in other words those who are autoantibody-positive, and patients after the onset of the disease. The short-term goal is to understand whether memory stem T cells play a role in the disease. The long-term goal is to find ways to target these cells to control autoimmunity.

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

Paolo Monti: The increasing incidence of type 1 diabetes in western countries is posing serious health, social and economic problems. There is general consensus that the immune system is the cause of diabetes, but also holds the key to finding a cure. However, despite massive efforts and research investments, the results of clinical trials are still modest. We believe that the identification of self-reactive GAD65-specific T cells with a stem cell memory phenotype will potentially provide a novel cell target to eradicate memory autoimmunity from patients with type 1 diabetes.

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

Paolo Monti: The work described in the proposal is largely based on flow cytometry. I will employ a panel of fluorescently-conjugated monoclonal antibodies from BD specifically designed for flow cytometry. Using these BD reagents, we set up a twelve-parameter flow cytometry panel working on the BD LSRFortessa™ cell analyzer to study blood samples from our patient's cohort. Memory stem T cells will be identified as CD3+CD8+CD45RA+CCR7+CD122+CD95+ cells.


Michiko Shimoda, PhD
Visiting Assistant Researcher
University of California Davis

Abstract Title:
Characterizing Individual Tissue-Infiltrating T-Cell Clones in the Setting of Autoimmunity


BD: What is your educational background?

Michiko Shimoda: I received my BS and MS degrees from the University of Tokyo in agricultural chemistry and immunology, respectively. I received my PhD from Tokyo University of Agriculture and Technology. I received research training as a visiting researcher in Dr. Garnet Kelsoe's laboratory at the University of Maryland at Baltimore.

BD: How and when did you become interested in science?

Michiko Shimoda: My father, an organic chemist, loved science and technology. He was very well read in the scientific literature, and would often relate to me his eccentric ideas on solving various scientific problems. So, it was natural for me to choose science. I became interested in experimental science during my master's course at the University of Tokyo. In this multidisciplinary setting, we worked on varied projects in soil, food, microorganisms, plant sciences, and immunology.

BD: How did you become interested in your broad field of study?

Michiko Shimoda: I became interested in immunology when I learned about antibody somatic recombination, somatic mutation, and class switching, the mechanism for creating diverse B-cell repertoires. My initial excitement became a more serious interest after I worked for Dr. Eli E. Sercarz in UCLA as a volunteer technician. Dr. Sercarz's novel ideas were far beyond my understanding at the time, but his approach to studying the immune system impressed me. The immune system is a network of "professional" immune cells and other types of cells plus commensal microorganisms, each of which recognizes and communicates with each other.

BD: Describe the project for which you received the BD grant.

Michiko Shimoda: Autoimmunity affects nearly 25 million people in the United States. Most of those diseases remain incurable. In many instances, the disease is mediated by a small population of pathogenic T cells, but no reliable method exists for identifying and characterizing those pathogenic T cells because their populations are too small to analyze conventionally, even after clonal expansion. In addition, there are often many bystanders, peripherally expanded T-cell clones in patients with autoimmunity, and even in healthy controls, which are not necessarily involved in the diseases. It is important to determine how these expanded but non-autoreactive T cells differ from the tissue-infiltrating T cells. We believe that a detailed characterization of the specific T cells driving an autoreactive inflammatory process will help us to understand the process of autoimmunity and to identify novel targets for future drug development.

In this project, I hope to characterize individual tissue-infiltrating T-cell clones by combining cell-sorting strategies with T-cell repertoire analysis. Here at UC Davis, Dr. Emanual Maverakis' has used this approach to identify T-cell clones that expanded in response to antigens in autoimmune patients. I will take advantage of his novel application to identify tissue-infiltrating T-cell expansions in patients with autoimmunity and then to compare how they differ from peripherally expanded but non-infiltrative T cells isolated from the same individual.

In the first experiments, I will detect clonally expanded T cells in patients' blood and skin by analyzing the rearranged T-cell receptor (TCR) sequences with next generation sequencing. Each T cell expresses a single TCR encoded by a unique TCR gene rearrangement. For example, if a single T-cell clone carrying TCR Vb7 gene rearrangement expanded during immune response, this TCR Vb7 rearrangement should exist at a significantly higher frequency in a patient compared to healthy controls. Since pathogenic T cells, but not other T cells, can infiltrate into skin, clonal expansions found in blood and skin are most likely pathogenic.

Using a magnetic bead separation strategy in combination with T-cell repertoire analysis, I will look for a population enriched with pathogenic Vb7-expressing T cells. I will then characterize their phenotype by flow cytometry analysis after staining with a fluorescently-labeled antibody specific to Vb7 in addition to a panel of fluorescently-labeled BD antibodies specific to multiple other molecules of interest.

BD: What are the long- and short-term scientific goals of this project?

Michiko Shimoda: Our long-term goal is to conduct genetic dissection of pathogenic T cells to identify mutations and epigenetic changes that may be associated with their resistance to current therapeutic drugs. Short-term, we hope to characterize the phenotype of pathogenic T cells so that we can reliably isolate them from patients. I eventually expect to identify unique combinations of cell surface markers for specific autoimmune diseases.

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

Michiko Shimoda: If successful, my project may give us a clue why certain autoreactive T cells are resistant to currently available therapeutic drugs. Also, this approach can be applied to a variety of autoimmune diseases, including scleroderma, psoriasis, and lupus.

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

Michiko Shimoda: I will use various fluorescently-labeled anti-human antibodies (CD4, CD8, CD3, CD62L, CCR7 and other cell surface markers) to characterize T-cell clones expanded in peripheral blood and skin of patients. I will also use BD IMag™ T-cell isolation kits to enrich T-cell subsets.


Dorothee Viemann, MD
Professor
Hannover Medical School

Abstract Title:
Illuminating Neonatal Sepsis as Age-Specific Systemic Inflammatory Response Syndromes Playing up between Neonatal Monocytes and Endothelia


BD: What is your educational background?

Dorothee Viemann: I passed my undergraduate A-level in Lohne, Germany, and then completed my medical studies at Ruhr-University of Bochum, Boston University, and the University of Strasbourg. I received my medical degree from Ruhr-University of Bochum, followed by a residency at the University Hospital of Kiel and clinical fellowship at the University Hospital of Muenster in pediatrics. My specialty is pediatrics, but I also hold certifications in neonatology, laboratory medicine, and infectious diseases.

BD: How and when did you become interested in science?

Dorothee Viemann: I realized quite early, perhaps by age twelve or thirteen, when I would pose questions about how and why things were as they were. A scientist is a person who wants to probe deeper into those questions, who wants to act on them.

BD: How did you become interested in your broad field of study?

Dorothee Viemann: I attended lectures in pediatric immunology by a brilliant scientist, Prof. Christian Rieger, who studied in the US and then went on to chair the Department of Pediatrics at the University of Bochum. He urged me, after I passed my first exams, to gain laboratory experience. So I moved to the Department of Immunology and Transfusion medicine at the University of Luebeck, which is where I conducted my first lab studies on B cells and myeloid cells. During my clinical fellowship in Muenster, I worked with immunodeficient patients, with a focus on inflammatory diseases.

BD: Describe the project for which you received the BD grant.

Dorothee Viemann: Neonatal sepsis is completely different from adult sepsis. In adults it usually takes two days before caregivers realize that the infection is systemic. Neonates can die within two hours of onset. As medical students, we are told that sepsis is a problem in newborns because their immune systems are weak or do not function as well as adults'. Yet, we see a hyper-immune response in these patients. I hypothesize that the neonatal immune system is not deficient, but rather incapable of self-regulation, which would explain systemic hyper-responsiveness.

Immune system cells are easy to isolate from cord blood, but most investigators studying neonatal sepsis completely ignore endothelial cells, which are involved in septic disorders and systemic inflammatory responses. I wondered if the underlying problem in neonatal sepsis might arise from interactions between endothelial cells and innate immune cells.

In preliminary work, I discovered that when endothelial cells are stimulated with bacterial products or are infected with bacteria or viruses, they over-produce cytokines; monocytes do not. Interestingly, endothelial cells, unlike myeloid cells, cannot be tolerized–they do not down-regulate their immune response during the course of microbial challenges. In adults, down-regulation of the systemic inflammatory response depends totally on myeloid cells such as macrophages and monocytes. However, we recently discovered that neonatal myeloid cells cannot down-regulate their own activation, or the activation of endothelial cells for which they are responsible. So, neonatal sepsis appears to be the result of a vicious cycle that plays between endothelial cells, which are not so different from those of adults, and myeloid cells.

I have found, surprisingly, that endothelial markers are sky-high in healthy preterm babies, which suggests some type of endothelial pre-activation, probably birth-associated, that might carry significant consequences when these patients encounter pathogenic bacteria.

We hope then to culture monocytes from neonates of different gestational ages, and use supernatants from these cultures to see if there's a dependence on gestational age or mode of delivery on endothelial pre-activation. We will determine what consequences such pre-activation has for the response of endothelial cells upon challenge with bacterial antigens.

BD: What are the long- and short-term scientific goals of this project?

Dorothee Viemann: We must first prove that endothelial activation in term and preterm babies is higher than in healthy and septic adults, and that activation is the crucial determinant of sepsis risk and course of the disease. Since we can't isolate endothelial cells from humans, we will use endothelial surface markers shed by those cells found in serum. We will determine myeloid-specific activation markers in parallel. So, short term, we expect to identify the interplay between endothelial cells and monocytes within the crucial pathogenic mechanism of neonatal sepsis. Having identified the main problem in neonatal sepsis compared with adult sepsis, the long-term goal would be to develop new therapeutic strategies.

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

Dorothee Viemann: Sepsis affects one to two percent of normal-term babies but up to ten percent of preterms, in whom disease severity is also much higher. While all newborns show massive increases in biomarkers indicating myeloid cell activation, these fall rapidly in healthy term babies but persist for more than two weeks in preterm infants, thus predisposing them to hyper-inflammatory responses when they encounter bacteria. A strategy that mitigates this response, for example by inhibiting production of critical cytokines like tumor necrosis factor, could eventually become routine in treating preterm infants.

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

Dorothee Viemann: We will rely on a wide range of BD reagents. For cytokine studies in serum probes we will apply BD™ Cytometric Bead Array (CBA) Human IL-8 and TNF Enhanced Sensitivity Flex Sets from BD Biosciences. BD IMag™ Monocyte Enrichment Set - DM will help in the isolation of monocytes from blood samples. The purity of these cells will be determined by flow cytometry using the BD FACSCanto™ II system after staining with BD Biosciences' lineage marker antibodies. Finally, we will conduct cytokine analyses with BD Biosciences' TNF- and IL-8 ELISA, and calcium influx with the BD™ Calcium Assay Kit.