Winner Interviews

Fall 2015 Research Grant Recipients Talk About Their Research


Kesley Attridge, PhD
Lecturer in Biomedical Sciences
University of Wolverhampton, UK

Abstract Title:
Cutting out the Middleman: Do Cancer Cells Communicate Directly with CD4 T Cells to Establish Immunosuppression?


BD: What is your educational background?

Kesley Attridge: I received my undergraduate degree (BSc with honors in biomedical sciences) from Keele University, in the UK. I then earned my PhD in immunology from the University of Birmingham, and was a postdoctoral research fellow in immunology at Oxford University. There I undertook detailed studies of the molecular interactions that occur between T cells and antigen-presenting cells. I am currently a Lecturer in Biomedical Sciences at the University of Wolverhampton.

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

Kesley Attridge: When I began high school I always looked forward to practical laboratory classes. The hands-on nature of learning elevated the sciences above the other subjects I was studying. Perhaps because of this, I always did relatively well in science subjects, which only served to bias me further. Over time, I became particularly fascinated with biology, in part due to a brilliantly animated human biology teacher I met during the two years before I applied to study biomedical sciences at university.

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

Kesley Attridge: During my undergraduate days we studied a module on protein structure and function. Antibodies were one of the major examples given. I found it amazing that these tiny structures could be crafted and honed to recognize a seemingly endless array of targets, in such a highly specific manner. It turned out that this was something of a theme for the immune system, with T cells and their receptors utilizing a very similar system to identify their specific targets. That scientists were working to exploit these systems to generate highly specific therapies for a myriad of diseases seemed incredible, with almost unlimited potential. This is where my interest in immunology really began. I later decided that I wanted to undertake a PhD, and it seemed unthinkable that I would pursue research in any other field. As a result, I applied to study at the MRC Centre for Immune Regulation at the University of Birmingham, working on T-cell biology.

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

Kesley Attridge: CD4 T cells are a major component of the immune response to cancer. They not only directly effect tumor killing, but also produce an array of cytokines and chemokines necessary for the recruitment and activation of other effector-cell populations. In particular, CD4 T cells differentiated into the Th1 lineage are thought to be critically important, because their numbers correlate with improved survival times in many cancers. However, to counteract anti-tumor immunity, it is now clear that many cancers maintain highly immunosuppressive microenvironments to curtail or modify T-cell responses. Indeed, tumor cells are thought to directly effect the suppression of CD4 T cells, either by inhibiting their activity or by promoting their differentiation toward the Th2 or regulatory T-cell lineages. However, whether direct manipulation of CD4 T-cell responses is a strategy that cancer cells commonly use, and indeed whether the mechanisms deployed are the same across cancers, is unclear.

To address this issue we will establish co-cultures of CD4 T cells with cancer cells or their supernatants to determine whether direct communication between the populations occurs. We will assess whether cancer cells can suppress CD4 T-cell activation, proliferation and cytokine production. In addition, we will investigate whether cancer cells influence CD4 T-cell differentiation towards a number of recently identified lineages. By performing these experiments across a range of cancer types, we will determine both the immunosuppressive mechanisms utilized, and whether they are broadly utilized or specific to each individual cancer.

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

Kesley Attridge: In the short term we aim to identify novel mechanisms utilized by cancer cells to suppress CD4 T-cell responses. It will also be interesting to determine whether these or other previously described mechanisms are shared by cancer cells, or whether each cancer develops its own unique immunosuppressive strategy. In the long term, a major goal is to develop strategies to interrupt these pathways, so that CD4 T-cell responses are unhindered and can proceed effectively.

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

Kesley Attridge: The implications of the project are twofold. If we can identify novel immunosuppressive pathways, then there exists the potential to produce antibodies or small molecules specifically to target them. In doing so, we would hopefully restore CD4 T-cell function. In the context of an ongoing cancer, the hope is to tip the balance in favor of the immune system, thereby promoting tumor killing.

In assessing multiple cancer types, the aim is to determine whether immunosuppressive mechanisms are broadly applied, or whether cancers take a more tailored approach, with each using its own unique strategy. If we are able to identify commonly utilized pathways, this would be highly significant in that we would then have a therapeutic target that could be applicable across a broad spectrum of human cancers.

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

Kesley Attridge: The grant will primarily be used to purchase fluorophore-labeled antibodies for flow cytometric analyses. These antibodies will be essential for analyzing all aspects of the CD4 T-cell response, including activation, proliferation, cytokine production and differentiation.


Joke den Haan, PhD
Associate Professor
VU University Medical Center

Abstract Title:
Development of a Novel Vaccination Strategy to Initiate Anti-melanoma Immune Responses: Antibody-Mediated Antigen Targeting to CD169+ Macrophages.


BD: What is your educational background?

Joke den Haan: I completed my undergraduate studies in biomedical sciences, with a specialization in immunology, at Leiden University in the Netherlands, followed by a PhD at the Leiden University Medical Center under the supervision of Prof. Dr. Els Goulmy. During my doctoral studies, I identified peptides presented by HLA molecules that were recognized by cytotoxic T-cell clones involved in graft versus host disease after bone marrow transplantations. After that, I did a post-doc at the University of Washington in Seattle under the supervision of Prof. Michael J. Bevan. During this appointment, I determined which type of dendritic cell is specialized in cross-presentation of cellular antigens. I returned to the Netherlands and am currently an Associate Professor at VU University Medical Center in Amsterdam, where I research antigen presentation by dendritic cells and macrophages.

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

Joke den Haan: I became interested in immunology during my undergraduate studies. The immune system is very complex, with cells that are always on the move and with many factors and cell types interacting. In addition to fighting off pathogens, the immune system is also involved in autoimmune diseases and can even be employed to fight cancer. As we understand the immune system better, we will use this knowledge to develop better treatments in all these diseases.

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

Joke den Haan: We are interested in developing a new type of vaccination strategy to activate the immune system to kill melanoma cells. Melanoma is a form of skin cancer that is, when discovered at an advanced stage, very hard to treat. Advanced melanoma patients often do not survive the first year after diagnosis. At the same time it is known that melanoma tumors express antigens that can be recognized by the immune system. When the immune system and especially the cytotoxic T cells become activated in the right manner, these cells can attack and kill the melanoma tumor cells.

Our goal is to develop a vaccination that activates melanoma-specific cytotoxic T cells. In our previous studies, we discovered that when we target antigens to a very specific type of macrophage that expresses the marker CD169, we can elicit very strong cytotoxic T-cell responses to these antigens. In our proposed experiments we will target melanoma antigens to these CD169-expressing macrophages and investigate the strength of the cytotoxic T-cell responses, the specific characteristics of these cells, and their capacity to kill tumor cells in vitro and in vivo. We will perform these experiments using mouse in vivo models and human in vitro models.

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

Joke den Haan: The short-term goal is to show that this novel type of vaccination results in strong anti-melanoma cytotoxic T-cell responses in mouse models and in vitro human models. It is well known that cytotoxic T cells can only be activated by dendritic cells and these dendritic cells have to take up the melanoma antigens and present in to the T cells. Previous vaccination approaches consisted of injecting melanoma antigens, cells or DNA which would be taken up by dendritic cells present in the vaccination area. Alternatively, dendritic cells were grown in the lab from monocytes from the patient’s blood, loaded with melanoma antigens, and then given back to the patient.

However, these vaccinations were not very successful in stimulating strong cytotoxic T-cell responses. More recent insights have indicated that it is possible to target antigens to dendritic cells in vivo and thereby activate T-cell responses. We have tried targeting to dendritic cells and to CD169+ macrophages, and found cytotoxic T-cell responses as strong or stronger when antigens are targeted to the macrophages. We have investigated the mechanism and it appears that the macrophages, which are situated at the sites of antigen entry in the spleen and lymph node, very efficiently capture these antigens from the blood or lymph fluid and then transfer these antigens to the dendritic cells that in turn stimulate the T cells. Since these CD169+ macrophages are also present in human lymphoid organs, our long-term goal is to apply this vaccination approach in humans to elicit anti-melanoma and other tumor antigen specific cytotoxic T cells that prevent outgrowth of these tumors.

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

Joke den Haan: More than one million cases of skin cancer are diagnosed in the United States every year. Melanoma that has not spread is highly curable. However, five-year survival for melanoma patients whose disease has spread locally is 40-78%, and for distant metastatic disease just 15-20%. At the moment new types of immunotherapy are used to treat melanoma. While these treatments seem to increase survival rates significantly, not all patients respond to immunotherapy. Our hope is to develop a novel vaccination approach that together with other treatments will help to manage or perhaps even cure this disease.

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

Joke den Haan: We will use many antibodies and MHC tetramers for the detection of mouse and human antigen-specific cytotoxic T cells and to characterize and isolate macrophages and dendritic cells. We will analyze antigen-specific CD8+ and CD4+ T-cell responses using MHC-antigen tetramer staining, and stains for intracellular cytokine production (IFN gamma, IL-2, TNF alpha). In addition, we will investigate the generation of effector versus memory T-cell development using staining for specific surface and nuclear markers (CD44, CD62L, CD127, KLRG-1, CCR7, Tbet, eomes). Furthermore, we will phenotypically characterize human CD169+ macrophages from different human organs (tonsil, spleen) using flow cytometric analysis for several surface markers (e.g., CD68, CD163, DC-SIGN, MHC class II, CD80, CD86).


Raffaella Gozzelino, PhD
Principal Investigator
University of Lisbon, Portugal

Abstract Title:
Immune Regulation of Bone Morphogenic Pathways in Axial Spondyloarthritis


BD: What is your educational background?

Raffaella Gozzelino: I received my MSc in chemistry and pharmaceutical technologies with a specialty in pharmacology from the Faculty of Pharmacy, University of Parma, Italy. I then studied at the Faculty of Medicine, University of Lerida, Spain, where I earned a Diploma in Advanced Studies in cell biology and neurobiology. I was awarded my PhD in cell biology and neurobiology from the University of Lerida as well. After seven years, as postdoctoral fellow at the Instituto Gulbenkian de Ciência in Oeiras, Portugal, I moved to the Chronic Diseases Research Center at the University of Lisbon to establish my own research group. In the Inflammation and Neurodegeneration laboratory I direct here, our investigations focus on the role of inflammation and immunity in the pathogenesis of chronic immune-mediated and neurodegenerative diseases. I also oversee the PhD thesis work of three students.

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

Raffaella Gozzelino: My interest in science began in childhood. One of my favorite games involved assuming the role of the director of an important pharmaceutical company. In my imagination, this company constantly released new drugs to cure the faked pathologies and/or possible discomfort invented by me and my friends. After turning ingredients, usually soft drinks and juices, into these miraculous drugs, it was also up to me, the person in charge, to investigate the potential benefit of these treatments that were subsequently sold to “patients” in our imaginary pharmacy.

Later, in high school, I had the chance to attend motivating and interesting science classes. Our teachers’ entertaining lectures were aimed at understanding, instead of simply memorizing important concepts, allowing me to realize how much I might enjoy working in science. This is why, following my teachers’ examples, I always try to transmit to my students the same passion and enthusiasm I have for this fascinating job.

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

Raffaella Gozzelino: My involvement and interest in immunology began during my doctoral studies, in particular through a minor project I took on in the Inflammation Laboratory at the Instituto Gulbenkian de Ciência, in Portugal. In those few months right before graduation, I began to understand the importance of inflammation and immune response activation in a variety of human diseases. The data we obtained were so interesting that I returned to this very laboratory for post-doctoral training. Indeed, inflammation and immunity remain the basis of my current research.

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

Raffaella Gozzelino: The project for which I received a BD Immunology Award focuses on understanding the molecular mechanisms involved in the pathogenesis of axial spondyloarthritis (axSpA), a chronic disease that leads to a progressive axial skeleton deformation. Immunity and inflammation are known to play a dominant role in the pathogenesis of axSpA. However, the mechanisms underlying the impairment of bone cell function caused by the chronic activation of immune cells remain unknown. In this project, we will investigate the protective effect of immune-regulatory proteins by assessing whether levels of these proteins are modulated upon the induction of axSpA, an alteration that possibly correlates with disease severity.

We will first assess this hypothesis in a mouse model of axSpA and then, we hope, confirm it in human patients. The roles of our proteins of interest in preventing the negative impact of exacerbated inflammation on bone cell function will also be evaluated by assessing the severity of the disease in the presence and absence of the proteins in question. We hope to achieve this through a loss-of-function approach, through which the relevance of the genes encoding for these proteins will be evaluated in the context of axSpA. In vitro, the isolation of primary cells will allow us to determine the ability of the proteins to inhibit the deleterious effect of the activation of pro-inflammatory immune cells on bone metabolism.

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

Raffaella Gozzelino: Short term, it will be crucial to test the assumption that the levels of proteins of interest are modulated during the course of axSpA and correlate protein levels with disease severity. If this is confirmed, a long-term objective involves assessing whether these proteins could act as potential markers for axSpA prognosis in humans.

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

Raffaella Gozzelino: Despite new advances in the treatment of immune-mediated inflammatory diseases like axSpA, currently available drugs aim to ameliorate the symptoms of disease, which is already well-established at diagnosis, rather than prevent symptom occurrence. In addition to providing new insights into the molecular mechanisms regulating immune cells activation–a critical factor in the development of axSpA–the potential positive implications for human health rely on identifying possible targets, the levels of which, if restored, could revert the negative outcome of this pathology and perhaps become the basis of future therapeutic approaches.

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

Raffaella Gozzelino: We will use BD antibodies directed against cell and surface markers to monitor the activation of various immune cell populations upon the induction and/or manifestation of axSpA in mice and humans. In addition to employing antibodies, we will monitor the release of pro-inflammatory cytokines using BD ELISA kits. Finally we will use BD recombinant cytokines to study the role of inflammation in bone metabolism.


Thomas J. Kean, PhD
Instructor
Baylor College of Medicine

Abstract Title:
Improving Immune Reconstitution with Peptide-Targeted Bone Marrow-Homing Stem Cells


BD: What is your educational background?

Thomas Kean: I grew up in England and attended sixth form college in the small town of Brigg. From there, I pursued an undergraduate degree in biochemistry and pharmacology at the University of Strathclyde in Glasgow, Scotland. I went on to a master’s degree in pharmaceutical analysis at Strathclyde, which included a project at the European Pharmacopoeia in Strasbourg, France. From there, I worked towards a PhD in the Pharmacy Department at Cardiff University, Wales, under the guidance of Drs. Maya Thanou and Ruth Duncan, on a peptide-targeted, chitosan-based gene delivery system targeting the urokinase plasminogen activator receptor, which is over-expressed in many cancer cells. I was then recruited by Drs. Arnold Caplan and James Dennis to Case Western Reserve University in Cleveland to work on the production of ligand targeted mesenchymal stem cells.

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

Thomas Kean: One day sticks in my memory: I was four or five years old and had just gone back to my grandparents’ house after a hard morning at pre-school. I was sat on my granddad’s knee, and I told him that there were three things that I wanted to be: a farmer, a jet fighter pilot or a scientist. From there, it was probably Mum and Dad supplying me with chemistry sets, microscopes and electronics kits that really catalyzed my interest.

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

Thomas Kean: I remember hearing about antibodies in school and wondering why they weren’t used to deliver drugs specifically to cells. Well now they are! The same was true when I heard in Cleveland that stem cells were their own drug store, and not only that, they are a responsive delivery agent. When our lab moved to the immune system-focused Benaroya Research Institute in Seattle, it became even clearer that therapies have to be considered on a whole organism level as well as at the site of action. Drugs, including stem cells, have to be in the right place, at the right time, and at the right concentration. We’re only now starting to understand where, when, and how much we need, dependent on the circumstances. The idea that we can regenerate a tissue has been around for a long time. Today, with the advances we’re making in understanding how the body works, identifying stem cells and how to direct them towards specific lineages, it could become a reality within our lifetimes.

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

Thomas Kean: Many patients suffering from hematological diseases such as leukemia are unable to find an appropriate bone marrow match. Umbilical cord blood represents a large, readily accessible, zero morbidity, stem cell source that could fill that gap. Unfortunately, stem cell numbers in banked cords are commonly only sufficient to treat patients who weigh less than thirty kilograms. I identified bone marrow targeting peptides using in vivo phage display where over a billion peptide sequences are screened against the tissue, and the ones that stick are enriched. These peptides were then engineered into peptide “paints” which we apply to the surfaces of the stem cells in the cord blood unit. So we painted these cells with a kind of molecular Velcro® which increases their stickiness within the bone marrow, making the cell engraftment quicker and more efficient. This project will study the engraftment of these stem cells and look at the cells that are produced, or repopulated, by the stem cells using multi-color flow cytometry.

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

Thomas Kean: Long term, we hope to develop a whole new way of treating patients, changing current treatment paradigms and opening up single unit cord blood transplants to adults. Short-term, we need to show that the peptide paints aren’t limiting the stem cells in their ability to repopulate all the cell types found in blood. We will also show that those preliminary results, demonstrating quicker engraftment, can be reproduced with more donors with no toxicities or other downsides due to the coating.

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

Thomas Kean: Again, this is long term, but it could mean that a much broader population is able to receive umbilical cord blood as a transplant. If these studies prove successful, this cell targeting methodology would open up the use of umbilical cord blood stem cells to the approximately 10,000 adult patients per year who lack HLA-matched donors. In addition, the technology is applicable to practically any cell type and the targeting ligand(s) can be identified in many different disease states. Broadly speaking that means that therapeutic cells, whether they are umbilical cord blood stem cells, mesenchymal stem cells or induced pluripotent stem cells, can be transiently coated with a targeting peptide (defined through a screen against the location/molecule where the cells are needed) that will enhance the delivery of that cell to the target tissue.

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

Thomas Kean: Hematopoeitic stem cells are capable of producing all blood cell types, from lymphocytes to red blood cells. These different cell types that are repopulated by the cord blood stem cells can be detected using fluorescently-labeled antibodies against cell surface markers and flow cytometry. This will allow us to show when and which cell types are repopulated and compare that with cells that were not coated with the peptide paints. In addition, the antibodies can be used for slide sections of bone marrow to look, microscopically, at where the cells are taking up residence giving us a better understanding of the repopulation event.


Yarne Klaver, MD
PhD candidate
Erasmus MC, Rotterdam, the Netherlands

Abstract Title:
Can a Peripheral T Cell Co-Signaling Signature Be Used to Predict Intra-Tumoral Immune Status?


BD: What is your educational background?

Yarne Klaver: I studied medicine at the University of Maastricht. During the last half year of medical school I went to the Amsterdam Medical Centre for a scientific internship in the Experimental Immunology department. After finishing this internship I applied for a job as a PhD candidate at the Erasmus Medical center in the Medical Oncology department in the Tumor Immunology group, where I hope to finish my PhD in the upcoming years.

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

Yarne Klaver: I’ve always had a broad interest in science. According to my parents, I used to ask the “why question” and did not stop asking until they gave me a satisfying answer. On my tenth birthday my father gave me a microscope which I had a lot of fun with. The ability to see the cells of tree leaves and other organisms fascinated me. During high school, my favorite subjects were biology, physics and chemistry. During medical school, I chose a scientific internship as opposed to a more clinically-based topic for my final thesis.

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

Yarne Klaver: That occurred over two semesters during medical school. During one semester, the topic was cancer, and the other was about the immune system. Back then, not much was known about the interplay between cancer and the immune system. However, the more I read about this interplay, the more I became interested in the field of cancer immunology and immunotherapy.

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

Yarne Klaver: We are trying to get a better understanding of tumor-infiltrating lymphocytes. We know that in several tumor types T cells exist with tumor-specific T-cell receptors, and these kill tumor cells. However, due to the immune-suppressive tumor environment these cells cannot attack the tumor. One reason is the presence of co-inhibitory molecules on the T-cell surface. These co-inhibitory molecules normally act as a negative feedback loop to prevent T cells to be overactive, which could result in auto-immunity. In cancer, however, tumors utilize these co-inhibitory markers by expressing the ligands for co-inhibitory markers. This prevents the tumor-specific T cells from attacking the tumor cells.

We therefore are interested in the phenotype and several co-inhibitory markers on tumor-infiltrating lymphocytes. Additionally, in parallel we plan analyze peripheral blood to determine whether the peripheral blood reflects the local immune status of the tumor.

We have defined and validated multicolor flow cytometry panels containing eight to ten different antibodies and fluorochromes for assessment of the following: T-cell co-stimulatory/co-inhibitory signature on peripheral blood T cells; general T-cell subsets; regulatory T cells and T-cell activation status; and T-cell differentiation state. To analyze these T-cell panels we use the three-laser BD LSRFortessa™ cell analyzer. This enables us to look at several markers, but also to see whether there are markers that are co-expressed on T cells.

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

Yarne Klaver: We have two primary short-term aims: to assess an extended T-cell co-signaling profile of TILs in different tumor types by flow cytometry, and a head-to-head comparison of a T-cell co-signaling profile of TILs versus blood T cells. Longer term we hope to determine whether the phenotype and co-inhibitory profile of TILs or T cells in peripheral blood could serve as a prognostic or predictive marker in cancer.

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

Yarne Klaver: In recent years, evidence has accumulated that tumor infiltrating lymphocytes significantly affect the clinical attributes of cancer. The type, density and location of T cells in tumors provide a prognostic value that was superior to, and independent of those of the UICC-TNM classification criteria (T describes the size of the primary tumor, and whether it has invaded nearby tissue, N describes whether regional lymph nodes are involved, and M describes the presence of distant metastasis). We think that a closer look at the T cells will give possibly superior prognostic markers than just the enumeration of T cells in tumors. With the emergence of new immune-therapeutic approaches, we think it will be important to monitor patients closely to get a better understanding of the effects of these novel therapies. In the end we hope to find prognostic or predictive markers in patients’ blood that will provide accurate prognosis or predict treatment outcome. This approach is facile since blood is easy obtainable, and in addition would enable longitudinal monitoring during the course of the disease.

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

Yarne Klaver: Since our primary analysis technique will be flow cytometry, we will mainly use appropriate antibodies and fluorescence markers, plus reagents for making T-cell staining panels. For example to determine the expression of the co-inhibitory molecule PD-1 on T cells, we use antibodies for PD-1 (CD279), CD3, CD4 and CD8. Using this approach, we can also detect the differences in expression on T-helper cells (CD3+/CD4+) and cytotoxic T cells (CD3+/CD8+). By adding other CD markers in this panel, we can also assess the co-expression of multiple co-inhibitory markers.


Chozha Vendan Rathinam, PhD
Assistant Professor
Columbia University Medical Center

Abstract Title:
Unraveling the Role of “Itchy” Basophils in Mouse and Human Immune Systems


BD: What is your educational background?

Chozha Rathinam: I received my master’s degree in life sciences in 2000 from Bharathidasan University, Trichy, India, and my PhD in immunology and stem cell biology in 2004 from Hannover Medical School, Germany. I spent four postdoctoral years at the Yale University School of Medicine, where I continued research in immunology and stem cells. Since 2011 I have been an assistant professor in the Department of Genetics and Development at Columbia University Medical Center in New York.

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

Chozha Rathinam: I didn't realize my passion for science until I went to college. After joining my integrated master’s program in life sciences I became involved in research. That’s when I realized that science is “everything,” the driving force behind all secrets. After understanding the laws and facts of modern science, especially biology, most of the mysteries and superstition about life and the universe began to unravel. This was a great trigger for me to get involved in research, and now science has been my passion, hobby and profession.

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

Chozha Rathinam: Among the all the fields of biology, immunobiology—especially the origin of the immune system from hematopoietic stem cells—is to me the most fascinating. The fact that hematopoietic stem cells are inexhaustible through self-renewal, and are capable of differentiating into any type of immune system cell, compelled me to explore this branch of biology.

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

Chozha Rathinam: It is estimated that more than 3 billion people worldwide are infected with parasitic worms called helminths, or suffer allergic disorders such as asthma, allergic rhinitis, food allergies and eczema. Helminths induce a strong type 2 immune response characterized by high serum IgE levels and increased numbers of IL4 or IL13 secreting effector cells, including T- helper cells, eosinophils and basophils. Basophils are rapidly mobilized after helminth infection and can be efficiently recruited into lymphoid and peripheral tissues, where they execute their effector functions. Basophils can be found in increased numbers in mucosal tissues of patients suffering from allergic rhinitis or atopic asthma. Studies that emerged from mouse models indicate that basophils have both a protective role in mounting immunity against gastrointestinal helminths and a pathological role in the onset of asthma and allergies.

Patients with genetic mutations in the coding region of the ITCH gene, resulting in truncation of the ITCH protein, develop a complex. Earlier studies, including our own, on itch-deficient mice, indicated that loss of the gene leads to the development of severe immunological and inflammatory disorders and constant itching of the skin (“itchy” phenotype), very similar to ITCH deficiency in humans. Note that the mouse version of the gene is “itch” and the human version is designated “ITCH.” Although these studies enriched our knowledge, functions of ITCH in the control of the innate immune system remain totally unknown.

To identify the immune component(s) responsible for initiating the inflammatory disorders in the itch-deficient mice, we focused on the possible involvement of basophils, since they play pivotal roles in allergies and autoimmunity. Our preliminary analysis indicated that itch deficiency leads to basophilia or hyperactivation of basophils, and elevated IL4 expression in basophils.

Based on the preliminary data, we hypothesize that a deficiency of the E3 ubiquitin ligase itch results in deregulated functions of basophils. In the proposed research, we will design experiments to identify if itch-deficient basophils are responsible for the “itchy” disease, decode the mechanism through which itch-deficient basophils induce the “itchy” disease, and finally decipher the role of itch in development and functions of human basophils. We expect that T- helper cells will be augmented in the presence of itch-deficient basophils and that transfer of itch-deficient basophils is sufficient to cause the itchy phenotype in wild-type recipients.

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

Chozha Rathinam: The long-term scientific goal is to understand the precise genetic, molecular and biochemical mechanisms through which autoreactive basophils are generated and ultimately perform therapies for patients with disorders caused by basophils. Short term we hope to establish a new mouse model and humanized mouse model to study the biology of human basophil development and functions.

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

Chozha Rathinam: Itch belongs to the HECT family; its protein has more than twenty targets. Itch-deficient mice develop a skin-scratching phenotype and severe immune dysregulation. Patients who are homozygous for a truncating mutation in the gene show characteristic clinical features, including dysmorphic faces, failure to thrive, hepatomegaly, splenomegaly, and multisystem autoimmune disease. Since ITCH mutations in humans lead to severe autoimmune disorders, understanding the cellular and molecular functions played by ITCH in the immune system will be of paramount importance to understand the pathophysiology caused by its deficiency. Our research aims to dissect the cellular and molecular mechanisms through which ITCH deficiency causes human pathologies.

Knowledge obtained from the proposed experiments will provide key insights into the cellular mechanisms through which loss of ITCH signals causes autoimmune disorders in humans. We believe that data obtained from the proposed research would significantly help to design effective therapies against inflammatory disorders caused by ITCH mutations in humans.

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

Chozha Rathinam: We primarily would like to use fluorochrome-conjugated antibodies for identifying both mouse and human basophil subsets. We would also use cell culture reagents for in vitro studies, and antibodies to perform ELISA and intra-cytoplasmic staining.


Sarah See, PhD
Postdoctoral Research Scientist
Columbia University

Abstract Title:
Role of Anti-Apoptotic Cell Antibodies in Antibody-Mediated Rejection of Kidney Transplants


BD: What is your educational background?

Sarah See: I received my BSc degree with honors in microbiology and genetics from the University of Western Australia. I remained there, at the Telethon Institute for Child Health Research, for my PhD in immunology. I am currently a postdoctoral scientist at the Columbia Center for Translational Immunology, Columbia University, where I study transplant immunology under the guidance of Prof. Emmanuel Zorn.

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

Sarah See: I became interested in science, and specifically medical research, after seeing an immediate family member experience the long-term side effects of cancer therapy. This made me aware of the complexities of human biology and how much is still to be learned. I realized that greater understanding in this field would improve treatment, diagnosis and prevention strategies for disease and would have the potential to vastly improve patients’ lives.

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

Sarah See: I immersed myself throughout my undergraduate to postgraduate years in several scientific disciplines, from immunology to molecular biology to drug discovery, and became fascinated by immunology because it is central to human health and disease. Equally important to me was research that can benefit patient care. This cemented my interest in pursuing translational immunology and led me to my current position, where our research focuses on human solid organ transplantation immunology.

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

Sarah See: Long-term survival rates for renal transplant patients have not improved significantly due to chronic antibody-mediated rejection resulting in late graft loss. Advancing our understanding of the complex pathophysiology of antibody-mediated rejection will improve treatment options and patient care. While donor-specific anti-HLA antibodies are critical in graft survival, the role of natural autoantibodies (NAbs) has recently emerged as an important factor. Our lab previously found that increased levels of circulating IgG NAbs reactive to apoptotic cells in pre-transplant serum were associated with long-term graft loss, suggesting an important role for NAbs in antibody-mediated rejection.

We aim to extend these findings by assessing longitudinal samples collected from collaborating transplant centers. We will also determine the mechanisms underlying the role of NAbs in rejection by studying how they contribute to graft damage.

We have already begun to assess pre- and post-renal transplant samples from 700 patients for reactivity to apoptotic cells. IgG was purified from patient serum and tested by flow cytometry. We are performing comprehensive statistical analyses on these results to determine any significant associations between NAbs and post-transplant complications, including graft dysfunction and graft loss. We will also determine the IgG subclass in the new cohort, assess the complement-fixing capabilities of NAbs, and the capacity of B cells isolated from patient PBMCs to produce NAbs.

For mechanistic studies into the role of NAbs in kidney-graft survival, we will investigate their interaction with monocytes and endothelial cells. Infiltrating monocytes are frequently found in the capillaries of kidney grafts with antibody-mediated rejection and are known to respond to immune complexes. We hypothesize that monocytes also respond to immune complexes formed between natural antibodies and apoptotic cell debris. To test this, we will isolate and stimulate monocytes with anti-apoptotic cell immune complexes and evaluate cytokine secretion.

We also hypothesize that endothelial cells, the first point of contact between the transplant patient’s immune system and the transplanted kidney, can be activated by NAbs in a manner comparable to donor-specific anti-HLA antibodies to produce inflammatory mediators such as cytokines and adhesion molecules. To test this, we will stimulate endothelial cells with NAbs and measure cytokine secretion. Additionally, to assess whether damaged endothelial cells can activate neighboring cells in vitro, we will induce apoptosis in only a portion of cultured endothelial cells, incubate with Nabs and then determine endothelial cell activation of the viable cells by intracellular staining of kinase phosphorylation.

Finally, we will determine the contribution of natural antibodies to the clearance of apoptotic cells by phagocytic cells. To do this we will incubate monocytes/macrophages with apoptotic cells and NAbs and perform phagocyte-engulfment assays.

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

Sarah See: We anticipate that this study will confirm the prognostic value of pre-transplant serum NAbs and provide insights into the mechanisms involved in antibody-mediated graft rejection. Long term, we aim to study the role of NAbs in other types of solid-organ transplants such as heart and liver. We also hope to further understand how and why NAbs are produced in the context of solid-organ transplantation by characterizing the B cells that produce them and how these B cells are activated to become NAb-producing cells.

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

Sarah See: This research has the potential to influence clinical strategies. A strong association between natural antibodies and antibody-mediated rejection of kidney grafts might warrant their inclusion in pre-transplant patient screening, together with the conventional donor-specific antibodies. In the post-transplant setting, NAbs might provide another biomarker to monitor and predict graft outcome to enable early treatment of high-risk patients. While our studies are focused on natural antibodies in the renal-transplant setting, we anticipate that the mechanisms revealed will be applicable to other solid-organ transplants. Our functional studies might uncover effector mechanisms that could be targeted for therapeutic strategies.

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

Sarah See: We will use a variety of antibodies for cell surface staining of monocytes/ macrophages and endothelial cells (CD11c, CD68, CD14, CD16 etc.), antibodies for phosphorylation intracellular staining to measure endothelial cell activation (NF-κB, Akt, PI3K) and cytokine ELISA antibody sets or BD™ CBA kits to measure endothelial cell activation (IL-6, IL-1, TNF-α, MCP-1, etc.). We will also use chromogen and streptavidin-HRP for ELISPOT development to assess NAb-producing B cells.