The tapestry of the immune response for protective immunity

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Nov 28, 2008 - performed using various soft and hard casting compounds to outline ... Key words: viral immunity, T cell response, immunological memory, ...
[Human Vaccines 5:2, 50-52 February 2009]; ©2009 Landes Bioscience

Portrait of a Leading Vaccinologist

Gabrielle Belz Walter and Eliza Hall Institute of Medical Research; Melbourne, Australia

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Clinical Training in the Whole Animal

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On completing high school, I enrolled in a Bachelor of Science at The University of Queensland. My course was, by choice, heavily laden with chemistry, mathematics, physiology and human anatomy. While a career in medicine initially appealed to me, arriving nearly an hour late for my first anatomy practical due to scheduling conflict with chemistry was a clear sign that dissecting humans did not suit my constitution. Around this stage, I also realized I had selected one of the heaviest workloads for a first year science course. Growing up in rural Queensland instilled a keen interest in animals. This eventually prompted me to enrol in a veterinary degree. Many of the subjects during the first few years of vet school were rather bland—they included subjects that established a fundamental understanding of disciplines that supposedly underpin veterinary medicine and surgery. It wasn’t until microbiology, pathology and the more applied clinics and surgery courses that I became engaged. Professors Allan Frost and Peter Spradbrow lectured us in microbiology and virology and began to shed light on the world of pathogens. At this time I also began to talk with Professor Trevor Heath who trained under the late Bede Morris (JCSMR; Canberra, Australia). His main interest was in understanding the architecture of lymphoid tissues and the flow of lymph and cells traversing organs like the spleen and lymph nodes. At the time, these studies were performed using various soft and hard casting compounds to outline the pathways within lymph nodes. This area of research has undergone a spectacular renaissance with the emergence of two-photon microscopy, which has allowed visualization of these pathways and the cellular interactions in real time. My close friend Stephen Nikles had already joined Trevor Heath’s team to complete a Bachelor of Veterinary Biology, an optional research year between third and fourth year of the veterinary course. I joined a year later and was set the problem of solving the architecture of lymphatic pathways in the lymphoid tissues of the upper respiratory tract, the tonsils. Tonsils were developmentally similar to lymph nodes, but architecturally very different. This, combined with the additional leadership roles taken on by Trevor Heath taking him a little away from direct veterinary departmental activities, allowed me an immense independence and freedom to explore new ways of looking at the tissues, particularly by electron microscopy. Prolonged hours sitting in the cooled rooms (usually about 4°C) housing our scanning electron microscope (SEM) that we used to trace over the surface of a tonsil or vessels of a lymph node would reveal the intricate detail of immune

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The lungs are critical for the exchange of oxygen and carbon dioxide necessary to sustain life. In an adult, the respiratory tract has a surface area of approximately 70 m2 exposed to the outside environment. The airways are colonized by a multitude of commensal microflora and multiple mechanisms exist to protect these tissues from infectious organisms. Every year, about 150 million new cases arise and more than 2 million children die as a result of acute respiratory infection. This staggering morbidity and mortality is driven by pathogens such as Haemophilus influenza, Streptococcus pneumoniae, respiratory syncytial virus and influenza virus. The emergence and spread of the virulent avian influenza A, H5N1 viruses throughout Asia, North Africa and Europe has heightened our concern about the possibility of another influenza pandemic. Understanding how the body responds to pathogens like influenza is critical for efforts to develop novel or improved vaccines. Born in Queensland, Australia I grew up with a healthy mix of city and country life on the Darling Downs. As the daughter of two teachers (and a genealogy of around five generations of teachers), my earliest childhood experiences often focussed around learning, discovery and creativity. Before venturing back into the workforce, my mother would spend endless patient hours with my brothers and sister teaching us the intricacies of routine disciplines such as reading and writing, but also artistic pursuits like origami and music. By the time my older brother was of school age, I had developed an insatiable desire to learn and spend time with children my own age. After much consideration my parents reluctantly allowed me also to attend school early making me a year younger than my peers. Primary school was largely one big playground of sport and learning; for my secondary education I moved to St. Saviour’s College, where I became interested in physics, chemistry and genetics. These disciplines were heavily focused on logically solving puzzles. Like my parents, I had always imagined I would fulfil the family prophecy and become a teacher; instead my scientific research career has come about in a rather convoluted manner.

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Key words: viral immunity, T cell response, immunological memory, antigen presentation, dendritic cells

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The tapestry of the immune response for protective immunity

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Correspondence to: Gabrielle Belz; Walter and Eliza Hall Institute of Medical Research; 1G Royal Parade, Melbourne, Victoria 3052 Australia; Email: belz@wehi. edu.au Submitted: 11/28/08; Accepted: 11/28/08

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Previously published online as a Human Vaccines E-publication: www.landesbioscience.com/journals/vaccines/article/7841

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cells in action. A wandering imagination resulted in sculpting of stories about how the lymphoid cells would use their uropods and tentacles to traverse lymphatic vessels, or crawl through the leaf-like opening of a terminal lymphatic. I was keen to study cells in motion, rather than the single snapshot captured in my electron micrographs.

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ABOUT DR. BELZ

Dr. Gabrielle Belz received her veterinary degrees in 1990 and 1993, followed by her Ph.D. in 1997 from the University of Queensland, Brisbane, Australia. She performed her postdoctoral work at the University of Calgary, Alberta, Canada and in the Immunology Department at St. Jude Children’s Research Hospital in Memphis, TN. In 2000 Dr. Belz returned to Australia to take a position at The Walter and Eliza Hall Institute (WEHI) of Medical Research, Melbourne, Victoria. As a laboratory head in the Division of Immunology, she is interested in (1) the factors that influence the quality and quantity of the CD8+ T-cell response to viral infection, (2) the functional specialization of dendritic cells in immunity and tolerance and (3) the transcriptional regulation of CD8+ T-cell differentiation during viral immune responses. In the course of her scientific career, Dr. Belz has received numerous awards and honors, such as the Gottschalk Medal awarded by the Australian Academy of Sciences in 2008. Dr. Belz has coauthored over 70 articles in peer-reviewed journals. She serves on the editorial boards of the Journal of Immunology, Immunology and Cell Biology. Dr. Belz regularly organizes, chairs and speaks at national and international meetings in the fields of Immunology and Vaccinology. In addition to her research, Dr. Belz is actively involved in postgraduate and undergraduate teaching as well as in projects communicating science to the lay community, such as the Science in Schools program.

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Suzanne Cory, the then very recently appointed director of The Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia) encouraged me to study abroad. After a brief period in Calgary, Canada extending my work on lymphocytes in mucosal lymphoid tissues, I was invited to join the program of Professor Peter Doherty in Memphis, TN. This was facilitated by an international fellowship from the National Health and Medical Research Council (NHMRC, Australia). I was eager to study immunology and was inspired by the enthusiasm of this recently appointed Nobel Laureate to engage with junior people. The prospect of working in his laboratory at the St. Jude Children’s Research Hospital (SJCRH) was motivation enough. However, another driving force to do great science was the vision of the hospital instilled by Danny Thomas to “cure terminal illness in children.” Indeed, the close proximity of the research and clinical areas and intermingling with patients and their families in the cafeteria, meant that for even the basic scientists, the underlying reason for their work was not far from their mind. Around the time of my arrival, Professor Rafi Ahmed (Emory Vaccine Center, Georgia) had recruited an outstanding scientist, John Altman, to Atlanta from Professor Mark Davis’s laboratory (University of Stanford). John had elucidated the final step required to form major histocompatibility class I (MHC I) tetramers—an idea that Mark had been developing for well over a decade. Remarkably, this technology opened the flood-gates for tracking virus-specific ‘killer’ T cells during infections and visualizing the exact cells with the machinery to kill virally-infected cells. At this time, the specificity of only a relatively small number of T cells for the influenza and herpesviruses were known. It became clear that the utility of this tetramer technology would only be widely useful if we elucidated many more of the peptide fragments (or epitopes) of viruses recognized by our T cells. Early in an immune response professional antigen presenting cells engulf and digest bits of pathogens and degrade them into peptide fragments that can easily slot into a major histocompatibility molecule. These peptide-laden molecules can then be tethered to the surface membrane of the antigen presenting cell where they are scanned by T lymphocytes. The prevailing view at the time was that viruses generated only one, or at most a very small number of peptides, that could be recognized by T cells. Despite the lack of enthusiasm for the notion that a large repertoire of viral peptides could be presented and recognized by T cells, we sought to directly test this hypothesis. At the time, the nucleoprotein epitope described by Alain Townsend (University of Cambridge, UK) was largely considered to be the only major influenza-derived peptide recognized by T cells. Together with Dr. Philip Stevenson, an English postdoctoral fellow in Peter’s laboratory we scanned proteins in herpesvirus (murine γ-herpesvirus) and influenza virus searching for additional viral T cell epitopes. Collectively, we identified a library that represented the major CD8+ T cell epitopes for these viruses

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The Australian Veterinary Connection: A Postdoc in Memphis?

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recognized in H-2b (C57BL/6) mice, and identified the major codominant epitope in influenza acid polymerase (the PA224-233) alluded to by Jack Bennink some 20 years previously. This gave us (and others) the capacity to examine, on a single cell basis, precisely how different populations of T cells were regulated in an immune response to produce protective immunity towards these infections. This work clarified that viruses actually generate a great number of antigenic peptides—a number of these could potentially be included in antibody-based vaccines to amplify T cell responses. In addition, MHC tetramer technology allowed us to uncover the unexpected requirement for CD4+ T cells during T cell programming in order

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CD8α+ dendritic cells—pivotal guides of T cell priming, differentiation and protective immunity. My international fellowship provided by NHMRC dually provided two years international training and a two year return component in Australia. In 2000 I returned to the Walter and Eliza Hall Institute (WEHI) of Medical Research, Division of Immunology to work with Dr. William Heath. Here I would embark on studies to understand how a rare population of these antigen presenting cells, called dendritic cells (DCs), were able to initiate an adaptive immune response to a viral infection. Recruitment of DCs to the lymphoid tissues provides the point of first engagement of the T cell with antigen and spurs the initiation of peripheral T cell activation and their subsequent differentiation into effector and memory T cells. Professor Ken Shortman (WEHI) has spent more than a decade isolating and defining various sub-populations of DCs within lymphoid organs like the spleen, lymph nodes and thymus. Despite this, very little was known about the function of these different DC populations. In 2000, I was awarded a Queen Elizabeth II Australian Research Fellowship to investigate how phenotypically different DCs were functionally distinct. In 2001 I was also the recipient of a generous Wellcome Trust Fellowship, which provided the funding to pursue an independent research program at the institute. This work focussed on utilizing complex viral models to track antigen-specific T cells, rather than simply monitoring the changes in cell numbers, the predominant approach of the previous two decades. We then began the tedious process of characterizing the DCs from lymph nodes collected from the lung following an influenza challenge, and regional lymph node following intradermal herpesvirus infection. These experiments involved harvesting the single lymph node draining the infected tissues, segregating the non-DCs and recovering the small number of each DC population that resided in these lymph nodes—generally only a few thousand per lymph node. We determined that different populations of DCs were critical for pathogen immune responses. Indeed, one subset that expresses the CD8α-chain, but not the β-chain, appeared to be critical for CD8+ T cell antiviral responses. This population of cells was also endowed with the capacity to “cross-present” exogenous antigens to CD8+ T cells —a process initially described in key experiments by Frank Carbone (now at University of Melbourne, Australia) and Professor Mike Bevan (University of Washington, Seattle, WA). Most recently, Professor Ken Murphy (St. Louis, MO; published in Science, November 2008) and his colleagues have identified a novel transcription factor, Batf3, that singularly regulates the development of CD8α+ dendritic cells. They conclusively demonstrated the critical role of this subset of antigen presenting cells for antiviral responses in vivo. This expanded cellular and molecular understanding of functional specialization of DC subsets perhaps

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opens the way for selective targeting of different DC populations for targetted vaccine approaches. During my work on DC subsets, I became a Howard Hughes Medical Institute International Scholar. This is an amazing program that provides investigators the opportunity to pursue difficult and important problems in science with relative freedom. It also exposed me to diverse disciplines and scientists that could fuel my interest in how cross-disciplinary contributions could facilitate scientific endeavor. It was at this time I also began to collaborate extensively with Andreas Strasser, Philippe Bouillet, Steve Nutt and Axel Kallies (all at WEHI) on molecules such as Bim, Puma and Blimp1 (B-lymphocyte-induced maturation protein 1) that genetically regulate the fate of T lymphocytes and other cells. The advent of fluorescent proteins [Green fluorescent protein (GFP) was formally awarded the Nobel Prize for Chemistry to O. Shimomura, M. Chalfie and R. Tsien in 2008] allowed incorporation of these molecules as reporter tags in mice. This use of this elegant approach in the Blimp1-GFP reporter mouse uncovered the unexpected finding that Blimp regulated not only B cell development, but also T cell differentiation and allowed dynamic tracing of the regulation of this molecule in T cells during a viral response. Because memory CD8+ T cells provide life-long immunity to a particular pathogen, understanding their development, behavior, and maintenance is crucial for the development of successful vaccines. The utility of these reporter molecules often make them ideal for understanding the developmental program of lymphoid cell differentiation. For B lymphocytes, much is known about the molecules that regulate different stages of development from a naïve B cell to a long lived memory cell that takes up residence primarily in the bone marrow. Dissection of this process has been greatly facilitated by the capacity to discriminate stages of development of B cells by their retention or secretion of antibody. By contrast, T cells lack such a genetic tag and much less is known about how molecular regulation T cell differentiation occurs in an immune response once they exit the thymus (about which a great deal is known). Thus, my research now focuses on understanding the molecular regulation of antigen presenting cells and their collaborations with T cells. This work should result in a broader understanding of the molecular control of protective immunity and the cellular and molecular molecules and signalling pathways that could be targeted for vaccine development. Over the past decade, my research has sought to tease apart how immune cells are organized in lymphoid tissues in such a way that they can communicate with each other and instruct the different armies of immune cells (T cells, B cells, NK cells and dendritic cells, just to name a few) to protect the body from invasion, or contain any pathogens that breach it’s barriers. Paradoxically, despite enormous advances in our understanding, we still lack a unified definition of the features of a protective T cell and how best to generate them. The various aspects of my work explore the precise cellular and molecular detail of the interactions between antigen presenting cells and T cells critical in generating protective immune responses for vaccine development.

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to generate effective memory CD8+ T cell responses. This feature of the immune response was not evident from classical limiting dilution analysis but has opened the door for developing a detailed understanding of the cellular and transcriptional steps in “programming” effector and memory killer CD8+ T cell differentiation in pathogen infections.

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