The remembrance of 56 years of research (2012)

Reminiscences published in honor of the occasion of Professor Ken Shortman's 75th birthday. A symposium in honor of Ken, ‘The Pursuit of Perfection by a Gentleman Scientist’, will take place at the Walter and Eliza Hall Institute of Medical Research on 19 July 2012. Having survived for 75 years, I have been asked to outline my career path, any wisdom I may have collected en route and my main research achievements. So I begin in 1956, when I discovered I had some talent for research. I concluded my science degree at Sydney University with an honors year in biochemistry, studying the respiration of barley leaves during infection with powdery mildew; yes, some distance from immunology! I finished with first class honors and a conviction that research was the career for me. Biochemists were at that time in short supply, and Gordon Ada at the Walter and Eliza Hall Institute in Melbourne (WEHI) needed one. I was accepted as a PhD student with him. I was probably the only applicant! The day of my arrival at WEHI for the PhD interview turned out to be a formative experience. The Director, Sir Macfarlane Burnet (AKA Sir Mac) gave on that day a rehearsal of his USA lectures that were to be the first public presentation of his Clonal Selection Theory. As a keen biochemistry student I had read the theories of Pauling proposing that antigen somehow acted as a template to direct the folding of antibodies to the correct specificity. Instead, I now heard a simple and logical model involving Darwinian selection at the cellular level. I was an instant convert. Perhaps that is why the cell, rather than the molecule, has remained the basic unit of my immunological thinking, despite my molecular training. Although Burnet had famously shifted the research focus of WEHI from virology to immunology, my PhD project was a relic of the virological past. Gordon Ada had shown influenza was an RNA virus. He asked how viral RNA, or even cellular RNA, could survive in the face of the RNAases that were clearly present in the cytoplasm. It had been claimed that an RNAase inhibitor was present to regulate RNAase activity, but no one could repeat these experiments. Under Gordon's guidance I showed that heavy metals in commercial RNA preparations inactivated this inhibitor, a problem solved with a little EDTA. I then purified and characterized this labile inhibitor, and so obtained my PhD at Melbourne University in 1961. What was research like in these ‘good old days’? Slow, for sure, but you learned to be resourceful. I need to do repetitive small fixed volume pipetting. There were no Eppendorfs then, but I made by glass blowing a series of small bulb pipettes, calibrated them, then operated them like a Pasteur pipette. There were no plastic disposables and I washed all my own glassware, a character-building experience. The three papers from my PhD were all published under my name as sole author. Not only was Gordon Ada a very generous person, his personal scientific ethics did not allow him to be an author on a paper unless he ‘got his hands dirty’ doing the actual experiments. Even Gordon could not survive as a laboratory head today with that philosophy; the NHMRC rejection letter would cite the lack of last author papers. Having obtained a PhD, I was told I should get post-doctoral experience in Oxford or Cambridge or Harvard or Stanford. So I went instead to Paris, with Piotr Slonimsky at Gif-sur-Yvette. Australia was at that time a monoculture of British origin, and I was seeking a broader cultural experience. This I then had, operating in a different language, living in a tiny single room, assimilating history, architecture, cinema, music and dance, and learning the French attitude to food and wine. The science was great too, seeking the messenger RNA directing mitochondrial synthesis during respiratory adaptation in baker's yeast; progress was limited because at that time we were not aware that mitochondria had their own DNA. But after France I went the prescribed route as a post-doc with Bob Lehman in Kornberg's Biochemistry Department at Stanford. I relished the scientific culture there: cutting-edge research on DNA synthesis, innovative, thorough and of the highest critical standard. No surprise that Nobel Laureates (Kornberg, Berg, Lederberg) were lined up in a single corridor. With this training I became a molecular biologist, before that term was even devised. I returned to a position at WEHI in 1964. Gus Nossal, soon to be Director, had a great research plan for me, the molecular basis of antibody synthesis. I deflected from this path, but fortunately two talented molecular biologists (Jerry Adams and Suzanne Cory) arrived at WEHI, worked for a while in my laboratory and made a major contribution in this area. They started with myeloma cell lines, but I was determined to work with normal cells directly isolated from mice. Having previously studied pure populations of yeast and Escherichia coli, I knew there was no point commencing biochemical studies on the mixed populations of cells found in lymphoid organs. At that pre-FACS time there were no methods for cell purification, beyond the separation of blood into erythrocytes and white blood cells and the isolation of some phagocytes by adhesion to glass or plastic. Accordingly, I set about developing a new technology for immune cell purification, using various bead columns and buoyant density separation in albumin gradients. I also incorporated the sedimentation velocity and electrophoresis techniques; others developed about the same time. I found I had become a biophysicist and a world expert in cell separation. A later reviewer of my work over these 10 years stated it was ‘ultimately futile’. This was an overstatement, but basically correct. The best way to separate lymphoid cells is the same way they recognize each other, using specific molecules on the cell surface. At the same time as I was developing these techniques, Herzenberg and others were developing the fluorescence-activated cell sorter (FACS). WEHI purchased one of the first FACS instruments, and wisely we hired a real biophysicist, Frank Battye, to run and develop it. FACS then became my primary cell separation tool, supplemented by simplified versions of my earlier cell separation techniques. The development of the cells of the immune system as an aspect of hematopoiesis then became a major theme of my research. My earlier cell separation procedures had given some insights into this area and prompted my interest; the advent of FACS now allowed a systematic study of the sequential developmental steps and the isolation of rare precursor cells. I began with a focus on B cells, where the work of Gus Nossal had placed WEHI as a world leader. Although we made good progress, particularly in the publications with Maureen Howard, our findings were never fully integrated with other studies in this field. It was the work from my laboratory on T-cell development in the thymus that finally had a major impact. To focus on this I reluctantly abandoned the B-cell projects. We started with the background of Jacques Miller's discovery of the thymus as the source of T cells and Don Metcalf's study of overall cell generation within this organ, and set out to map the intrathymic steps in T-cell development. After Roland Scollay joined me following his work with Irv Weissman on T-cell migration out of the organ, I finally had a serious research team, which included Anne Wilson, Wei-Feng Chen, Li Wu, Martin Pearse and Howard Petrie. We made some key contributions to T-cell development in the thymus. Our balance sheet approach showed that most cortical thymocytes were destined to die in the thymus, confirming Metcalf's earlier conclusions. There was at the time remarkable resistance to this ‘wasteful’ concept that we now accept as the price of positive and negative selection. We began to track back to still earlier developmental steps, joining Dale Godfrey, Albert Zlotnik, Rob Macdonald and others in outlining a developmental pathway within the early ‘double negative’ thymocyte fraction. This provided the cellular signposts for later molecular studies. My eventual aim was to link T-cell development in the thymus to hematopoiesis in bone marrow. Finally, following clues from Roland Scollay and Gerry Spangrude, Li Wu as part of her PhD research isolated the earliest thymus precursor cell, the first lymphoid precursor cell to be identified. It was over 10 years before other laboratories began to explore the same ground. We can be proud of our pioneering work, which opened a door to the in depth molecular approaches of Harald von Boehmer, and to the recent insights in lineage choice from the gene expression studies of Ellen Rothenberg. Why then did I leave this rewarding field of T-cell development to study dendritic cells (DC)? In part it was because T-cell studies now required a molecular approach, and I was no longer a molecular biologist. In part it was the attraction of the exciting new field recently opened up by the work of Ralph Steinman, involving cells that no one at WEHI was studying. In part it was because I had the skills needed to tackle the isolation of these rare cells and still rarer precursors; the difficulties and expense certainly limited the competition. But mostly it was because every step I took in research on T-cell development led to DC. When Carlos Ardavin and Li Wu transferred our early thymus precursor into recipient mice, this lymphoid precursor made, as well as T cells, a subtype of thymic DC; the implications of this are still being debated. To our initial consternation, we found our T-cell-specific reagents, anti-CD4 and anti-CD8, labeled certain DC subsets. When Frank Sotzik and I searched for an early T precursor expressing CD4 in the human thymus, we isolated instead human thymic DC. When Richard Boyd, David Vremec and I began isolating thymocyte stromal cell interaction complexes using my new elutriation technique, we discovered rosette structures with thymocytes surrounding a central DC. And finally when I tried to develop in culture T-lineage cells from single early thymic precursors (before we understood the need for Notch ligands), the only cells that grew out with a high cloning efficiency were DC. I finally accepted that the hand of fate was directing me to DC research. DC have certainly proved a rewarding research field for me. Importantly, DC also led to fruitful collaborations with such fine scientists as Li Wu, Bill Heath, Frank Carbone, Gabrielle Belz, Meredith OKeeffe, Hubertus Hochrein, Jose Villadangos, Andrew Lew and Len Harrison. This work depended on the technical skills and continuity provided by David Vremec and Jo Pooley. We have trained some remarkable PhD students, including Shalin Naik, Anna Proietto and Priyanka Sathe. We made some important findings. Although no one was initially enthusiastic about our discoveries of yet another DC subset, the specialized immunological roles we ascribed to individual DC types are now well accepted. We have mapped some of the pathways of DC development, a novel aspect of hematopoiesis. With Irene Caminschi, Mark Wright and Mireille Lahoud, we discovered some novel molecules on the DC surface. And at last some part of my basic research may have a direct medical application. Irene Caminschi and Mireille Lahoud have shown that targeting vaccine antigens to one of our novel DC surface molecules, Clec9A, is a remarkably effective way of boosting the efficiency of vaccines. The Reis e Sousa lab in UK has come to the same conclusion. This new approach to vaccine development, taken up by the Burnet Institute, provides me with a great incentive to continue research. What might be learnt from my research experience? It is exceptional to have an entire career centered on one institute, as mine has been. It only worked because of the exceptional range of skills available at WEHI, and the collaborative and creative environment WEHI offers. Even with this I needed sabbatical periods elsewhere to learn new skills and think of fresh approaches. Notable were several short sabbatical periods in Lausanne with Brunner, Cerottini and Macdonald, in Basel with von Boehmer and in Zurich with Suter; skiing in Switzerland was also refreshing. The French input into my work was maintained with a year in Marseille-Luminy with Pierre Golstein and training in human DC with Banchereau's team in Lyon. For me it has been rewarding and even essential to change research fields several times. But today this must be done with caution, as there is a lag before you are accepted as a useful contributor in a new area and there is a lag in publication output; it is hard to sell all this to a NHMRC review committee. Perhaps the best strategy is to maintain some of the previous publication generating work going while developing the new focus. And it is worth considering Don Metcalf's advice to one jaded researcher who wondered about changing fields after 7 years in one area. As I remember his advice was to stay with it a bit longer, you might learn something. One thing I have observed is the amazing diversity in research style and personality among excellent scientists. This is probably essential to scientific progress, and certainly adds to the joy of a research career. I have had the pleasure of working at WEHI under four Directors: Burnet, Nossal, Cory and Hilton. All have been great directors, but they represent totally distinct personalities and directorial approaches. I have worked in the shadow of some remarkable WEHI scientists, including Nossal, Miller, Metcalf and Mackay. They were true giants in their research fields, but again all totally different in personality. And I endorse a comment made by the ever-wise Peter Colman, who stated ‘you need some mad bastards in research’; I will refrain from listing these. I hope I have contributed to this essential diversity, even if some may consider I fit the adjacent category.