1975-1977, Monash University, Melbourne.
After qualifying in Medicine I was appointed Senior Tutor in the Department of Pathology and Immunology at Monash University Medical School. During this time, guided by Dr. Ban-Hock Toh, I became interested in auto antibodies directed to the cytoskeletal protein actin. After two years at Monash, I obtained a National Health and Medical Research Council (NH&MRC) Medical Postgraduate Scholarship, which allowed me in 1977 to enrol as a PhD student at the Walter and Eliza Hall Institute, University of Melbourne.
1977-1980 Walter and Eliza Hall Institute, Melbourne.
For my PhD, I worked in the laboratory of Dr Alan Harris, a cell biologist attached to the Cancer Research Laboratory of Dr Don Metcalf. The title of my PhD, which I obtained in 1980, was "Studies on the clonal origin and development of cytotoxic T lymphocytes". One major aspect of this work dealt with the ontogeny of cytotoxic T cell precursors (CTL-P) in the developing foetal mouse thymus. It had been known from the experiments of Jacques Miller in 1961 that removal of the thymus from newborn mice of certain strains had a profound effect on the immuno-competence of the animal later in life. I was able to show that at the clonal level, CTL-P first appeared in the foetal thymus at about 19 days of development and that during the next 48 hours, their frequency increased by about 100-fold. In collaboration with Dr. Tom Mandel, we developed an in vivo model system to study colonisation of lymphoid organs by CTL-P.
1980-85 Ludwig Institute for Cancer Research, Lausanne
For my post-doctoral studies, I went to the Ludwig Institute in Lausanne, working with Dr. H. R. MacDonald. We were one of the first groups to combine flow cytometry and sorting with limiting dilution frequency analysis of lymphocyte function. We were also the first to identify CD4-/CD8- (double negative, or DN) thymocytes and also that such DN cells expressed CD25. The main themes of research were:
1) Phenotypic and functional analysis of mouse thymocytes
2) Differentiation of foetal mouse thymocytes.
3) Role of IL-2 in thymocyte differentiation.
4) Identification of CD4-/CD8- thymocytes and their expression of CD25.
1985-1989 John Curtin School of Medical Research, Canberra.
In 1985, I left Lausanne to take up a position as Senior Research Fellow in the Department of Experimental Pathology headed by Prof. Peter Doherty, at the John Curtin School of Medical Research, Canberra, Australia. Here, I had a small research group. I was also responsible for establishing a functioning cell sorting (FACS) facility. The work I carried out in Canberra involved five main themes:
1. Mouse foetal thymus development.
2. FACS analysis of the inflammatory infiltrate in mice infected with lymphocytic choriomeningitis virus (LCMV). This work was done with the group of Prof. Peter Doherty.
3. Studies of the immunobiology of Pgp-1, or CD44, work done in collaboration with Dr Ian Walker.
4. Effects of anti-oxidants on T cells activation, a project in collaboration with Drs Ian Clark and Bill Cowden.
5. Studies of pancreatic islet xenograft rejection in diabetic mice, a project in collaboration with the transplantation immunology group of Dr Charmaine Simeonovic.
All these projects resulted in numerous publications.
In 1989, I moved to Strasbourg where I set up a Cellular Immunology laboratory in the Institut de Chimie Biologique directed by Prof. Pierre Chambon. There, I was nominated Professor of Immunology at the Université Louis Pasteur where I taught at the European School for Biotechnology Strasbourg (ESBS). The research interests of the cellular immunology group were focused on different aspects of mouse thymus development. The themes I chose for analysis were:
1. Phenotypic analysis of developing foetal thymocytes by three colour FACS?.
2. Studies of an IL-7 transgenic mouse line developed in my laboratory.
3. Development and receptor repertoire analysis of gd and abTcR-expressing cells.
4. Transcription factor (particularly NF-kB) expression in the developing thymus.
5. Studies of the regulation of CD44 gene expression.
This work has been supported by grants from Association pour la Recherche contre le Cancer (ARC) (IL-7), Boehringer Ingelheim Fonds (gd repertoire analysis) and from the French Multiple Sclerosis Foundation (CD44).
In 1994 and in order to relieve me of an increasing teaching load, I applied for and obtained a Directeur de Recherches (DR2) position within the Institut National de la Sante et de la Recherche Medicale (INSERM). With our IL-7 transgenic mice, I had already developed collaboration with the laboratory of Dr. Ton Rolink at the Basel Institute for Immunology. Together, we had published our studies on the development of B cells in these mice. In order to learn more about B cell development, I requested a leave of absence from INSERM in order to work at Basel where I became a Member of the Basel Institute from 1996-1998. There were two main projects I undertook there.
The first project was to introduce the IL-7 transgene into transgenic and mutant mice in which B or T cell development was perturbed. The idea of these inter-crosses was to see what impact IL-7 over-expression would have on B cell development. The second project involved studies of B cell development in the mouse embryo. No in vitro culture system was being used which efficiently reproduced the sequences of events known to occur during B cell development in vivo. Therefore, I decided to resurrect a foetal liver organ culture (FLOC) culture system that had been described by Owen some 25 years ago. Here, B lymphopoiesis could be manipulated in vitro and the B cell repertoire studied.
For family reasons, it was decided to move to the UK. I obtained an MRC Group Leader Position at the MRC Center for Immunoregulation, Birmingham University Medical School, directed by Prof. Ian MacLennan. As a returning scientist, the MRC provided me substantial start-up funds and the University of Birmingham a personal chair in Developmental Immunology. However, before being able to set up a fully-operational laboratory, the family had decided that they would prefer returning to France.
I returned to France to an INSERM Unit within the French Atomic Energy (CEA) research campus in Grenoble. I continued to be interested in the use and applications of flow cytometry. In 2001, I obtained €350,000 from ARC to purchase a Mo-Flo high-speed cell sorter. I have continued to focus my research on the role of IL-7 in lymphopoiesis, and in particular its role in maintaining the pool of lymphocytes in peripheral lymphoid organs.
From November 2003 to September 2008, I worked in Prof. Ton Rolink's laboratory in Basel. In 2002, a culture system was reported by Zúñiga-Pflucker (Immunity, 17: 749-756 2002) whereby mouse T cell progenitors can be grown in vitro. T cell progenitors cultured on OP9D in the presence of IL-7 efficiently develop into functional T cells. This so-called OP-9 DL1 culture system has far-reaching implications for studies of T cell development. With Ton Rolink, I have been addressing the following questions using the OP9D cell culture system:
Defining, by limiting dilution analysis, the T, B, NK and myeloid developmental potential of ?conventional? progenitor thymocytes.
Identifying and analysing the developmental potential of a subpopulation of bone marrow cells with T progenitor activity. These studies have revealed that a cell, of similar phenotype and developmental potential to that described in Pax-5 KO mice, is also present in normal, wild-type animals.
Analysing the in vivo reconstitution potential of in vitro-cultured T cell progenitors using RAG/gamma-c KO recipient mice.
Studying the role of regulatory T cells in bone marrow transplantation.
These experiments stem from a long-term collaboration I have had with Ton Rolink and expressed in a Nature Reviews Immunology article.
NUI Galway 2008-present
Since September 2008, I have been Science Foundation Ireland Stokes Professor of Immunology at the National University of Ireland in Galway (NUIG) where I am in charge of the flow cytometry facility and have a small research group in the Regenerative Medicine Institute at NUIG working on the immunology of mesenchymal stromal cells. I continue to collaborate with Prof Rolink in Basel.