Professor Jacques Miller, pathologist

Professor Jacques Miller. Interview sponsored by the Australian Government as an ongoing project from the 1999 International Year of Older Persons.

Jacques Miller attended St Aloysius' College in Sydney then went on to study medicine at the University of Sydney. During his medical studies, he took a year off in 1953 to do a BSc in a bacteriology laboratory. Miller did his medical residency at Royal Prince Alfred Hospital, Sydney and was then awarded a Gaggin Fellowship to do medical research in London. At the University of London, he completed a PhD in 1960. In 1963 he spent a year at the National Institutes of Health in Maryland. He returned to London for several years and in 1966 moved to Australia to become head of the experimental pathology unit at the Walter and Eliza Hall Institute of Medical research. Miller served on the International Research Agency for Cancer, and was president of the Scientific Council. He served on the World Health Organization in the area of eradicable diseases and had a term on the International Union of Immunological Societies.

Interviewed by Professor Frank Fenner in 1999.


An intercontinental childhood

Jacques, you had an interesting and exciting childhood and early adulthood, moving between France and China before coming to Australia. Could you tell us about that?

My father was the manager of the Franco-Chinese Bank in Shanghai, staying in China and Japan for about 25 years. One of my sisters was born in Paris, before my parents left for Shanghai, another sister was born in Shanghai, and I was conceived in Shanghai but born on holidays in France, where my mother had gone for health reasons. When she was on the boat – that is how people went in those days – she found herself pregnant, so she decided to have the baby in Nice, next to Marseilles, where the boats used to go.

After less than a year we went back to China but about 3 years later my mother returned to France with the family – again for health reasons and because she wanted my elder sister to be educated there, not in China. Unfortunately, my elder sister contracted tuberculosis, and so the family moved to Switzerland for her to be cured. (That was before the days of streptomycin.) My father eventually came to Switzerland for long service leave, but then World War II started and we left in a hurry for Shanghai. After about a year and a half, knowing that the Japanese were going to be involved in the war, we went to Australia. That was about 3 months before Pearl Harbor.

I didn't know much English at all and had to learn very quickly. But children do learn very quickly. Because my father had thought the Jesuits were wonderful in Shanghai, he decided that in Sydney I would go to a Jesuit school, St Aloysius', and my sister went to Loreto Convent. Both were in Milsons Point. Actually, Gus Nossal was a year ahead of me at St Aloysius'. He was always enthusiastic and very outspoken. He loved debating and was very articulate, just like now.

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Into medicine and on to research

You did pretty well at school, I gather.

I was lucky, I did very well at school and therefore I got an Exhibition, which in those days was a kind of fellowship which took you to university. I had always wanted to do medicine. One reason was that having escaped from the war twice, I was dead against violence. I didn't think I could escape being drafted but I thought I might be a doctor in the army rather than a soldier. I was interested in medicine also because when my sister got tuberculosis they didn't know anything about how infectious diseases were conquered by the body – what pathogenetic mechanisms dealt with such infections. That always interested me and I wanted to learn something about it.

In 1956 I did my residency in the Royal Prince Alfred Hospital, which was the main hospital in Sydney. That is where I met my wife, who was a nurse at the time, and we got married the year I was a resident. During my medical studies I took a year off in 1953 to do a Bachelor of Science, because I hoped to do a little bit of research into pathogenetic mechanisms. In medical school you don't learn much about that – you learn the various signs and symptoms but you don't go into depth. I chose to do a BSc (Med) year in Pat de Burgh's bacteriology department, at the University of Sydney, because they were studying the response to ectromelia virus and that sounded very interesting.

An old friend of mine, ectromelia virus, which I worked on with Mac Burnet in the late 1940s. So you gained a taste for research and knew it was what you wanted to do?

Even more than before, yes. After my medical training and internship, I saw an ad in the Medical Journal of Australia about a Gaggin fellowship to be given by the University of Queensland for 2 years' medical research in London and then 1 year in Brisbane. I applied for that fellowship and was very pleased that it enabled me to do a PhD at the University of London – based at the Chester Beatty Research Institute, the institute of cancer research in South Kensington.

When I arrived at the institute I didn't really know exactly what I wanted to do as a PhD subject and I was told to look around. At the main institute in London they were studying chemical carcinogenesis at the chemical and nucleotide levels. I was not terribly interested in this – I was rather depressed, actually. But the institute had satellites, one in Surrey and one in Chalfont St Giles at Pollards Wood, and at Pollards Wood I met R J C Harris, who was interested in the Rous sarcoma virus and its effects. He suggested that I study another tumour virus, such as the leukaemia virus (Gross, in the United States, had just isolated it in tissues) and I decided that the induction of mouse leukaemia by Gross virus would be the subject for my PhD.

So that kept you in virology, but in a cancer research environment.

Yes. Having worked in virology in de Burgh's laboratory, I was very pleased.

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Leukaemia virus and newborn mice

In those days, viruses were not generally accepted as a cause of cancer. Gross had been able to induce leukaemia in mice by using filtered extracts of mouse leukaemic tissues. He had never isolated the virus as such, but the filtered extracts were able to induce leukaemia in low-leukaemic strain mice, that is, strains of mice which normally don't get leukaemia. Leukaemic extracts from high-leukaemic strain mice, which normally do get leukaemia, were injected into newborn mice at birth – it had to be at birth and not later – and the leukaemia developed 3 or 4 months later.

Rather than develop my own virus from the mouse leukaemic extracts, I wrote to Gross saying, 'Would you mind sending me some of your filtered extracts? I have to do a PhD, I've only got 2 years and it would take me too long to start from scratch, whereas if you sent me your filtered extracts I could induce leukaemia and then repassage that through a series of mice.' Luckily for me, he was kind enough to do it.

I remember that Ludwik Gross wrote the book Oncogenic Viruses.

In those days you had to inject the extracts in newborn mice – if you injected into adult mice nothing happened. That was interesting in itself. Knowing that people like Sir Peter Medawar had discovered immunological tolerance by injecting cells into newborn mice, I wondered whether that had anything to do with the induction of leukaemia by injecting these kinds of extracts to newborn mice, and I was very interested in the phenomenon of immunological tolerance. I had not met Medawar but I decided to see if I could learn something from him.

In 1960 Medawar gave the Tercentenary Lecture of the Royal Society. Afterwards, in a quick chat, I asked whether he would be kind enough to teach me a few things about how to induce tolerance in mice. He said, 'Come along one day,' and very nicely he arranged for one of his co-workers, Leslie Brent, to teach me how to inject mice intravenously, how to skin-graft, and other techniques.

Brent later became Professor of Microbiology at St Mary's, didn't he?

That's correct. I was then able to do some experiments on immune tolerance, particularly to see whether I could induce leukaemia in low-leukaemic strain mice simply by putting in them a thymus graft from a high-leukaemic strain. That was a naive idea, but all these techniques that I learned at the time became very useful later.

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Immune tolerance: what was the role of the thymus?

How did you get onto thinking about the thymus?

Lymphocytic leukaemia induced by Gross virus began in the thymus and then spread elsewhere. Many other types of lymphocytic leukaemia in mice do exactly the same thing: you can induce leukaemia in low-leukaemic strains by various agents, such as irradiation or chemical carcinogens like dimethylbenzanthracene. All these lymphocytic leukaemias begin in the thymus and then spread. In fact, the spontaneous leukaemia of high-leukaemic strain mice, which are called AK, also begins in the thymus. In America and other places it had already been shown that thymectomy (removal of the thymus) in older mice would prevent both spontaneous leukaemia and leukaemia that had been induced by the various agents I mentioned, provided it was done at about 1 or 2 months of age – so preventing leukaemia from starting at 9 months of age, as it normally would.

That had not yet been done for the virus-induced leukaemia, so one of the first things I did was to see if thymectomy prevented virus-induced leukaemia. I injected the virus at birth and then thymectomised the mice when they were a month old, and it worked – it prevented leukaemia. The next question was obvious: put the thymus back with a graft and see if leukaemia begins again in the thymus. And it did. That was to be expected, but what was not expected was that when I put the thymus back 6 months after adult thymectomy, thinking that by then surely leukaemia could not start again, it did. That suggested that the virus must have remained latent for all that time.

The next experiment was simply to thymectomise adult mice that had been inoculated at birth with the virus, wait for 6 months – not give them a thymus graft – take their tissues, make an extract of their normal tissues and see if that induced leukaemia. And it did. So that was the logical outcome of the experiment.

If it showed that the virus was generalised throughout the body, not localised to the thymus, was the thymus the critical place where it multiplied, presumably because the lymphocytes there were the ideal cells in which to replicate and produce leukaemia?

Probably that is totally incorrect but it is exactly what I thought. You would have to give the virus at birth because the thymus at that particular stage was producing very large lymphocytes – people did not know then why it produced lymphocytes – and perhaps those lymphocytes were the ideal medium for virus replication. So if you inject the virus at birth, it goes to the thymus, replicates and then migrates to the rest of the body. My idea was that if you thymectomised first, at birth, and then injected the virus immediately afterward, leukaemia would not occur – that was obvious – but when you gave the thymus back later on, as I had done, leukaemia should still not occur because the virus would not have had a chance to multiply.

I can see that. That's what my experiment would have been.

So I had to learn the technique of neonatal thymectomy. I could do it in the adult but it is a bit different in the newborn. Finally I worked it out, and actually it's quite easy.

I remember meeting you at Pollards Wood in 1962 and learning from you how to do a neonatal thymectomy. I never used it in my subsequent work, though.

The one problem about neonatal thymectomy is cannibalism by the mothers, so I had to do a lot of mice. I must thank my wife: she helped me very nicely by making sure that the mothers would not eat the babies after thymectomy, or changing them around until she found some that did not eat babies.

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Why did the mice waste away? An unexpected finding

I became worried because although all the mice that survived the operation were perfectly healthy with their mothers and until after weaning, about 4 to 5 weeks of age, they then suddenly wasted and became sick. This was never recorded after adult thymectomy – you can take the thymus of an adult mouse and it lives perfectly well into old age – but after neonatal thymectomy, generally at 6 to 8 weeks depending on the strain, they started wasting away and many of them died. I couldn't understand that.

I did a post mortem, of course, and I found several things. One was lesions in the liver, which looked as if they were infected by a virus – hepatitis virus, for example. But also I found a deficiency of lymphocytes in the lymph nodes and spleen, which was quite remarkable. At that time Jim Gowans and Peter Medawar had both shown that lymphocytes in the spleen, in the lymph nodes and in the recirculation pool – that is the lymph and the blood – were the cells which could initiate immune responses. (They were called immunologically competent cells.) My mice had a deficiency of those lymphocytes, which led me to think they must surely come from the thymus.

Yes, because the thymus is filled with lymphocytes.

Correct, but in those days it was thought that because the thymus had a lot of dying lymphocytes it was a graveyard for them. Not only that, but thymus lymphocytes were not able to induce immune responses in appropriate recipients as the lymphocytes from the spleen and lymph nodes or the recirculating pool were. And also because adult thymectomy did not interfere with immune capacity, people did not believe that the thymus had anything to do with immunity.

Yet, because of the results that I had obtained, I really wondered whether the thymus was the seat of production of those lymphocytes which would eventually become competent. Maybe they were just immature in the thymus – they had to mature and go out, and then become competent. I wrote that idea up in the Lancet in 1961, after being pushed to write it by Sir Alexander Haddow, who was officially my mentor.

It would have been a great help to have somebody like that to push you to write it up and to make sure that you ended up in a journal like the Lancet.

I had to check my idea, and one way was to see whether these mice were totally incompetent. Having learned the technique of skin-grafting from Medawar's group, I gave those mice skin grafts from many different types of strains and even rats, and they accepted the skin grafts. They never rejected them.

The other thing was to see if those mice could be rescued immunologically, to be able to respond as normal mice and reject the skin grafts, by giving lymphocytes – and by giving a thymus graft. Lymphocytes from the circulation rescued them perfectly well, or putting in a thymus graft also rescued them. Putting two and two together, it looked very much as if the thymus was the source of those lymphocytes.

That totally unexpected outcome of investigations of mouse leukaemia must have changed the direction of your life.

Exactly. It changed my life and I actually had to learn immunology.

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Spreading the word

You were not only learning immunology but creating it, to a large extent. Three people brought developments within a decade: Medawar with immunological tolerance, Gowans with the lymphocyte circulation, and you – the youngest – with neonatal thymectomy and the role of the thymus in the production of lymphocytes and therefore immunologically competent cells. When did you first talk in public about it?

There were some meetings at the British Society for Immunology in Oxford and in London, but the first international meeting at which I gave my results was in Perugia, in 1961 – even before the Lancet paper came out. This was a meeting on tumour viruses rather than immunology and actually my written paper is on my work on leukaemia. But what I gave viva voce was on the effects of neonatal thymectomy, because it was so interesting, and the published discussion of my work was on that and hardly at all on leukaemia. People were interested but still very sceptical.

The second international forum was in February 1962, in New York. I had applied to go to the New York Academy of Science and had been accepted, so I gave very detailed results on neonatal thymectomy and the immune response, to an audience mainly of immunologists and transplantation biologists. That made quite a few headlines – even in Time magazine, as a matter of fact!

That's going a long way in America.

In general people accepted the data, but quite clearly some were still very sceptical because of the knowledge that thymus lymphocytes are not able to induce immune responses and that adult thymectomy does not do anything at all. They wondered whether my mice might be so worn down by infections for one reason or another – perhaps they were dirty mice, or something like that – that it would only happen in my case. People in Holland tried to reproduce the work, I remember, but couldn't. That worried me. By that time I had a technician, so I sent her along to that place in Holland where she taught them how to do the thymectomies and of course it worked.

People in America reproduced my work partly but could not cross barriers like the strong H2 barriers. If mice which were thymectomised had skin grafts put on them from mice which were very similar to them in genotype, they accepted those skin grafts, but they would reject skin grafts from mice which were very distant or from other species like rats. Finally it turned out that all those people were not doing complete thymectomies but only partial ones. Once they were able to do complete thymectomies, all the work that I had done was reproduced exactly.

Such a delicate operative technique has to be performed skilfully to succeed. A newborn mouse is a tiny object, and the thymus is a tiny object within that again.

Yes. And it is very interesting that partial thymectomy doesn't have any effect at all. It looks as if the piece of thymus that remains behind has all the necessary equipment to do whatever the whole thymus has to do.

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So why did the mice waste away?

Did you stay on in America?

No. After the February meeting of the New York Academy of Science I returned to London, but I decided I must do a year in a place somewhere in the world which had germ-free mice, because of the problem with infection. My idea was that my mice got infected because they were immuno-incompetent, not for some other reason. They were immuno-incompetent because they were thymectomised at birth, so if I could only do thymectomy in a germ-free situation, I would never get wasting disease but yet I would get evidence of immuno-incompetence, like failure to reject skin grafts. I found that the National Institutes of Health in Bethesda had germ-free facilities; I applied for an Eleanor Roosevelt Fellowship in 1963 and got it; and I was able to do the work in a germ-free tank at NIH. I showed there that these mice lived on perfectly healthily but yet could not reject skin grafts.

So wasting disease essentially had been due to their lack of control of infection?

That's right.

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Testing a theory – and a very testing procedure

NIH is a wonderful place to work in and your work in the germ-free unit must have been a good experience for you.

It was very good. Also, I did some work with Lloyd Law, who was on the same floor as that unit, and showed that neonatally thymectomised mice were much more prone to cancer-inducing agents than normal mice. I had done that before with chemical agents, but we used the polyoma virus and showed it to be true. Burnet had a theory of immuno-surveillance, that the immune system was essential to survey the body against mutations which occur, for example, in cancer and to get rid of cancer cells. So it was very good to show that if you were immuno-incompetent your threshold for developing carcinomas or different types of neoplasm was very much reduced.

It had been on the right track but didn't have the experimental evidence?

That's right, so it was useful to be able to do all these experiments in NIH. After the Eleanor Roosevelt Fellowship I went back to London in 1964, with a few visits to America – to Denver, for example, where they wanted to thymectomise opossums in the pouch! In those days people only thought of one type of lymphocyte, and the very fact that neonatal thymectomy in mice was still associated with some lymphocyte production suggested that perhaps you should take the thymus out before birth, to make sure that all the lymphocytes have been removed. That is impossible in mice, but in opossums you could do it in the pouch. They had opossums in Denver and were working on them, so I went there twice to do it. It was very difficult and I never got very far. They were like balloons filled with nothing but blood.

It's bad enough on a neonatal mouse, but on something only a few millimetres long!

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Could thymus lymphocytes become immuno-competent?

In 1965 Gus Nossal succeeded Sir Macfarlane Burnet as Director of the Walter and Eliza Hall Institute, in Melbourne. And he remembered his schoolboy friend.

Yes. Being an immunologist he had been interested in my work, and he invited me to the institute. I decided to go back to Australia partly because my wife, being Australian and having a sister there, would like to go back, and also because I realised that Gus would give me tremendous encouragement and a lot of facilities, many inbred strains to work on and so forth.

You haven't regretted that decision?

No, not at all. The Walter and Eliza Hall Institute is a magnificent institute and a great place to be. The infrastructure is stupendous and it is just wonderful that you can talk to any scientist, whether or not they are in your field. Gus is to be congratulated very much for having built an institute in such a way.

Even when I was there, in Burnet's day, it was a wonderful place to work in and very bright visitors would come out from Europe and America to work with him for a year or so, but it was really tiny in numbers of people – about the size of a small department of a modern institute or university. Gus's first idea was to build it up, and he did that very effectively.

It was still quite small when I first joined, but they built two floors and then one floor on top, and then finally you had a completely new building.

When you joined the Hall Institute in 1966, what did you embark on?

I was pleased to find that Gus had already chosen Graham Mitchell, who had just graduated in veterinary science from the University of Sydney with first-class honours, to be my first PhD student. We made it our first priority to see what sort of cells in the lymphoid and haematopoeitic system could restore immune responsiveness to thymectomised mice, either neonatally thymectomised or adult-thymectomised, irradiated and marrow-protected.

To explain: the effects of neonatal thymectomy can be reproduced in the adult, provided after adult thymectomy you get rid of all the lymphocytes that are present in the body, by irradiation, for example. If you give very high doses of irradiation you have to inject bone marrow, of course. But in the absence of the thymus, the stem cells coming from the marrow can no longer generate lymphocytes, at least thymus-dependent lymphocytes. So you could reproduce the effects of neonatal thymectomy in the adult, provided you followed that up with irradiation.

We decided to have a very, very good look at the types of lymphocytes which would restore immune responsiveness to immuno-incompetent mice. We used thoracic duct lymphocytes, removed from the lymph – and the spleen, of course, and bone marrow, thymus and lymph nodes – and studied their effects.

It was good, because thoracic duct cannulation was a very powerful technique which taught us a lot of things, for example that neonatal thymectomy – or adult thymectomy, irradiation and marrow protection – was associated with a tremendous diminution of the number of cells that could recirculate. Those recirculating cells were the cells which Gowans had shown to be immuno-competent, so again this showed that the thymus had something to do with the buildup of immuno-competence. But was it an effect of putting lymphocytes out to the circulation, or was it a humoral effect? It could have been an endocrine effect, as Medawar believed – that the thymus did not produce lymphocytes that went out in the circulation as competent cells but produced some kind of hormone which activated lymphocytes.

I remember talk of the 'thymic hormone', yes.

We really had to show whether thymus lymphocytes became immuno-competent. One of the experiments we did was to inject thymus lymphocytes which had been passaged twice in irradiated mice, together with antigen, to try and boost them up. But even those thymus lymphocytes which had been passaged with antigen into irradiated mice were unable to induce responsiveness. They were certainly not as good as lymph-node lymphocytes. But when these lymphocytes were given to adult-thymectomised, irradiated and marrow-protected mice, then the response was fantastic. In the presence of bone marrow, thymus lymphocytes were able to restore responsiveness, which in those days we measured by antibody production, on a plaque technique.

Yes, one thought immunity and antibody went together.

Yes, except for my skin-grafting, which depended on cellular immunity, not antibody. It had been shown that only lymphocytes (not antibody) could reject skin grafts. Thymus lymphocytes had never been shown to produce antibody, but when we showed that in mice that had been thymectomised and protected with bone marrow were able to restore antibody responses, we thought, 'That's fantastic.'

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Differentiating lymphocyte subsets

I said, 'Now we're going to show that thymus lymphocytes are the precursors of antibody-forming cells. We have to have a genetic marker.'

In those days they didn't have any known markers like theta or Thy-1, or CD4 or CD8 – they had none of the markers we have now. We had only the markers which were determined by histocompatibility genes, so we simply used an F1 hybrid thymus into a parental genotype recipient. In other words, we had a cross between two strains of mice, A and B, which can be distinguished by histocompatibility genes. The thymus came from an F1 and the recipient mouse was a thymectomised, irradiated, parental genotype strain mouse which had been protected again with parental genotype marrow. Into these mice we injected the F1 thymus, and we got a tremendous number of plaque-forming cells. And now, because we had antisera against both parents, we could determine whether the origin of the plaque-forming cells was thymus or bone marrow. The results were absolutely spectacular, no question about it. One plate was complete with plaques and the other plate had nothing.

We knew that one of these would tell us whether it was thymus lymphocytes that became antibody-forming, and I bet it would be thymus. It wasn't. It was bone marrow. But the thymus-derived cells were absolutely essential in order to allow the bone marrow-derived cells to make antibody, so there must have been some kind of interaction between thymus-derived cells and bone marrow-derived cells. We were able to publish those spectacular findings during 1967–68 in a very short statement in Nature; a paper which Burnet sent to the National Academy of Science, in the United States; and four papers in the Journal of Experimental Medicine.

That was the very first unequivocal demonstration, I think, that in mammalian systems you have two major subsets of lymphocytes, one of which is not derived from the thymus but becomes the precursors of antibody-forming cells. The other one is derived from the thymus but is needed to help in some way the antibody-forming cell precursors to become antibody-formers. That is somewhat similar in birds, which have both a bursa and a thymus.

I remember the bursa of Fabricius in the bird. Is the system the same as in mammals?

It is exactly the same, but the bursa doesn't exist in mammalian species. Its function, we know now, is taken over by bone marrow.

In hindsight it turns out very useful that 'bone marrow' begins with a 'b', so that you could talk of B cells in both birds and mammals.

Yes. We called them 'bone marrow-derived cells' and 'thymus-derived cells', which was quite a mouthful. For a long time I tried to work out short terms for them but it was Roitt, in London, that coined the term 'T and B cells'. I wish I had done that.

Roitt is a very good populariser of science who writes general textbooks for non-immunologists to understand, as well as immunologists.

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Eventual acceptance, but new complexities

To make two such major discoveries – the interaction of B and T lymphocytes in antibody production, and the role of the thymus itself – is quite an achievement.

Thank you. Actually, the concept of two cells interacting in the immune response was not accepted by the immunological community for a long time, for several years. First of all, Gowans had shown that the small recirculating lymphocyte can either produce antibody or reject skin grafts. To him the small lymphocyte was a type of cell and there need not have been two subsets with any difference between small lymphocytes that caused cellular immunity, or skin graft rejection, and those that became antibody-formers. It was well ingrained in the literature that one type of lymphocyte can do both.

Secondly, both T and B cells had to be specific for a given antigen. In fact, we proved that by using the antigen-suicide technique of Gordon Ada. We showed that we could suicide thymus cells with a specific antigen that was heavily radio-iodinated. So they had specificity of their own. And of course the bone marrow-derived cells had specificity because the antibody they formed was specific. So how could two very rare, clonally individuated cells ever meet each other in order to collaborate? People said, 'It is just not possible.'

How did you get over that? What was the next step in the investigation?

People repeated our work and eventually it came out to be absolutely true: there are two types of cells. Then came the antigen Thy-1, which someone had discovered in America – I wish I had used it before – and which was shown to be on some lymphocytes but not on others. In London, Mitcheson and Martin Raff used an anti-Thy-1 antibody to show that you could eliminate the type of lymphocyte that was thymus-derived and responsible for skin-graft rejection. It did not eliminate the other lymphocytes.

The other thing which came out was that one type of lymphocyte had immunoglobulin on its surface but not the other, and the one that had immunoglobulin on its surface was the bone marrow-derived one. As all this came together, people started believing in two subsets.

Among the interactions between T and B lymphocytes, essentially, a subset of T lymphocytes is important in its effect on the precursors of B lymphocytes.

Yes, that is correct. After this work, it was shown in America – not by us – that even T cells can be divided into two major subsets, CD4 and CD8. The first helps B cells form antibody and the other becomes the cytotoxic T cell, which kills off virus-infected cells and may be involved in graft rejection and so on. So even that is more complicated than people thought in the early days.

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Studying delayed-type hypersensitivity: could MHC restriction be shown in vivo?

Perhaps harking back to your early family history of tuberculosis, you started looking at the delayed-type hypersensitivity reaction.

Yes. That is a type of cellular immune response which we wanted to study in vivo. Many people at that time were studying cytotoxic T lymphocytes in vitro, in tissue culture. I was keen to study the interaction which takes place in vivo, so we decided to study delayed-type hypersensitivity (DTH, as I call it). With Matthew Vadas, another PhD student at the time, we devised a new technique for measuring DTH which relied on the radioactivity that you can measure in a particular site, in fact in the ear of a mouse. I didn't like to measure the thickness of the ear. It seemed to me that it is not so accurate, because it depends on the person who measures it. We devised a method of injecting a radioisotope in the ear so that the ear would have a certain amount of radioactivity and we could measure that effectively in a counter. It would not be dependent on the investigator.

Zinkernagel and Doherty had discovered the phenomenon of restriction by the major histocompatibility complex (MHC). This phenomenon had been demonstrated in vitro, and using this particular technique we were able to show that it also occurred in vivo in the transfer of delayed-type hypersensitivity. When you transfer DTH in vivo, you cannot transfer across an H2 barrier. You have to have H2 identity, which is similar to MHC restriction in vitro. I remember ringing Zinkernagel and speaking to Doherty about it in those days. They were very pleased that another system showed that they were correct.

If you had showed they were wrong, they wouldn't have been so happy! But if 'discoveries' are made in vitro, in cultured cells, it is very important to check them in an intact animal. Otherwise you may be led up the garden path.

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Immune-responsiveness genes: a masquerade

How did you go on from there?

At the time, 'immune-responsiveness genes' were in fashion. These were supposed to allow certain strains of mice to respond to certain types of antigen. For example, in the laboratory you could make synthetic antigens like a polyamino acid of glutamic acid, alanine, tyrosine, and you could show that certain strains would respond perfectly to these synthetic antigens but other strains would not respond at all.

When you say 'respond', do you mean produce antibody?

By delayed-type hypersensitivity or by antibody responses, by any criteria. The supposed immune-responsiveness genes were thought at one stage to be the receptor on T lymphocytes – the T-cell receptor for antigen – because they generally acted in the case of cell-mediated immunity or helping antibody formation. But our experiment with the transfer of delayed-type hypersensitivity showed that the immune-responsiveness genes were just the MHC-restricting element of Zinkernagel and Doherty – they were identical. And this turned out to be true.

So the term 'IR genes' has disappeared from the literature as a false conception. Sometimes things get simpler rather than more complicated! Since cellular immunity can be effective against intracellular parasites, did you think of investigating the idea that it might be effective against cells that had changed by becoming malignant?

Yes. Also, in my early work I had shown that neonatal thymectomy allows mice to develop cancers much more frequently than non-thymectomised mice. It was evident that this must be true to some extent but we did not investigate it in great detail. A lot of people in George Klein's laboratory, among others, were investigating tumour immunity, and it has been shown that there is such a thing as immuno-surveillance. But now not just T cells but NK cells, natural killer cells, are involved as part of the immune system.

That again shows the complexity of things we call lymphocytes, when NK cells are also morphologically just like the others. The cells that circulate in the lymph are really a pretty complicated lot.

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Where was that elusive T-cell receptor?

Where did you go after the work on DTH?

We did quite a lot of different things. It would be hard to single one out. I will tell you one of my great failures. We decided to look for the antigen receptor on T lymphocytes. It was clearly known that it was immunoglobulin on B cells which allowed B cells to make antibody. Several groups in the world thought, without actually knowing, that there was some kind of immunoglobulin molecule on T cells. In fact, in the Hall Institute, Jack Marchalonis coined the term 'IgT' for immunoglobulin T, being on T cells. I had never thought that was correct.

We really tried to find immunoglobulin on T cells, but I just wasn't able to show any evidence of it. With the help of a molecular biologist, David Kemp, we showed at the RNA level that there could not be any RNA expressed to make immunoglobulin on T cells. That was a negative result, which was written up in the Journal of Experimental Medicine. Trying to look for the T-cell receptor without much molecular biology knowledge, I failed completely to find it. The Americans found it!

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Why are we tolerant to our own tissues? More complexities

You have certainly done a wide range of different things. You did so much work in vivo, in the best experimental animal there is, the mouse – which now has been rendered vastly more valuable than ever before, even with SPF pure-line mice, by the possibility of making transgenic mice.

Yes. That, I think, rescued me from the T-cell receptor. When transgenic mice became available I immediately realised that this is the ideal system to study tolerance to 'self' – not tolerance, as Medawar and others had studied, to antigens introduced into the animal, but tolerance to one's own-self tissues. Why we are tolerant to self is still unresolved. When transgenic mice became available we were able to inject into the fertilised egg a gene which would make a gene product in a particular tissue. We chose a gene product against which we had an antibody, so that we could find its product. It is like having a marker for self, a self marker. All our work since 1986 or '87 has been done on transgenic mice and on self-tolerance. That has been very productive.

We have shown that there are several mechanisms of self-tolerance. In transgenic mice, both we and Zinkernagel have shown that some of it is due to ignorance. We have also shown that some tolerance is due not just to the self-reactive cells being eliminated in the thymus. If they escaped from the thymus they could be eliminated in the circulation as well, as a result of antigen driving the cells to elimination – now shown to be due to a death gene called Fas, or CD95. These studies have been productive but have led to more complications, involving signalling in the cells by various types of molecules. Other people studied CD95 as one which signals the death of the cells, but we have looked at signalling molecules with names like CD30, CD40 and so on, which are very important in the immune response – or in shutting it off. That has great potential for the study of autoimmunity – autoimmune diseases, for example – and perhaps also neoplasms. If, instead of shutting the cells off, we could activate them to kill cancer cells, that would be very useful.

How is the shutting off done in the thymus?

Probably the cells which encounter self antigens in the thymus are killed through some mechanism dependent on the avidity of the T-cell receptor for its target. If the T-cell receptor has a very high avidity, then the T cell is killed. If it has a low avidity, the cell can escape or it can respond to other antigens. We have worked on that, but it was not discovered by us.

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Contributing to the scientific community

Would you tell us about some of your activities in national and international bodies?

I served on the Academy's Council and Sectional Committees, and a few other things like that. Also, I was asked to serve on several international agencies.

For 3 or 4 years in the 1970s I was on the International Agency for Research on Cancer, whose headquarters are in Lyon, and I eventually became the President of the Scientific Council. It was interesting to see what sort of research is done in that field, and I benefited from that.

I served on WHO for quite a number of years, on eradicable diseases. WHO wanted to eradicate five parasitic diseases by the end of the millennium, including malaria, schistosomiasis and leprosy. It was very ambitious: I don't think malaria will be eradicated by the end of the millennium. I was one of several immunologists on that committee.

So it was strongly oriented towards vaccine production?

Definitely towards vaccination, yes. We were also trying to boost research by checking grant applications and deciding on various methods of attacking those diseases. I also served on WHO every now and then to summarise certain aspects of medical research – immunological research, in my case – writing books that the WHO put out on certain topics.

You did a term on the International Union of Immunological Societies, and you have regularly attended conferences on immunology, from way back in 1961 up to the present.

For many years, with three or four international trips each year. I have been quite active in that respect.

You have given us a very good picture of your career, Jacques. Thank you very much.

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