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F U L L T E X T S O U R C E : Cell
Image 1: Photo of J. Miller in 2019 at WEHI.
I grew up partly in China, partly in Switzerland, and partly in Australia. We lived in China because my father was manager of the Franco Chinese Bank. We moved to Switzerland because my eldest sister, Jacqueline, contracted tuberculosis; and that country, in the 1930s, was supposed to be the best place to manage this illness. Finally, the threat of Japanese invasion during World War II led my father to take his family away to Australia, where we arrived barely 3 months before the bombing of Pearl Harbor.
My curiosity for how the body responds to infection began as a child. My younger sister, Jeanine, and I had often been playing in the same room as Jacqueline, even when she was coughing blood-stained sputum, but we never developed the disease. I had overheard the doctor explaining to my mother what tuberculosis was and how little was known about how the body resisted such infections. This inspired my interest in medical research. Furthermore, the fact that I grew up while World War II was raging both in Europe and Asia made me wish to avoid military engagements and study medicine.
After my residency as an intern in the Royal Prince Alfred Hospital in Sydney, Australia, I got a scholarship that enabled me to study for the PhD degree at the Institute of Cancer Research in London, UK. Many of the researchers there were involved in studying chemical carcinogenesis, but this did not really interest me, as I would rather investigate a model where pathogenetic mechanisms had to be worked out. Fortunately, the Institute had a satellite outside London in Pollards Wood, Buckinghamshire, at an estate that had previously belonged to Bertram Mills and his circus animals. The rooms in the magnificent Tudor-style mansion had been converted to well-equipped laboratories; and the horse stables had been transformed to mouse-, rat-, and rabbit-holding rooms. As a PhD student, I was under the supervision of Dr. Robert J.C. Harris, who was working on the development of sarcomas in turkeys induced by the Rous sarcoma virus. He suggested that I might be interested in investigating the pathogenesis of lymphocytic leukemia induced by what was presumed to be a virus discovered by Ludwig Gross in the USA. Within a few weeks of my arrival, Dr. Harris left because he was offered an excellent position at the National Institute for Medical Research in Mill Hill, London. I was therefore left without a supervisor but was lucky to inherit his animal space.
Gross had made filtered extracts of lymphocytic leukemic tissues from “high-leukemic-strain” Ak mice that are prone to develop the disease spontaneously at around 9 months of age. He then injected the filtrate intraperitoneally into mice of the “low leukemic strain” C3H, which do not normally get leukemia, and observed the development of this disease but only if the filtrate was injected into newborn C3H mice, not into adults. I wrote to Gross telling him of my intention to investigate the pathogenesis of this type of leukemia; to my great relief, not only did he approve, but he even sent me some C3H mice that had been inoculated at birth and some normal ones.
We had some hints on the physiological origins of lymphocytic leukemia from the studies of numerous investigators. In addition to the lymphocytic leukemia arising spontaneously in Ak mice that was studied by Gross, lymphocytic leukemias were also shown to arise following ionizing irradiation in (low leukemic strain) C57BL mice and also following treatment with some chemical carcinogenic compounds in another low leukemic strain (DBA/2). From these studies, we knew that lymphocytic leukemia began in the thymus and then spread to other lymphoid tissues, and that it could be prevented by thymectomizing adult mice at 6–8 weeks of age.
Following the results obtained with other types of leukemias, I inoculated baby C3H mice with leukemic filtrate (herein after termed “virus”) and thymectomized them after weaning. These failed to develop leukemia but did so when subsequently grafted with a neonatal thymus, the graft being introduced subcutaneously in the axilla or under the kidney capsule. What was most fascinating was my finding that grafting the thymus as late as 6 months after thymectomy still resulted in leukemic transformation of the thymus graft. Clearly the virus must have remained latent and indeed I could recover it from the non-leukemic tissues of thymectomized mice previously inoculated at birth with virus. The question therefore arose as to why the virus had to be given at birth, and where did it multiply. One of my hypotheses was that leukemic transformation occurred only if the virus could multiply in the developing thymus of newborn mice. Thymectomy performed after weaning would have removed the source of the malignant cells but not the virus which would have spread to other tissues and would thus be available to transform cells whenever its target, the neonatal thymus, was grafted. One obvious way to test this hypothesis was to remove the thymus from newborn mice, inoculate the virus immediately after neonatal thymectomy, and then graft a newborn thymus subsequently at different intervals. If the hypothesis was correct, neonatal mice lacking a thymus from birth should no longer be susceptible to virus infection and should therefore not develop leukemia when later grafted with thymus tissue.
The neonatally thymectomized (NTx) mice fared well at first, but many wasted and died several weeks after weaning, regardless of whether they had been inoculated with virus. Mice thymectomized as adults, as had been performed by numerous investigators, had never shown any signs of ill health, such as weight loss, or any obvious pathology. This led me to conclude in a paper on leukemia published in Nature in 1961, “that the thymus at birth may be essential to life.” Examination of the tissues of my NTx mice revealed a marked deficiency of lymphocytes in blood and lymphoid tissues, and many wasted mice had liver lesions, suggesting infection by an endemic mouse hepatitis virus. As circulating lymphocytes had recently been shown by James Gowans, using rats, to be immunologically competent cells, able to initiate both cellular and humoral immunity, it was clear that I should test my mice for immune functions. I did so before the onset of wasting. The results were spectacular—a eureka moment! The mice failed to reject allogeneic skin grafts, even those from donors that differed at the strong histocompatibility locus H-2, and even from rats (Figure 1). They also failed to produce antibody to some, but not all, antigens I tested such as sheep erythrocytes. I also showed that thymus grafts restored immunocompetence, but the crucial finding was that foreign thymus grafts did restore competence, though not to skin from the same donor as the thymus graft, even if that donor was H-2 disparate. That skin was never rejected. The foreign thymus must thus have induced tolerance to itself, and by implication, the thymus must have the ability to induce self-tolerance. My own words in the paper in which these published data appeared were: “Antigenic material might make contact with certain cell types differentiating in the thymus and in some ways prevent these cells from maturing to a stage where they would be capable of reacting immunologically” and I used the words “selective immunological thymectomy” to describe this negative selection. These results were published first as a preliminary communication in The Lancet in 1961 and soon after in a detailed paper in the Proceedings of the Royal Society series B in 1962.
The foreign thymus must thus have induced tolerance to itself, and by implication, the thymus must have the ability to induce self-tolerance.
Figure 1Immunodeficient Neonatally Thymectomized Mouse
Neonatally thymectomized (AkXT6)F1 mouse bearing skin graft (SG) from another mouse strain (C3H) and from a rat.
I concluded from all these data that the developing thymus was the source of lymphocytes that would mature to become fully immunocompetent. To bolster this conclusion, it was essential to show that some lymphocytes must leave the thymus to populate the lymphoid system. However, with no known cell surface markers and no flow cytometry at the time, I could only do this using thymus grafts and a strain of mice that happened to have a chromosome marker (T6). Thymuses from C3H or AK newborn mice were grafted into 5- to 7-day-old NTx (AKXT6)F1 mice which were immunized with foreign skin 2 to 4 months later. Up to 15% of the cells in metaphase in the spleen were found to lack the T6 marker and thus were thymus-graft-derived cells (now called T cells).
As stated above, adult thymectomy had never been associated with any defects, and that was one of the reasons why most investigators believed that the thymus was a vestigial organ with no useful function. However, I hypothesized that the adult thymus was a source of immunocompetent lymphocytes, and immune system reconstitution following irradiation provided an opportunity to test this idea. Since lymphocytes are radiosensitive, an adult mouse can be made lymphopenic by irradiation; however, the immune response can recover within around 8–10 weeks in irradiated (euthymic) mice if they are provided with a source of stem cells (i.e., a bone marrow transplant). It therefore occurred to me that if the thymus was important for the differentiation and function lymphocytes, then a prior thymectomy in adult mice should prevent recovery of their immune response after irradation. So, I thymectomized and sham-operated CBA mice at 8 weeks of age, irradiated them at 10 weeks and immediately gave them syngeneic bone marrow (these mice are herein after named “ATxXBM” mice). They were grafted at 16 weeks with skin from different donors. All the sham-operated mice were able to reject foreign grafts, but 70 to 77 percent of the ATxXBM mice could not. Hence, I concluded that the adult thymus must still have the potential to produce lymphocytes that can act as immunocompetent cells. These results were first published in Nature in 1962 and subsequently in much more detail.
…most investigators believed that the thymus was a vestigial organ with no useful function.
It seemed worth investigating whether the thymus might produce lymphocytes that would be able to restrain the growth of tumor cells. To this end, I applied 3,4-benzopyrene to young adult mice that had been NTx or sham-operated. Papillomas occurred in both sets of mice but reached a larger area in the NTx mice. Most importantly, by 180 days 12 percent of skin tumors in these mice became malignant in contrast to only 4 percent in the controls. This was published in 2 papers in Nature, one in 1963 and another in 1965. My words in the conclusion of one of these papers are: “Interference with the cellular immune mechanism may be necessary, in some cases, to allow the full expression of a carcinogenic process.”
In the 1960s, immunologists were reluctant to accept the idea that the thymus had an immune function. This was not only because adult thymectomized mice showed no defects, but also because thymus lymphocytes, unlike lymphocytes located elsewhere, failed to adoptively transfer either humoral or cellular immune responses. Furthermore, in contrast to other lymphoid structures, the thymus did not show, after immunization, any histological evidence of an immune response, such as plasma cells or germinal centers. When I presented my results at various meetings, the data were not criticized, but the interpretation was questioned. At a meeting of the British Society for Immunology in 1961, it was argued that what I had observed must surely have occurred only in the strain of mice that I had been using; that whatever the thymus might have been doing in my mice, it could not possibly do in humans; some argued that perhaps my mice, having been raised in converted horse stables, must have been exposed to so many intercurrent infections that the additional trauma of neonatal or adult thymectomy followed by irradiation precipitated immunodeficiency. This latter suggestion prompted me to go to the National Institutes of Health in Bethesda in 1963, courtesy of an Eleanor Roosevelt Fellowship, to repeat the work in germfree tanks. Germfree C57BL mice were neonatally thymectomized or sham operated in the tank and grafted with H-2 disparate BALB/c skin. None became sick, and none of the NTx mice rejected the skin. By showing that the thymectomized mice did not reject the foreign skin even when isolated from any infectious agents that could compromise immune function, I convinced most immunologists that the thymus had an immune function.
In addition to the thymus, birds have another thymus-like organ, the bursa of Fabricius. It was known since the work of Bruce Glick in 1956 that testosterone injection in ovo caused impairment of bursa development and defects in antibody production in the mature bird. Nobel Laureate Sir Frank Macfarlane Burnet and his colleagues pursued this work further and showed that those testosterone-treated birds in which the atrophy extended to the thymus, in addition to the bursa, failed to reject foreign skin. Since, however, mammals do not have a bursa, and my NTx mice failed to produce antibody to some antigens, Burnet argued that cells derived from the mammalian thymus must perform the dual role of humoral and cellular immunity.
In 1965, I was invited to lead a new laboratory at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia. There, with the help of my first PhD student, Graham Mitchell, I wanted to determine what quantitative differences existed between the recirculating lymphocyte pool of NTx and sham-operated mice. This we did by cannulating the thoracic duct 5–6 weeks after birth and draining the lymphocytes continuously for a period of 48 h, after which no further lymphocytes drained out. The cumulative total number of thoracic duct cells drained reached close to 108 cells in sham-operated mice, but only slightly more than 106 cells in the NTx mice. Since I had shown that NTx mice failed to reject foreign grafts and often failed to make antibody, I surmised that the cells making up the difference in the two sets of mice were thymus derived and responsible for both cellular and humoral immune responses, whereas the few cells found in the thoracic duct lymph of NTx mice were derived from another source.
We then designed experiments aimed at definitively determining whether thymus derived cells were indeed important for both cellular and humoral immune responses. Since no cell markers were then available, we used the H-2 disparate strains CBA and C57BL and their F1 hybrid. Thoracic duct lymphocytes from (CBAXC57BL)F1 mice were injected intravenously into immunoincompetent adult thymectomized and irradiated (CBAXC57BL)F1 mice to restore immunocompetence. As there are no stem cells in thoracic duct lymph, we used bone marrow from one of the mice in the parental strain (CBA) to protect the heavily irradiated adult thymectomized F1 mice. After these mice had fully recovered, we injected them intravenously with sheep erythrocytes and removed their spleen (at the height of the known antibody response) to determine whether the antibody forming cells came from the thoracic duct lymphocytes that were presumed to be thymus-derived in our quantitative experiment. The number of antibody-forming cells was detected by an established plaque assay, in which spleen cells, sheep erythrocytes and complement were poured onto agar plates, incubated at 37°C and clear plaques resulting from an antibody forming cell lysing the surrounding sheep erythrocytes were counted. As the spleens were obtained from the (ATxXBM) F1 mice that had their immunocompetence restored by syngeneic thoracic duct cells, a normal antibody forming cell response was obtained as expected (Figure 2). Now, we could determine from which cell source were the antibodies derived: if they were derived from the F1 lymphocytes, they would be destroyed by anti-C57BL antibodies made in CBA mice immunized with C57BL tissues; if they were derived from the bone marrow donor (CBA in our experiments), they could not be destroyed by such antibodies. The results were spectacularly clear each time the experiment was repeated. Anti-C57BL antibodies had no substantial effect (lysing only from 0%–12% of the antibody forming cells) whereas, as expected, anti-CBA antibodies lysed close to 100% (86%–96% percent).
Figure 2Schematic Representation of the Experiment that Proved the Existence of T and B Cells
Lymphocytes obtained from the thoracic duct (TDL) of normal F1 hybrid mice were inoculated intravenously (IV) into immunodeficient, thymectomized (ATx) and irradiated (X), syngeneic F1 mice, together with bone marrow cells (BM) from one of the parental strain (CBA) and challenged with sheep erythrocytes (SRBC). The number of antibody-forming cells in the spleen was determined by a plaque assay, in which spleen cells, sheep erythrocytes and complement were poured onto agar plates, incubated at 37 degrees Celsius and clear plaques, resulting from an antibody forming cell lysing the surrounding sheep erythrocytes, were counted before and after further incubation of an antiserum directed against the histocompatibility antigens of the parental strains.
These results proved beyond any doubt and for the first time that thymus-derived cells (later called T cells and accounting for more than 90% of the thoracic duct cells found in mice) were not antibody-forming cell precursors. These initially came from the bone marrow. Why, then, did NTx mice fail to respond to some antigens? Graham Mitchell and I postulated several possibilities, one of which was that antibody forming cell precursors (later known as B cells) could only respond to those antigens if they collaborated in some way with “helper” T cells, as a result of which they received from the T cell a factor (I called it a pharmacological factor) which somehow turned on antibody production.
How did the immunological community react to these findings? Burnet expressed reservations “about the significance of results obtained in such biological monstrosities as pure line mice thymectomized, lethally irradiated, and salvaged by injection of bone marrow from another mouse.” James Gowans argued that small lymphocytes were morphologically identical and that two rare clonally individuated cells would never find each other to interact at close range. “If we have two cell lines that are collaborating, then we have specificity residing in two cell lines, one thymus-derived and the other marrow-derived. The problem is to bring these two specific cell lines together. Does this necessity for the two cells to find one another raise problems? It seems an inefficient mechanism if it rests only on chance contacts.” Robert Good was “concerned at separating thymus-derived from marrow-derived cells” since the former “are in fact marrow derived-cell” (meaning derived from stem cells). He also claimed to “have evidence that in the rabbit [the bursa equivalent] resides in the ilial lymphoid tissue and in the lymphoid tissue of the appendix”. The then-Professor of Immunology at the John Curtin School of Medicine in Canberra was less diplomatic and stated that B and T cells represented only the first and last letter of the word “bullshit.”
The then-Professor of Immunology at the John Curtin School of Medicine in Canberra was less diplomatic and stated that B and T cells represented only the first and last letter of the word ‘bullshit.’
Experimental results obtained by others using mice in the mid and late 1960s could be interpreted as showing that more than one cell type operated in immunity. For example, Henry Claman in Denver showed that irradiated mice given syngeneic bone marrow cells and syngeneic thymus cells produced more antibody than when given either cell source alone. As no antibody or genetic markers were used in his experiments, he could not identify the origin of the antibody-forming cells. Arnold Reif and Joan Allen in NIH, developed an antibody which marked thymus cells and only a proportion of lymphocytes elsewhere. Delphine Parrott and Maria East in the UK showed that NTx mice had a deficiency of lymphocytes in some areas of the lymphoid tissues, but not in those areas where antibody-producing cells (identified as “plasma cells”) appeared.
Soon, almost every immunologist joined the band wagon and helped work out the intricate details describing cell types, pathways, factors, molecules, etc. Immunology exploded! It is very exciting for me to see that the thymus, once believed to be a useless vestigial organ populated with cells—which, in 1963, were considered by Nobel Laureate Sir Peter Medawar “as an evolutionary accident of no very great significance” —is producing T cells involved essentially in the entire spectrum of tissue physiology and pathology. T cells are now known to have roles not just in reactions considered to be bona fide immunological, but also, to cite just some examples, in metabolism, in tissue repair, in dysbiosis, and in pregnancy. I also find it most rewarding to see that basic research on thymus function—first published in one of my papers in 1961, and on T and B cells a few years later—has sown the seeds that spawned the new era of immunotherapy, which can now claim a seat in the therapeutic pantheon of oncology, next to and perhaps about to supersede surgery, radiotherapy, and chemotherapy.
Before I end, I want to thank the Lasker Foundation for celebrating basic medical research and for having chosen Max Cooper and me for this prestigious award.
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Edited by Engadin, 10 September 2019 - 08:23 PM.
