THE DISCOVERY OF THE INTERLEUKIN 2 MOLECULE

By

Kendall A. Smith

INTRODUCTION: THE DEFINITION OF DISCOVERY

Louis Pasteur’s now famous phrase in describing scientific discoveries, “chance favors the prepared mind” is apt for the story of the discovery of the interleukin 2 (IL2) molecule. One often hears of the serendipity involved in scientific discovery, one of the most famous of which is the discovery of penicillin by Fleming. He noticed that bread mold growing on a discarded bacterial culture dish had a zone of absent bacterial growth surrounding it. Subsequently, Fleming teamed up with the chemist Florey and in a series of experiments, they painstakingly identified the active antibacterial chemical. Also, one often hears of a person “being in the right place at the right time” to make a discovery, which appears obvious once the discovery is made known. Sir Peter Medawar classified discoveries into “synthetic and analytic”. A synthetic discovery is one that is made as if by accident, when there was no intention “to find” the thing discovered. Thus, in this instance the discovery amounts to the realization that something new has occurred. For if the investigator does not realize that a discovery has been made, it is as if nothing has happened. By comparison, an analytical discovery results from a direct quest to uncover and make known the characteristics of something that is suspected or known to exist, but is unknown as to the particulars. Thus, the discovery of penicillin was both a synthetic discover by Fleming and an analytic discovery by Florey. The discovery that genes were comprised of nucleic acids by Avery, McCloud and McCarty would be an example of analytic discovery, in that their experiments were a focused effort to discover the chemical composition of the genetic material. Medawar pointed out that one often gets more credit for the synthetic than the analytic discovery. However, this is not always the case, in that another analytic discovery was Watson’s and Crick’s discovery of the double helical structure of DNA.

More often than not, the preparation of the scientist in making a discovery is more important than luck. Ben Hogan, upon winning yet another US Open back in the 1950s was accused of being lucky with his putting by his radio interviewer. His response: “You know it’s strange, but I have found that the more that I practice, the luckier I get”. Accordingly, Ben Hogan is saying the same thing that Louis Pasteur said. Thus, like most scientific discoveries, the story of the discovery of the IL2 molecule is a combination of chance and preparedness. As I tell the story, I will leave it up to the reader to decide which was the more important.

THE 1960s: CELLULAR IMMUNOLOGY & BLASTOGENIC FACTOR

The story begins  more than 40 years ago, in 1960, the year that I matriculated at the university. Peter Nowell, a young scientist from the University of Pennsylvania changed the view of immunology forever, and created a whole new field, that of cellular immunology. Nowell made the startling accidental discovery that an extract from the kidney bean containing a molecule termed phytohemagglutinin (PHA) could stimulate lymphocytes to divide[1]. Prior to this, no one knew what lymphocytes were for, and most thought that they were boring, end-stage cells that were incapable of dividing. Nowell would never have made his discovery had he not left the PHA-separated plasma enriched for the leukoctytes, or White Blood Cells (WBCs) in the incubator over a weekend. When he looked at the cells after a few days in the incubator, he found that they were no longer small, boring lymphocytes. Instead, they had transformed into large “blastic” cells that actually resembled leukemia cells. There were multiple cells undergoing division (mitosis), and Nowell thought initially that he had stumbled on the cause of leukemia.

Nowell soon realized that he had not discovered the cause of leukemia, because the cells were not immortalized. They ceased proliferating after only a few days in culture. However, he had discovered something equally as important, i.e. that lymphocytes are capable of proliferating. An explosion of scientists began culturing lymphocytes, and one of these investigators was Ross McIntyre, a young hematologist at Dartmouth Medical School, who was to become my mentor 10 years later. Actually, Ross’ claim to fame was that he was the second person to report that PHA activated lymphocytes to proliferate.

The mechanisms responsible for the ability of lymphocytes to multiply rapidly remained obscure for more than 20 years after Nowell’s discovery. However, in 1965 there were two reports of the discovery of an activity found in the culture media of stimulated lymphocytes that promoted their proliferation. Termed “Blastogenic Factor” by Kasakura & Lowenstein[2], and Gordon & McLean[3], who reported their results in two back-to-back papers in the journal Nature, this activity in retrospect was probably due to the IL2 molecule. However, it would be another 15 years before the activity in the culture supernatants could be actually ascribed to a single molecule

Subsequent to the discovery of Blastogenic Factor, numerous reports appeared over the next 10 years that described variations of this basic observation, i.e. that there are soluble factors produced by proliferating lymphocytes capable of promoting the proliferation of lymphocytes. However, the molecular characteristics of these factors remained obscure, and it was not clear whether there were many molecules that functioned to stimulate cell growth, or whether there was just a few, or even only one molecule responsible. Moreover, the cellular source of these activities remained obscure.

In 1967, when I was a third year medical student, the Vietnam War was escalating. I was particularly concerned about this, as I had grown up watching WW II movies on TV, and the last thing in the world I wanted was to go to war. One day, as I was crossing the quadrangle, I met one of my friends, who was a fourth year student. He asked me where I was thinking of going for my internship. At the time I had only one clinical rotation under my belt, and I had no idea what kind of a doctor to be, let alone where to go for my internship. However, I knew where I did not want to go, and that was Vietnam. He said, that was no problem, provided that I could get into the National Institutes of Health (NIH). They had a two year program in medical research training that would satisfy the requirement of the armed services, which at the time was mandatory for everyone, especially all doctors.

I identified Dr. Charles Mengel, Chief of Hematology and Oncology, who had been to the National Cancer Institute (NCI). I went to see Dr. Mengel, and told him that I was positive that I wanted a career in academic medicine, and I asked him to help me get a good internship and a slot at the NIH. It helped that I was at the top of my class. Dr. Mengel said that he would help me, provided that I come to work in his laboratory in my 4th year. This was my first real experience with research, and I immediately took to it. It satisfied my quest to know, and it challenged my creative and competitive juices. I loved it!

Subsequently, Dr. Mengel was true to his word, and he helped me secure an internship at Yale, and as well, I was accepted as a Clinical and Research Associate at the NCI. I will never forget June of 1968, flying into Washington for my interview at the NIH. Vietnam War protestors were demonstrating in Washington, and there were thousands camping out on the Mall, while soldiers patrolled the city in armored vehicles with automatic rifles. I could only marvel at the whole spectacle and count my lucky stars that I was on my way to New Haven after my interview.

THE ‘70s AND THE CREATION OF MONOCLONAL T CELLS

I spent the next 2 years at Yale-New Haven Hospital, working every other night and every other weekend. After 6 weeks of this “hard labor”, I really made the decision that research was the life for me. Therefore, when I reported to the NCI in Baltimore in 1970, I was primed for a life in research, and felt lucky not be in Vietnam. This time was a turning point for me, in that I was invited to join the lab of Dr. Michael Mardiney, an immunologist who had trained at the Scripps Research Institute. Michael was heavily involved in the intricacies of growing lymphocytes in the lab. Through his training and with the help of my bench mate, Len Chess (now at Columbia), I began to know and love lymphocytes.

At the NCI, my projects focused on activating human lymphocytes with viruses, to see whether we could use this assay to detect immunity to viruses. My first papers dealt with lymphocyte proliferation assays after stimulation by mumps and rubella viruses. In addition, Len and I worked together on experiments examining the effects of interferon inducers on the capacity of lymphocytes to respond to antigens. Interferons are cytokines very similar to the interleukins, but they were discovered because of their capacity to interfere with virus replication, hence the name. Also while at the NCI, I was fortunate to attend the First International Congress in Immunology, which was held in Washington, D.C. in 1971. The number of people involved, even at that time, amazed me, and the excitement of everyone was palpable. I knew that I had chosen correctly, and I could not wait to begin.

In 1972 I left the NCI, assuming a postdoctoral fellowship with Ross McIntyre at Dartmouth Medical School in Hanover New Hampshire. I was really searching for a mentor, because my little exposure to science had taught me that I needed training to become a scientist, in much the same way that I had been trained by experts to become a doctor. Unfortunately, just before my arrival at Dartmouth, Ross lost his grant support. Consequently, I had left a lab at the NCI, where money was no object, to a lab where the entire budget was $5,000. Ross did not want me to do clinical work, so I did the next best that I could, and we performed clinical trials, administering to cancer patients the same interferon inducers and immune stimulants that I had worked with at the NCI. However, the very first thing that I did was to write letters to other labs, primarily in Europe, to secure another postdoc for the next year.

I was accepted for a fellowship by Professor Georges Mathe from Paris, who had achieved fame a few years earlier by showing that he could prolong remissions in childhood leukemia by administering immunotherapy in the form of a tuberculosis vaccine (BCG). My family and I left for France on the SS France in July, 1973, just as the Watergate hearings were in full swing. We all felt relieved to be getting out of the country. The Vietnam War was still going strong, and the atmosphere at home was definitely gloomy.

The City of Light was true to its name and for me it was love at first sight. In Paris, I could not get enough of everything French, including all of the French mannerisms, which I studied and copied, incorporating them into my persona. L’Institut de Cancerologie et d’Immunogenetique in Villejuif, France, is situated on a bluff just outside of the southern Paris city limits, and with a view all the way to the Sacre Coeur, north of Paris, I felt as if I was home at last. At the Institut, I began work trying to develop the assay for killer T cells, so that I could study the mechanisms whereby the BCG enhanced immune responses against leukemia cells. Also, I made the acquaintance of another American scientist, Torgny Frederickson, who was on sabbatical leave from the University of Connecticut. Torg was an expert in mouse hematopathology, and he was interested in Friend leukemia virus, which causes leukemia of the RBCs.

Torg asked me to help him develop an assay for erythropoietin, which is now more commonly known by its acronym, EPO. EPO is the growth factor that promotes the proliferation of the precursors of Red Blood Cells (RBCs), and is now known to be in the same molecular family as IL2. Together, Torg and I devised an assay based upon my experience of measuring the proliferation of T cells[4]. To obtain EPO-responsive target cells, Torg showed me how to take the livers of 10-14 day old fetuses from pregnant mice. At this stage of the embryo, the liver is comprised of almost all RBC precursors, most of which are EPO responsive.

To develop the assay, we used the methods familiar to me and to most immunologists, of culturing the cells in 96 well plates. We also used the principles of serial dilutions of our EPO samples, which was derived from my experience performing interferon assays in Ross McIntyre's lab at Dartmouth. This allowed us to compare the concentration of EPO in one sample vs. another by determining how much it could be diluted before losing activity. The end point in the assay was the incorporation of a radiolabled precursor to DNA into newly synthesized DNA molecules. As it happened, the assay was very rapid, in that maximal activity occurred after only an overnight culture. Therefore, we had a sensitive, rapid and most important, a quantitative assay for EPO. We used the assay to perform absorption experiments, search for the capability of the fetal liver cells to absorb the EPO activity. The experiments worked and in our first paper we speculated that this absorptive capacity was due to EPO receptors on the cells. In addition, we used the EPO assay to test for the anti-proliferative activity of interferon, which we reported in a companion paper[5]. The reason to go into this history will become evident as we get into the IL2 assay, later on.

I had fully intended on staying in France for 2-3 years, to really learn my craft. However, Professor Mathe’s BCG therapy was becoming questioned and consequently he was having financial problems. The Institut was without money for several months of my stay. Therefore, I reluctantly returned to the U.S. after only 13 months in Paris. The return was just as memorable as our departure. The SS France docked in New York on August 8,1974, and the headlines screamed “NIXON RESIGNS”.

During my absence, Ross had his grant funded again, so that we went back to our house in Hanover, and resumed our lives as Americans. This time, we came back waving the flag, with a new appreciation of the benefits of being in our own culture again. With Nixon out of office, and with the end of the Vietnam War in sight, I hoped that the craziness would soon be over and that we could all move on to the important things, which for me was getting on with science.

Back at Dartmouth, now on the faculty as an Assistant Professor of Medicine in the Division of Hematology and Oncology, I proceeded to carry on with my studies begun in France. Thus, I continued the EPO experiments that Torg and I developed, and applied for a grant to purify EPO, using our new assay to monitor the purification process. In addition, I continued my experiments to generate leukemia-specific cytotoxic T cells (CTL) using mouse model systems.

Early in 1975 Ross invited Robert Gallo to speak at Dartmouth. Bob had isolated a human leukemic cell line that was producing a retrovirus. After his talk, at dinner I proposed to collaborate, to see if we could identify leukemia virus-specific CTL. Bob was enthusiastic about my ideas and invited me to come down to Bethesda to set up the initial experiments. Therefore, I loaded up my Fiat Spider with all of my crucial gear and reagents and set off for the NIH. I spent 2 months in Bob’s lab and learned a great deal and met many people. However, just before my arrival, there had been a freezer accident and they had lost a crucial cell line that was necessary to make a growth factor that stimulated the growth of the leukemic cells that made the virus. Therefore, I was never able to do the experiments that I had intended.

One of the persons that I met in Bob’s lab was Frank Ruscetti. Frank had been recruited by Gallo to bring expertise to the group in the growth of blood forming cells, which he had perfected during his postdoc at the University of Pittsburgh. Frank and I hit it off right away, and saw eye-to-eye on many things. Having grown up in the first generation of an Italian immigrant family in the South side of Boston, Frank told it like it was and then some. I always describe him as a cross between Leonardo Da Vinci and Rocky Marciano, because he is truly a pure intellectual, probably the most well read scientist I know, and he never pulls punches.

Several months after my return to Dartmouth from Gallo’s lab in early 1976, I got a call from Frank. He told me that in the process of trying to rediscover the leukemia cell growth factor that was lost in the freezer accident, Doris Morgan, one of the other young scientists in the group, found that culture medium from PHA-stimulated lymphocytes contained a growth factor activity that curiously promoted the extended growth of cells that looked like lymphocytes, not leukemic cells, and they wanted to see if they made interferon. This growth factor activity was most probably the same activity that had been described as Blastogenic Factor 10 years previously, although there is no way to know for sure, because the molecule(s) responsible for the activities had not been identified. Anyway, Frank knew that we had an interferon assay up and running at Dartmouth, and they suspected that they were actually growing T cells with the aid of the growth factor activity in the lymphocyte conditioned medium, which they termed LyCM. One of the known characteristics of T cells at that time was the capacity to make interferon. Frank sent me some supernatant samples from the cultured cells that were coded, so that I did not know what they were. The very first experiment revealed that the cells did indeed make interferon activity when activated by PHA.

In other experiments Doris and Frank found that the cells expressed markers that are characteristic of T cells and they showed that the cells needed the LyCM to survive and grow. If the LyCM was replaced by fresh medium, the cells died within 24 hours. All of these experiments indicated that the cells growing were T cells, and not B cells, which were well known to grow if infected by the virus that causes infectious mononucleosis[6]. I was intrigued by these findings, because I was focused on generating CTL that could kill mouse leukemic cells. I proposed some experiments that we could do in collaboration, to see if we could grow mouse CTL using the PHA-stimulated LyCM, which we dubbed “Franky’s Factor”. Frank immediately agreed and we set off on the planned experiments.

Not soon thereafter, I got another call from Frank. Gallo had already made arrangements for his lab to collaborate with Ron Herberman, one of the immunologists at the NCI. Essentially, he told Frank not to collaborate with me, but to cooperate with Herberman’s lab. To Gallo this made sense, in that Herberman had a much larger lab than I had, and he was right there on the NIH campus. Moreover, Gallo told Frank that he did not want him to work on the T cells any more. Rather, he wanted Frank to focus on growing leukemia cells, the task for which he was hired originally. Gallo was not interested in lymphocytes, because he was trying to isolate viruses from leukemia cells that arose from an entirely separate cell type.

Since Gallo did not want Frank to work with me, and Herberman’s group was focusing on trying to grow human T cells, I decided to switch everything to the mouse system, and to try to select for leukemia-reactive T cells. To do so, we tried immunizing T cells with leukemia cells in the culture flask, which we called a mixed tumor-lymphocyte culture (MTLC). A new graduate student, Steve Gillis, had joined my lab a year previously and I also had a new postdoctoral fellow, Paul Baker, who had been referred to me from my friend Torg Frederickson. I also had 4 technicians working with me that I had trained over the preceding 2 years. By this time, I was convinced that growing T cells was very important, so I put everyone in the lab on the task of trying to grow T cells from MTLCs with Franky’s Factor.

As it happened, Steve was the first to successfully grow T cells in the lab. He was successful because, like Doris Morgan, he kept the cells at a very low density. Subsequently, we discovered why this is so important. As the cells grow, they use up the IL2 in the medium and when they are at a high density they use it up very fast. As soon as they run out of IL2 they rapidly die. Anyway, we were very pleased with ourselves, because we found that we could grow Cytotoxic T Lymphocyte Lines (CTLL) for several months and they would retain their capacity to kill the leukemia cells to which they had been selected via the immunization and MTLC.

We submitted our paper to the prestigious British journal Nature. Almost in the return post we had it back, un-reviewed and rejected. I decided that the editors just did not “get it”, because it was so new. Therefore, I sent it right back to them, with an explanatory letter, as to why it was important that they send it out for review. They did review it, and it was accepted, and it was published on Bastille Day, July 14, 1977[7]. Being the Francophile that I am, I always liked the fact that it was published on July 14th.

Although we could grow these T cells in the LyCM, we had no idea whether each batch of LyCM had the same amount of growth factor activity. Therefore, having already created a growth factor assay for EPO, it was straight-forward to adopt the same methods for measuring the T cell Growth Factor (TCGF), a term that we coined to identify the activity, and that we first used in our paper that was published in The Journal of Immunology in 1978[8]. We used the TCGF-dependent CTLL as the target cells and created an assay that was identical to the EPO assay that Torg and I had created in France 3 years earlier. The key characteristic of this assay was the quantification of the TCGF activity. This allowed us to compare one sample with another and to standardize the amount of TCGF that we used to maintain the growth of the CTLLs. Until the TCGF assay, the various cytokines that had been described since 1965 had never been quantified. This ability was critical for our later successful purification of TCGF.

Once we had the CTLLs growing for several months, we next decided to try to clone the cells. The word clone means the asexual progeny of a single cell. As stated by the Nobel Laureate Sir McFarland Burnett in 1959, until we had the capacity to derive clones of cells, we would really never be able to sort out the tremendous complexity and diversity of immune recognition. Paul Baker, the postdoc from Torg’s lab decided to take on the cloning project. There were basically two methods used to clone tumor cell lines, and he tried them both. His very first experiments yielded fruit, and he was able to rapidly perform a series of experiments that essentially proved that we had derived monoclonal cytotoxic T cells. Paul’s paper appeared in the January issue of The Journal of Experimental Medicine in 1979, 20 years after Burnett’s prophecy and only 5 years after I had started my own lab at Dartmouth[9].

THE DISCOVERY OF THE IL2 MOLECULE

Also in 1979 there was a now famous meeting that took place in Switzerland on Lake Constance in a town called Ermatingen. At this meeting it was proposed to create a new nomenclature to describe the various activities that we were all working with. At the time, there was evidence that a mitogenic activity produced by macrophages and termed Lymphocyte activating Factor (LAF) could also make T cells proliferate[10]. However, we had data indicating that LAF functioned to enhance the production of TCGF, which explained its mitogenic activity[11, 12]. Therefore, we changed the name of LAF to interleukin1 (IL1), because it functioned first in the sequence, and TCGF became IL2 because it seemed to come second. The term interleukin was meant to designate that the molecules functioned to carry messages between leukocytes. However, this nomenclature change was really premature, because we still had not identified and purified either the IL1 or IL2 molecules. Actually, we were successful with IL2 several years before IL1 was identified and purified, so that the numbering is backwards: IL2 is actually the first interleukin molecule to be identified, characterized and purified to homogeneity.

Our report on monoclonal T cells created a tremendous stir within the immunologic community. It seemed that everyone wanted to create monoclonal T cell lines. At the 4th International Congress of Immunology, which was held in Paris in 1980, I chaired a workshop on T cell clones. There was standing room only, and the enthusiasm of those who had already made heir own T cell clones was incredible, and matched only by the questions and skepticism of those who had tried, yet had not yet been successful. It was evident that we needed to understand the variables inherent in the generation of T cell clones, because it was difficult, even for us to create new monoclonal T cell lines at the drop of a hat. The most troublesome problem was that we really had no idea about the molecular nature of the IL2 activity that was so critical in initiating and maintaining the growth of the clones, once the initial cultures had been started.

We certainly recognized the importance of monoclonal T cells themselves as reagents that would unlock the mysteries surrounding the nature of T cells. In our first papers we succinctly stated that with these new cellular reagents, the molecular nature of the T cell receptor for antigen, and the molecular mechanisms responsible for T cell cytotoxicity could be unraveled for the first time[9, 13]. However, it was also clear that these cells would permit us to investigate the molecular nature of the IL2 activity, and that we desperately needed to understand this before we could reproducibly generate more T cell clones.

Accordingly, by 1980, I had made the decision to focus the entire efforts of the lab on identifying, characterizing and purifying the molecule or molecules responsible for the IL2 activity. At the time, the field had rapidly become very competitive, and there were at least a half dozen other groups that were trying to do the same thing. There were many unknowns. Although we knew that we needed to stimulate the cells with PHA or similar agents to promote the production of IL2 activity, we still did not know whether T cells were the source, or whether monocytes/macrophages were the source. Removal of each of these cells from a mixed population of cells markedly reduced the yield. Also, others at the time suggested that there were multiple molecules that combined to generate the IL2 activity, based upon the initial biochemical analysis. If this were true, then any attempt to isolate a single molecule capable of mediating the IL2 activity would fail.

Just at the end of 1979, Steve and Paul moved on to their next stations in their research careers, Steve to a postdoc in Seattle, and Paul to a new faculty position in Montana. I was looking for a postdoc with biochemical expertise that could help us to purify the molecule. Rich Robb, who had been trained by Jack Strominger at Harvard, appeared to be ideal, for his doctoral work had focused on the purification of membrane molecules. Therefore, armed with the rapid and quantitative TCGF bioassay, when Rich came, we set out to purify the molecule responsible for the IL2 activity. The monoclonal T cells were critical in this quest, in that they eliminated any idea that more than one cell was necessary to produce the effect, which was a theory forwarded at the time by some of our competitors.

In our initial experiments it became evident that it would require large amounts of starting material, if we were to successfully isolate enough molecules so that we could characterize them biochemically. For guidelines, we had the experience of the biochemists who had been isolating enzymes for the past 20-30 years. They routinely would start with kilograms of tissue that they obtained from the slaughterhouses. Obviously, we could not do that, in that I decided to focus on isolating human IL2, for I felt that human material would be infinitely more valuable than animal material. Consequently, I decided to try to use human tonsils as a source of cells from which we could make liters of PHA conditioned media. From a single tonsil obtained at surgery, we could derive a billion cells, from which we could make a liter of LyCM. I hired a high school boy to come in after school and mince up the tonsils that we obtained every morning, and we then stockpiled as many liters of IL2-containing media that we could.

Another key player in the story was a technician, Maggy Favata, who was instrumental in performing the IL2 assays and generating the LyCM along with myself. It was therefore a small team that worked feverishly to produce the starting material that Rich meticulously tried to purify, using the methods and expertise that he had perfected in his doctoral training. Over the course of the next year, we had generated data that allowed us to publish a paper in Molecular Immunology in 1981 that showed that a single molecule and not a mixture of molecules was responsible for the IL2 activity detectable by our assay[14]. Thus, all of the heterogeneity that others had detected could be ascribed to variable amounts of sugar moieties attached to a single protein molecule. This paper was really the first identification of IL2, as we know it today. Simultaneously, we labeled IL2 with radioisotopes, then Rich purified the radiolabled molecules and demonstrated that all of the labeled molecules were identical to the unlabeled molecules we had purified, and resulted in a protein of uniform size of 15,500 Daltons, with a characteristic single charge. Finally, these purified, radiolabled molecules would bind to IL2-responsive cells and stimulate their proliferation. This was the first demonstration of the IL2 receptor, which was also published in 1981, in The Journal of experimental Medicine, but this is the subject of another essay[15].

Although we had identified and characterized the IL2 molecule by these analytical biochemical experiments, we had only miniscule amounts of the molecule, so that we could not go to the next steps in its characterization, and we therefore still could not prove that we had purified the molecule. We needed to generate even larger amounts of starting material, so that we could end up with enough purified IL2 protein that we could use to demonstrate with chemical tests that we had isolated a single protein with all of the IL2 activity. At this time, Steve Gillis in Seattle discovered a T cell leukemia cell line that produced ~ 100-fold greater quantities of IL2 than we could derive from human tonsils. Obviously this meant that 1 L of conditioned media from this cell line equaled 100 L of tonsil media, and allowed us to scale up the amount of purified IL2 protein that we could make.

Maggie and I then went into the IL2 production mode, and Rich purified as much IL2 as he could. Eventually, we produced enough purified material that we could immunize mice with human IL2, to try to make a monoclonal antibody reactive to the molecule, so that we could use it to streamline the purification process. Over the course of several months in 1981 we tried several times to make the antibodies, but all attempts failed. No one had reported the production of monoclonal antibodies to a cytokine before, so that we did not really know whether it was possible at all. However, using our IL2 assay, we could detect antibody activity in the serum of immunized mice, so that I felt that it should be possible if we just kept trying.

In the fall of 1981, I went to spend several months at the Basel Institute of Immunology. Just before I left, I instructed Maggie to keep trying to make the antibodies, and within a few weeks, Maggie phoned the lab in Basel, announcing that they had identified hybridomas that produced anti-IL2 activity. These antibodies turned out to be the first IL2-reactive monoclonal antibodies, and they functioned to bind IL2 molecules tightly enough that they could be used to purify IL2 in one step.

Over the course of most of 1982, Maggie and I produced and stockpiled LyCm, and purified the IL2 molecule using small (1ml) antibody affinity columns. These columns were a significant step up from the 100ml gel filtration columns that Rich had been using. We were able to purify milligram amounts of IL2 molecules and to demonstrate that the material was homogeneous, uncontaminated by any other proteins. We reported these results in a paper that appeared in 1983 in The Journal of Immunolgy[16].

The characteristics of the IL2 molecule that we uncovered in 1983 subsequently proved to serve as the prototype of a whole new class of immunological molecules that are now recognized as the “hormones of the immune system”. These molecules, which now number 29, serve to communicate between the cells of the immune system, directing their proliferation, differentiation and migration. Because these molecules come into play after the invasion of foreign microbes, they serve to convert the recognition of this invasion by the external environment to control by an internal mechanism that in itself has all of the characteristics of the classical hormones of the endocrine system.

CONCLUSION

From the first description of “Blastogenic Factor” in 1965, to the generation of milligram amounts of pure IL2 molecules in 1983, many investigators contributed to this process of discovery. There were both synthetic and analytical discoveries along the way, and I cannot begin to rank their importance in the final outcome. However, with these discoveries, immunology entered a new era, one that could move beyond the theories and whole animal experiments of the 1950s and the cellular experiments of the 60s and 70s. The era of molecular immunology, which evolved after 1980, has made possible for the first time rational therapeutic approaches to the enhancement and suppression of the immune system. Consequently, a new subspecialty in medicine, that of Medical Immunology, promises to finally fulfill the aspirations of the many immunologists who are trying to harness and manipulate the immune system. Discovery in science proceeds in fits and starts, but always eventually progresses.

REFERENCES

1. Nowell PC: Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Research 1960, 20:462-468.

2. Kasakura S, Lowenstein L: A factor stimulating DNA synthesis derived from the medium of leukocyte cultures. Nature 1965, 208:794-795.

3. Gordon J, MacLean LD: A lymphocyte-stimulating factor produced in vitro. Nature 1965, 208:795-796.

4. Fredrickson TN, Smith KA, Cornell CJ, Jasmin C, McIntyre OR: The interaction of erythropoietin with fetal liver cells I. Measurement of proliferation by tritiated thymidine incorporation. Experimental Hematology 1977, 5:254-265.

5. Smith KA, Fredrickson TN, Mobraaten LW, DeMaeyer E: The interaction of erythropoietin with fetal liver cells II. Inhibition of the erythropoietin effect by interferon. Experimental Hematology 1977, 5:333-340.

6. Morgan DA, Ruscetti FW, Gallo R: Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 1976, 193:1007-1008.

7. Gillis S, Smith KA: Long term culture of tumour-specific cytotoxic T cells [letter]. Nature 1977, 268:154-156.

8. Gillis S, Ferm MM, Ou W, Smith KA: T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol 1978, 120:2027-2032.

9. Baker PE, Gillis S, Smith KA: Monoclonal cytolytic T-cell lines. J Exp Med 1979, 149:273-278.

10. Gery I, Gershon RK, Waksman B: Potentiation of the T-lymphocyte response to mitogens. J Exp Med 1972, 136:128-142.

11. Smith KA, Lachman LB, Oppenheim JJ, Favata MF: The functional relationship of the interleukins. J Exp Med 1980, 151:1551-1556.

12. Smith KA, Gilbride KJ, Favata MF: Lymphocyte activating factor promotes T-cell growth factor production by cloned murine lymphoma cells. Nature 1980, 287:853-855.

13. Gillis S, Baker PE, Ruscetti FW, Smith KA: Long-term culture of human antigen-specific cytotoxic T cell lines. Journal of Experimental Medicine 1978, 148:1093-1098.

14. Robb RJ, Smith KA: Heterogeneity of human T-cell growth factor(s) due to variable glycosylation. Mol Immunol 1981, 18:1087-1094.

15. Robb RJ, Munck A, Smith KA: T cell growth factor receptors. Quantitation, specificity, and biological relevance. J Exp Med 1981, 154:1455-1474.

16. Smith KA, Favata MF, Oroszlan S: Production and characterization of monoclonal antibodies to human interleukin 2: strategy and tactics. J Immunol 1983, 131:1808-1815.