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Harden: That is worth knowing.
Gallo: That is the truth.
Harden: We want to ask you a series of questions related to the work on interleukin-2 and the first human retrovirus, but, to begin, we would like to set the larger scientific context. You have been involved with the great leaders of the molecular biology revolution–you mentioned Howard Temin a few minutes ago–and with the discovery of reverse transcriptase and so on. Could you make some general comments about the scientific climate for the rise of molecular biology? This would help set the context for discussing your work within it.
Gallo: Are you asking me how did the developments of molecular biology chronologically and productively affect my own research in cancer or in AIDS?
Harden: Yes, and whose great ideas influenced you the most.
Gallo: I am almost afraid to answer such a question because inevitably I will leave out some of the most important people. I may just block them out and not think of them at the moment. Then later I will say, “My God, I forgot the most important person.” So, starting with apologies for what I am going to forget and for what I will not have time to mention, the impact of molecular biology on our work and on basic immunology as well was, of course, enormous.
There was no impact early on from molecular biology and not very much from immunology on the discovery of interleukin-2. That is a different story. But for the developments in virology, the development of blood tests, the rapid developments in the understanding of the role of HTLV-I in leukemia and the kind of leukemia it causes, and the role of HIV in AIDS, I cannot give enough credit to the advances in molecular biology and basic immunology.
Interleukin-2 was partly an accident. We were looking for a growth factor. We were not looking for a T-cell growth factor; we were looking for one for myeloid cells. That is pertinent, for reasons that are in my book and that I will not repeat here. We were surveying and screening things very widely. So it was an empirical, intensive, old-fashioned Paul Ehrlich kind of research in the sense that we were screening and testing.
You remember that I told you about the PHA-stimulated lymphocytes that were my control for leukemic cells? Well, once upon a time, in 1971, I think, I happened to look into the medium. We used to throw the medium away. But I began to look into the medium for growth factor and I found [Dr.] Leo Sachs's GM-CSF [granulocyte-macrophage colony-stimulating factor]. This important cytokine, or lymphokine, which makes myeloid cells grow and differentiate, is, in fact, in that conditioned medium. That was one of the first discoveries of a lymphokine. That is, although that molecule was known before, it was one of the first discoveries that T cells were making molecules that were irrelevant for T cells. I remember first telling Leo Sachs, who is from the Weitzman Institute in Israel, about it and that it did not make any sense. Why would T cells be involved with neutrophils? There should be a feedback loop from the neutrophil. That was an empirical observation.
When I was looking for a different growth factor in the middle 1970s, I went back to the T-cell conditioned medium, the medium that we PHAstimulated T cells for, and made the discovery of T-cell growth factor, now known as interleukin-2. But that discovery would not have been made without the intensity, and the mothering and nurturing aspect, of a woman scientist, [Dr.] Doris Morgan, who had joined me just a short time before. She was doing the experiment as I had outlined it, but I was looking for myeloid cells. She kept coming to me with these lymphocytes and I said, “They will be found to be Epstein-Barr virus transformed B cells. So what?” It happens occasionally, that adults will have immortalized B cells growing, and it is because of EBV [Epstein-Barr virus] infection. Looking at the cells, you cannot tell a T cell from a B cell.
Now comes the next accident. Somebody else in the laboratory, I think it was [Dr. Robert] Bob Gallagher, who is now a clinician, sent the cells to Building 10. I do not know why he did that, but he did. I think it was [Dr.] Ethan Shevach in Building 10 who did the analysis and found that the cells were EBV-negative, immunoglobulin-negative, and therefore they were not B cells. They were lymphocytes. My God, were they T cells? At that time there were very few assays for T cells. T and B cells had only been distinguished a few years earlier, as I recall. But the cells were positive for something called E-rosetting, and they had some other marker that indicated they were T cells. We realized that we had grown T cells for the first time.
I had been, only a few months before, at a lecture by a clinical immunologist from Yale [University], who was talking about how we should not have blood going from older people into younger people because the T cells would be deficient as T cells cannot grow. I mean, with PHA stimulation, one round and it was over. I was sitting there knowing that I was able to grow T cells long-term, but Doris Morgan had to mother those cells very carefully and kept them for a long period of time. Then we realized that there was an active factor as well, that there was a protein. That is the discovery of IL-2. It did not depend on molecular biology or modern immunology. I do not know what it depended upon, probably a series of chance events, a woman who was willing to stay with cells, and some other fortuitous accidents.
But the discovery of HTLV-I, HTLV-II, and HIV, those are different stories. All of those depended powerfully on the developments in molecular biology and immunology. For example, the molecular biology developments that allowed gene cloning, gene sequencing, and learning the structure of the viral genome, were applied to animal retroviruses, so we knew and had a framework for what the genome of a retrovirus was like. Then we discovered HTLV-I, and that made its understanding, its cloning, and the discovery of new genes that were not known in animal retroviruses readily doable. Finding the sequence was due to using molecular hybridization technology in the tumor cells in a clonal fashion that had been all worked out earlier in animal models–the techniques were there–so we could actually just follow them pretty easily. All that we owed to molecular biology.
Who? The names are obvious. The people who developed gene cloning. There were multiple West Coast scientists, particularly at Stanford [University], and people who did the endonuclease restriction patterns, the people who got the Nobel Prize for it, [Dr.] Ham[ilton] Smith, and so on. I do not know where to stop. Reverse transcriptase. Without that I could not have found the virus. So interleukin-2 plus reverse transcriptase, Temin and [David] Baltimore. Without reverse transcriptase, we could not have done a lot more of the gene cloning from the cDNA. We could not quantitate virus readily without the reverse transcriptase assay. We would not have known the virus was there without it, because it was a new virus and there were no probes for it. That assay was absolutely essential. The most anybody had ever done was to see it for an instant. But how to follow it regularly when it is fast, you could just take small aliquots and know when the virus peak was coming out. The AIDS virus comes out with a burst and a peak, and you would miss it if you did not have reverse transcriptase assays. You cannot do EMs [electron micrographs] every 10 minutes.
The blood test technology? Well, we had to mass produce the virus. That was not something from molecular biology or immunology; that was something from old-fashioned virology, by trial and error. Much of it was developed in Eastern Europe, but there was nothing, as I said, from molecular biology in that.
But, then, in doing the test itself, the ELISA assay [enzyme-linked immunosorbent assay] came out of basic immunology. The Western blot also came out of basic science and we applied it to clinical medicine for the first time. To my knowledge, no one else had done it before, but the test really came out of basic immunology.
I should also have mentioned the people who distinguished T cells from B cells. I forget at the moment who that was. I know it, but I forget.
Harden: There is also the instrumentation that was developed, such as the FACS [fluorescence-activated cell sorter] machine.
Gallo: Yes. I am forgetting the FACS machine, which was obviously helpful in the studies of pathogenesis. Also the people who made the monoclonal antibodies for CD4. Start with that. How did we know to look in CD4 cells if clinicians did not say CD4 went down? How did they know CD4 went down if they did not have an assay for CD4? You do not have an assay unless you have the monoclonals. Who developed those monoclonal antibodies first? There was [Dr. Stuart] Stu Schlossman and the other man with a Chinese name, from the Ortho Company, who did that pioneering work in the mid-1970s, I think, or perhaps the late 1970s. All those monoclonals were critical. Who developed the technique of monoclonal antibodies? You can go back to César Milstein and Georg Kohler. That is the story of science, is it not, scientists standing on the shoulders of earlier scientists.
Rodrigues: To shift gears somewhat, we have been hearing recently about the use of embryonic tissue in research. You touched upon this in your book, saying that you had discussions with Phil Markham in the early 1970s about the ethics of using human embryos in research. Given that there are currently more limitations and restrictions on the use of these types of tissues, if those had been in effect at that time would that have been a stumbling block in your work?
Gallo: Yes. The answer to the question of whether limitations in the use of human embryo tissue would have restricted our research in the early period of time is yes, because the first growth of the myeloid cells–this is not immediately relevant to AIDS–but the first growth of the myeloid or granulocytic cells in our laboratory was based on a growth factor from a first trimester spontaneously aborted human fetus. We do not know the cell that produced it. That is what led me to the discovery of IL-2. We are searching for that factor again, because we could not get more human embryos in time. In fact, we have never found the factor that we were looking for again. We never went back to that question. That is another story. There is no question that we would not have been able to establish the cell line HL-60 with the restrictions now in effect, which would not have led to the retinoic acid story and the treatment we have today. There is a continuum, but it is not relevant to the issue of AIDS right now, nor for my work in AIDS or leukemia viruses, but it would have been relevant for that retinoic acid story.
Harden: Dr. Harvey Klein told us, when we interviewed him, that in your IL-2 work you made use of the buffy coats that had been spun off from donated blood in the Clinical Center. Would you talk about NIH intramural interaction on this work and the subsequent application of it by Dr. Steven Rosenberg?
Gallo: Yes. The question of intramural interactions in the process of discovery and its applications to clinical medicine are nowhere illustrated better, I think, than at NIH, and especially–well, I do not know if it is especially because it is still going on now–but, anyway, in the period I can recall it was most beneficial for my colleagues, myself, and for our work. It was over and over again. But a good example would be in the interleukin-2 story. Yes. Dr. Harvey Klein was correct. The Clinical Center was routinely sending us the buffy coat material that was not being used for anything else–that was the source of white blood cells. We could set up countless columns and PHA-stimulate them to separate lymphocytes from the granulocytes. If we then take those lymphocytes and PHA-stimulate them, we get the conditioned medium, which is where we found the interleukin-2. We had much contact with the Clinical Center in those earlier years and we obtained a large number of specimens from the Clinical Center. This happens, I might add, much less today. The reason is that there is far more competition for a dwindling number of specimens. If you are not right there in the building, it is much more difficult than it was to obtain them.
At that time, there was almost a search by the Clinical Center for a scientist to be interested in those specimens, more so than today when there is much more competition. As I said, even the Ph.D.s have become more interested in clinical medicine. But, in addition to the clinicians providing material and patient information for us, and providing the specimens–not just normal buffy coats, but those from patients that they were caring for–they often gave us insights. For instance, in AIDS, knowledge of the CD4 decline came purely from patients. Then there was Steve Rosenberg and [Dr. Ronald] Ron Herberman taking the interleukin-2 story from us to another level by thinking about it clinically, or in mouse experiments that only a clinical immunologist could dream about at that time. There was also our getting the information from Shevach that the cells we had were not B cells. I did not even know that came from him until I was writing my book. I found that out by talking to people in my laboratory. Nobody had ever told me that that was where the information came from. Quite frankly, once I knew that, I thought he should have been a co-author of the paper. He never made a complaint to me. He probably does not even know. But if he reads my book he will know.
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