The Compatibility Gene, page 9
With the benefit of hindsight, knowing that transplantation can work and that genetic matching is so important, it is fascinating that, in fact, the discovery of HLA was not a watershed moment for most scientists at the time. The pioneers – Dausset, van Rood, Payne and the teams who swapped cells and ideas in the series of meetings which began at Duke University in 1964 – had the field to themselves for many years before others recognized its importance.
The reason it took a while for others to catch on was that enormous variation in human compatibility genes didn’t immediately provide a new concept about the nature of humanity. Without a full knowledge of what these do, it took a leap of faith to think they were important. Quite simply, nobody knew at first why these genes were so different between people. Everyone agreed that these genes couldn’t exist just to make transplantation difficult. So what is the true role of our compatibility genes? The answer required a different era of biological science, in which molecular and cellular details drive progress. And only then, through the work of a different group of scientists digging deep into how our immune system works, do we finally gain a clear picture of what compatibility genes really do.
4
A Crystal-clear Answer at Last
On 15 November 2001, the American microbiologist Don Wiley disappeared into thin air. His car was found near Memphis, on the Hernando De Soto Bridge, which spans the Mississippi river, unlocked, with its keys still in the ignition. For weeks, the Memphis police scoured the area and found nothing. It was only two months after the 9/11 attacks, and the FBI was called in to investigate, because Wiley was one of the world’s leading authorities on dangerous viruses – he was familiar with HIV, Ebola, smallpox, herpes and influenza – and his disappearance was very strange. The fact that his wife and kids had just arrived in Memphis for a holiday they had all looked forward to hinted that it wasn’t suicide. And, unlike many bridge jumpers, Wiley hadn’t taken his shoes off first.
He had last been seen earlier that night, at a banquet for the Scientific Advisory Board of St Jude Children’s Hospital in Memphis. Peter Doherty, the renowned Australian immunologist and Nobel laureate, recalled that Wiley had been in good spirits.1 The chairwoman of the board, Patricia Donahoe from Massachusetts General Hospital in Boston, told the New York Times that she ‘certainly saw no signs of depression’ on Wiley’s part, adding that she was ‘very suspicious that there was some form of accident or foul play’.2 Harvard and St Jude Hospital offered a $10,000 reward for information as to what had happened.3
Wiley was a candidate for a Nobel Prize for his work with Jack Strominger and Pamela Bjorkman at Harvard during the 1980s.4 Like the trinity of Billingham, Brent and Medawar before them, this trio changed for ever how we think about the immune system. Bjorkman, Strominger and Wiley worked together for one thing: to get a picture of the HLA protein made by compatibility genes, to see what it really looked like. In 1987, after eight years of research, they obtained a portrait of HLA-A*02, the same HLA that Dausset had discovered and named MAC in 1958. He didn’t know how it worked, but Bjorkman, Strominger and Wiley’s image vividly revealed precisely how HLA works. It became an icon, instantly recognizable to any medical scientist or student in immunology.
Why is a picture of a protein so important? A gene is essentially an instruction that cells can use to make a particular protein; and proteins are long chains of atoms: mostly carbon, mixed with a few other elements like nitrogen, hydrogen and oxygen. These atoms connect together in a string but, crucially, for each individual protein the string folds up into a particular complex structure. We are interested in this structure, because that often explains what that particular protein does, and how it does it. An analogy is in how structural beams of a bridge are arranged to support a platform going from one side to another; simply the look of a bridge makes it clear what it does.
As the famous physicist Richard Feynman once quipped: ‘It is very easy to answer many of these fundamental biological questions; you just look at the thing.’5 The double-helix shape of DNA is a prototypical example of how the structures of biological molecules reveal how they work. The model of DNA built by Watson and Crick, in February 1953, showed that the components of DNA (the bases) linked in pairs across two helical strands. This meant that, if the strands separate, each can enable the addition of a new second strand, thereby making two copies of the double helix with the same sequence of bases. The celebrated structure of DNA is not ornament: it shows how DNA is able to copy itself, something central to our molecular understanding of heredity.6 For understanding our immune system, the shape of the HLA protein – hard won by Bjorkman, Strominger and Wiley – was as revelatory as the DNA double helix.
At Harvard University in 1978, Pamela Bjorkman began her graduate studies aged twenty-two. Her parents, surprised at her having such a strong interest in science, thought it would be good for her to go to college so that she could meet a nice man there. Bjorkman herself said she felt like Harvard must have accepted her through some kind of clerical error, and her insecurity made her determined to prove her worth. Her destiny began to take shape when she heard Wiley give an inspiring talk at the department’s autumn retreat at Woods Hole in Cape Cod.7 Woods Hole, on the sea, worlds apart from the hubbub of Harvard, is a place where aspirations come easy. Wiley spoke about the structure of proteins, and his infectious, childlike enthusiasm hooked Bjorkman.
Stylish, tall and often dressed head-to-toe in black, Wiley was a rising star. After his PhD with the Nobel-Prize-winning chemist William Lipscomb, Wiley skipped several rungs of the usual career ladder, missing out years of lab work under somebody else’s wing, to join Harvard’s Department of Biochemistry and Molecular Biology in 1971. Despite this success, Wiley felt lost starting his own research lab so young – so much so that his motivation waned, and he thought of leaving science.8 It wasn’t that he lacked ideas; but the difficulty came from his burning desire to only work on something really important. Finding something he thought worthy enough to work on was the difficult part.
After three years at Harvard, in 1974, Wiley came up for promotion from an assistant to an associate professor, and Harvard hesitated, deciding to delay its decision for another two years in order to see whether or not his research programme was going to take off.9 Eventually, Wiley found a worthy project: to determine the structure of influenza hemagglutinin, a protein that coats the ’flu virus and helps it stick to human cells. The outcome sealed his reputation for brilliance, as he discovered that the protein dramatically changes its shape to force an entrance for the ’flu virus to get inside our cells.
Wiley suggested to Bjorkman that she could continue with research on the influenza protein, but Bjorkman’s friend Jim Kaufman – today a renowned scientist in Cambridge University but then working nearby in Jack Strominger’s lab – told her that he couldn’t think of anything more boring to work on, because the big discovery for that protein had already been made.10 He suggested that instead she could try to get a structure of the HLA protein,11 and so, in June 1979, Bjorkman began her work with Wiley and Strominger on HLA.
Strominger, by then aged fifty-four, had begun his career as an undergraduate at Harvard. He had been surprised at being accepted, because there were quotas in place to limit the number of Jewish students admitted. He majored in Psychology, which he often said later was great training for running a research team. He went to medical school at Yale and witnessed there, in 1946, the first patients in the US to be treated with penicillin, something that made a deep impression on him.12 During the early 1950s, he had a brush with McCarthyism – because he owned books by Marx and Lenin that were actually reading material for a political science course he took while in medical school – before he was cleared and went on to work in the National Institutes of Health (NIH), which was small at that time. Like Wiley, Strominger demonstrated a precocious ability and, aged just twenty-six, he was given money and space to start his own research programme.
Strominger left the NIH because McCarthyism had left a bad taste in his mouth for government service. After briefly working in Europe, he went to Washington University in St Louis, Missouri, where he then made seminal discoveries about how penicillin works. Strominger, among others, successfully identified the way that bacteria made their cell walls, and as the process was unravelled, each step was tested for its sensitivity to penicillin. The very last step in the process was the key – penicillin works by interfering with bacterial protein needed to strengthen the cell wall, the final step in its construction. As a result of this discovery, Strominger was hot property. He moved to the University of Wisconsin, Madison, in 1964 and was then head-hunted back to Harvard, joining its faculty in 1968, the same year as Barnard’s successful heart transplant, and, crucially, set up his lab in the same building as Wiley.
Arriving back at Harvard, Strominger, who had just turned forty, was ambitious to take on a big new research problem.13 With his background in the chemistry of bacteria, he hadn’t yet read anything by Burnet or Medawar, but it was a time when transplantation biology was in the air.14 Everyone knew it was an important research field with many unsolved problems. Strominger needed a way into it, and his chance came in a flash of inspiration while he was at a meeting in Paris.15
One of the speakers, Allan Davies, from the Microbial Research Establishment at Porton, UK, gave a talk suggesting that differences in sugar molecules may underlie HLA compatibility – just like the basic blood groups that Landsteiner had discovered, but more complex. Strominger was listening, enthralled. For years, he’d worked on unusual sugars found in bacterial cell walls, so – he reasoned – he could apply his knowledge of sugar chemistry to attack the transplantation problem. HLA would be his new research priority.
Over the following few years, through the mid-to-late 1960s, Strominger found out that, in fact, Davies was wrong: sugars have nothing to do with our differences in HLA. But by then there was no turning back; his lab was focused on HLA, and his work on penicillin had wound down. During the decade before Bjorkman started working on HLA in 1979, Strominger’s lab had purified the HLA protein, figured out its composition, identified which parts varied and isolated the DNA that encoded it. When they met in the stairwell of the Fairchild Building, where they worked, Wiley and Strominger discussed working together to get the structure of the HLA protein; a researcher in Strominger’s team, Peter Parham, had worked at the project for a while.16 But it wasn’t until 1979 – with the trio of Bjorkman, Strominger and Wiley assembled – that work on the problem started in earnest.
Wiley actually thought at first that the project probably wouldn’t work.17 The problem was, as Wiley knew, HLA proteins have sugars (formally called carbohydrates) attached. Although the sugars aren’t anything to do with the enormous variation in HLA that humans have, as Strominger had found out, sugars can vary somewhat in their composition. Wiley thought the presence of a sugar attached to HLA would create problems for them trying to identify the specific structure of an HLA protein – indeed, most protein structures solved at that time were ones that didn’t have carbohydrates associated with them. So Wiley told Bjorkman that she could start the project with the proviso that, if it didn’t look hopeful after a year, she should give up and do something else. Bjorkman accepted the condition and, as a joint graduate student between Wiley and Strominger, set out to find the structure of HLA protein.
Proteins are generally around 10 nanometres long – a million lined up would reach a centimetre – and each contains about 20,000 atoms. Since the 1950s, scientists have used X-ray crystallography to pinpoint the position of these atoms and reveal a protein’s structure. The process requires first growing a crystal of pure protein. A beam of X-rays is fired at the crystal and then, by detecting the positions at which X-rays come out after passing through the crystal, the shape of the protein can be calculated.
The process to first get crystals of the protein involves adding the HLA protein to liquids containing varying concentrations of salt and other components in the hope that crystals form. It was mostly luck as to when Bjorkman would hit upon the right combination for crystals to form. She also needed to have the HLA protein in enough quantity for screening all the conditions in which crystals might grow.
Strominger’s lab had already worked out a way to obtain reasonable amounts of the HLA protein.18 For this, they used cells derived from a person living in an Indiana Amish community who had inherited the same A and B class I HLA proteins from each parent – so, unlike the usual level of variability, they just had one type of HLA-A and -B.19 Bjorkman could cut the HLA proteins off from the surface of these cells (by adding an enzyme called papain) and then just had to separate the HLA-A from the -B protein, which was relatively easy (using a standard procedure called ion exchange chromatography). With this, Bjorkman obtained a pure HLA-A protein, which happened to be HLA-A*02 in this particular person. To everyone’s surprise, the first time she tried to make crystals, it worked. These first crystals weren’t good enough to obtain a structure from – the crystal needs to be of a certain size and quality for X-ray analysis – but nevertheless, it was a start. Then her luck ran out – she couldn’t get the right quality of crystals to grow.
For seven years, Bjorkman was in the lab each day from around 10 a.m. right through until the early hours of the next morning.20 Harvard had an atmosphere that made scientists feel like they wouldn’t make it unless they gave it their all. But she was giving it her all, and still the crystals were too small and too rare. She began to accept that it might never work.21 Persevering, she had the ‘romantic and stupid idea that I’ll either do this or I’ll fail’.22 She had managed to fathom some details about the symmetry of the protein, and how its constituent parts were broadly arranged. To the outside world this wasn’t much, but when a big discovery doesn’t happen, you take refuge in the small ones.
It at least helped Bjorkman that there wasn’t such intense competition as there is in this kind of science today. The small, self-contained community of researchers in protein structures knew that Wiley was working on the structure of HLA protein and so, by and large, others left the problem alone. The pressure on Bjorkman was largely of her own volition.
Bjorkman switched from using X-ray sources at Harvard to more powerful beams that had become available in a type of particle accelerator being built around the world at that time. Each trip – often to one in Cornell near New York and later to another in Hamburg, Germany – was intense, because her experiments would take second place to the high-energy physicists’ research, and from one moment to the next she could never tell when the X-ray beam for her experiment would be switched on or off. On one occasion, she spent five days waiting for a beam to come on for her experiment, being told the whole time it would be on again in about an hour. On another trip, the X-ray camera was broken and – since each crystal could only be exposed to the beam once – she lost a whole year’s worth of samples when the camera didn’t record the X-ray pattern.
There was no eureka moment, but, by combining snippets of data from many experiments, Bjorkman squeezed out some success and she solved 90 per cent of the structure. But the final 10 per cent was strange and elusive. A monumental discovery was concealed in this enigmatic 10 per cent of protein that Bjorkman couldn’t fathom – but the secret would stay hidden for another year. To understand what happened next, we need to back up a bit in time, because a lot had occurred since the pioneers in HLA – Dausset, van Rood and Payne – had begun the quest that Bjorkman, Strominger and Wiley were about to shed light on.
In the mid-1970s, Rolf Zinkernagel, a medical doctor from Basel, Switzerland, and the Australian Peter Doherty carried out a series of experiments which revealed the real biological importance of the HLA proteins. Working together in the Australian city of Canberra, as part of Burnet’s ‘school of immunology’, the pair’s research would later be ubiquitously celebrated, detailed in every immunology textbook and would earn them a Nobel Prize.
Doherty had a PhD in Neuropathology from Edinburgh University and had been working in Canberra since the end of 1971 – well over a year before Rolf Zinkernagel arrived in January 1973. Zinkernagel, on the other hand, had struggled to get a job and only landed a place in Canberra through personal discussions he had with scientists visiting the institute where he worked in Europe.23 He moved to Australia with his wife and two small children – then aged two and a half years and eleven months – only to find out that there was little room in the crowded lab where he was due to work. He was directed to share workspace with Doherty, a small decision that turned out to have huge ramifications.
Zinkernagel and Doherty worked together for the next two and a half years, and their names are forever linked by this relatively brief moment of spectacular achievement. In hindsight, Doherty considers it a big advantage to them that Australia was still somewhat remote from the scientific mainstream – despite the legacy of Burnet and others at the Hall Institute – giving them the space to think differently. At the time, although HLA proteins were important for their role in transplantation, nobody had any idea what their real function could be in the body. There was evidence available to indicate that immune responses were influenced by specific genes, but research had focused on immune responses to chemically synthesized molecules.24 Zinkernagel and Doherty, interested in these studies, thought it important to study an immune response against a real danger, like a virus.
