Sixteen Years
In 1998, a hospital in Glasgow performed two kidney transplants, one in a man and one in a woman. These are complex operations followed by demanding treatment, but fortunately both kidneys took and functioned perfectly normally. A year and a half later, however, a routine examination revealed a small tumor in the woman’s breast. A few weeks later, several more small tumors appeared, this time under her skin. Tests showed that they were melanoma metastases. In March 2000, the patient died. Two months after her death, a tumor was found in the transplanted kidney of the man who had received his transplant at the same time. Once again, a biopsy showed melanoma.
The Scottish donor registry connected the two stories. Both kidneys had come from the same woman, who had died in May 1998, apparently from a hemorrhage beneath the membranes surrounding the brain. And here was the strange part: she had had melanoma sixteen years before her death. She had undergone surgery and treatment, then lived for fifteen years without signs of disease, attending follow-up appointments at a local clinic. Eventually, she was discharged as apparently healthy. A year after that discharge, she died, and the story of the transplanted kidneys began.
Sixteen years. Could melanoma cells really have survived somewhere in her body all that time, then traveled into the recipients with the transplanted kidneys? Where had those cells been hiding? What had kept them in check? And why did they wake up in someone else’s body?
At about the same time, across the Atlantic in St. Louis, a young researcher named Vijay Shankaran and his colleagues were investigating this question experimentally. They compared tumor development in mice with intact and impaired immune defenses. The immune defect arose because the mice lacked RAG2, an important protein needed for T and B lymphocytes to mature. All the mice received the same dose of a carcinogen, but sarcomas developed more often in the group with impaired immunity: 30 of 52 immunodeficient mice developed sarcomas, compared with 11 of 57 ordinary mice. Without mature lymphocytes, tumors appeared more frequently and sooner. The immune system really did influence their fate.
But how does a mobile immune cell recognize that one of the body’s own cells has become dangerous? And where does its order to act come from? To reach that order, we need to go back thirty years, to a cell that did not even have a name yet.
A Cell Without a Job
In 1970, a young physician named Ralph Steinman joined Zanvil Cohn’s laboratory at Rockefeller University. Cohn studied macrophages, large cells of the innate immune system that engulf microbes and cellular debris. Steinman was interested in what happened next: once a macrophage had swallowed something, what happened to the cellular remains and proteins, and how did an encounter with foreign material activate lymphocytes?
When he joined Cohn, macrophages were considered likely intermediaries between an antigen and a lymphocyte. One idea at the time was that a macrophage captured a foreign protein, preserved it, and presented it to lymphocytes for recognition.
For two years, Steinman watched macrophages and ran all kinds of experiments. He wanted to find a foreign protein on a macrophage’s surface. But the results did not fit the original idea comfortably. The protein could indeed be detected inside the cell, but once inside, it ended up in vesicles called lysosomes, where it was broken down. Macrophages readily engulfed the material under study, yet the expected transfer of an intact protein to lymphocytes could not be seen. The authors described this difficulty in the paper’s conclusion.
Steinman began looking for another cell and turned to the spleen. Earlier work had reproduced immune responses in cultures of spleen cells, but purified lymphocytes needed help from additional cells that adhered to their surfaces. In 1972, in this mixture, he noticed unusual, mobile cells with branching extensions.
These strange cells stretched out thin processes, drew them back, and changed shape again. According to a biographical account, the first observation took place in 1972. Steinman called Cohn over to his microscope. Cohn’s wife, Fern, later recalled her husband coming home with news of Ralph’s important discovery.
A year later, Steinman and Cohn published their description of the new cell type. Because of the branching processes, they called the cells dendritic, from the Greek word for tree.
Colleagues doubted that such a rare cell could play a decisive role. But in 1978, Steinman and his colleague Margit Witmer showed that dendritic cells were at least a hundred times better than other spleen cells at initiating a lymphocyte response.
By then, Steinman had a student of his own: Michel Nussenzweig.
Passing On the Find
A hundredfold advantage in a test tube did not yet establish a leading role in a living spleen. Dendritic cells made up only a small fraction of its cells, and any result could be attributed to their neighbors. To find out who was actually doing the work, the researchers had to remove dendritic cells, and only dendritic cells, from the mixture. The laboratory produced an antibody called 33D1 that bound specifically to dendritic cells. Together with complement, blood proteins that puncture the membranes of antibody-coated cells, it killed dendritic cells right in the test tube while leaving the others intact. The treated spleen cells were mixed with lymphocytes from a genetically different mouse. In such a mixture, lymphocytes respond to foreign tissue markers, much as they do during transplant rejection. In the 1983 study, removing dendritic cells reduced this reaction by 75–90 percent. Adding purified dendritic cells restored it, and an addition amounting to just half a percent of the stimulating cells was enough.
Gradually, a picture emerged of how the body assembles its immune defenses. Among the many T lymphocytes, only a small fraction can attack other cells. They must receive a signal, multiply, and develop into cells that can reach the relevant tissue. A dendritic cell connects these events: it collects material, presents fragments of it to a lymphocyte, and, given the appropriate signals, initiates a response.
The Display Window
But what exactly does a dendritic cell show a lymphocyte? A T lymphocyte cannot look inside another cell. It sees only what is displayed on the surface, where molecules called MHC play a central role. In humans, these molecules are called HLA. How this encounter worked remained a mystery for a long time.
The answer came from crystallography. At Harvard, in Don Wiley’s laboratory, Pamela Bjorkman and her colleagues spent several years trying to obtain crystals of the HLA-A2 molecule. In 1987, they published its structure. The top of the molecule turned out to be a groove, with two long helices along the edges of a flat floor. And inside that groove was something nobody had put there: on the electron density map, the cleft was occupied by foreign material that the molecule had picked up while it was still in the cell. Later, this material was identified as short protein fragments: peptides.
The display window had come into view. A cell continually cuts up some of its proteins into peptides and displays them outside, in the grooves of MHC class I molecules. A T lymphocyte checks what is on display without looking inside. If a viral or altered protein appears in the cell, a fragment of it can appear in the window.
A dendritic cell can do more: on MHC class I, it can display material captured and processed from another cell, as well as material from inside itself. This pathway was named cross-presentation. It allows killer T lymphocytes to be prepared for an encounter with a tumor before they reach it themselves.
How important this was for tumor defense was tested in 2008. Kai Hildner and his colleagues used mice lacking the Batf3 gene. These animals had almost none of one branch of dendritic cells specialized for cross-presentation. A fibrosarcoma that ordinary mice rejected grew in these animals; at the same time, the initiation of specific cytotoxic T-cell responses was impaired. In this transplantation model, the defenses failed to assemble without the necessary intermediaries.
From a strange cell under a microscope came an explanation of how many cells begin to act together: one finds a clue, another receives an order, and their descendants go on the attack.
But remember that the tumor arose from the body’s own cells. What happens if the body decides to attack itself? When that happens, a particular group of diseases develops: autoimmune diseases. But that is not our case. We know that something has changed inside the malignant cell. What on its display window might reveal that change?
A Clue from the Body Itself
In the late 1970s, while Varmus and Bishop in San Francisco were discovering that the viral oncogene src was present in the normal cells of birds and mammals, Lloyd Old’s group in New York, on the other side of the country, was studying features on the outside of tumor cells. They were looking for molecules on human malignant cells that the patient’s own immune system could recognize. The method was called autologous typing. Researchers took tumor cells and lymphocytes from the same patient and watched to see whether one would attack the other. The patient’s own cells against the patient’s own cells, with no foreign tissue and no mice.
A young German physician named Alexander Knuth took part in this work. Among the thirteen melanoma patients the group studied, the strongest reaction came from a man identified by the initials A.V. His T lymphocytes killed cells from his own tumor, grown in a culture called SK-MEL-29. In 1981, Knuth returned to Mainz and continued the same work. There, a second such patient was found, a woman identified in the scientific papers as MZ-2. She had stage IV melanoma, with metastases in the kidney, ovaries, lymph nodes, and spleen. Repeated operations and chemotherapy had not produced complete remission. But her T lymphocytes, like A.V.’s, recognized her tumor cells. Remember both patients: in each, the immune system had found its own clue in the tumor.
They recognized it, but what exactly were they recognizing? To find out, in 1984 Alexander Knuth followed the advice of his former mentor Lloyd Old and traveled to Brussels to meet another scientist, Thierry Boon. Boon’s group had developed a method that used activated T cells to find the tumor gene responsible for activating those same lymphocytes.
Boon and Knuth began the search. They took two types of cells from patient MZ-2: killer T cells and melanoma cells. From the killer T cells, they grew clones that were best at recognizing and destroying the melanoma cells. In the second stage, they continued to bring these selected killer T cells into contact with melanoma, but now selected only the rare melanoma cells that escaped their killers by losing the target. There were now three kinds of cells: killer T cells, ordinary melanoma cells, and escape variants. The third stage was the largest. They made a library from the ordinary melanoma: DNA was chopped into large pieces and inserted into hundreds of thousands of plasmids, circular DNA molecules used as carriers. They introduced these plasmids in batches into the escape cells. The idea was this: if introducing a plasmid caused a killer T cell to recognize and destroy the escaping melanoma again, that cosmid must contain the gene that produced the molecule displayed on MHC-I. And they found such a gene.
What would you expect from the gene they had found? After everything we know about mutations in cancer, the answer seems obvious: an altered protein would give the tumor away. In 1991, Pierre van der Bruggen, Alexander Knuth, Thierry Boon, and their colleagues published the gene. They named it MAGE, from melanoma antigen. It was the first gene for a human tumor antigen recognized by the patient’s own T lymphocytes. And the surprising thing was that it was not mutated! It was exactly the same in the patient’s normal cells. Only four years later would the mystery begin to clear: in a normal body, this gene is silent almost everywhere except in immature sperm cells in the testis. In melanoma, it had switched on at the wrong time and in the wrong place. What gave the tumor away was not an altered protein, but one of the body’s own proteins appearing where it should not.
And what about the first patient? Work with his cells continued in Mainz, and in 1995 Thomas Wölfel’s group found what A.V.’s lymphocytes were targeting. This time, it really was a mutation, in the CDK4 gene, whose protein helps a cell move toward copying its DNA. At position 24, cysteine had replaced arginine. The substitution was present in the melanoma and absent from the patient’s lymphocytes. Inside the cell, it interfered with CDK4 binding to the inhibitory protein p16, weakening one of the brakes on cell division. Outside the cell, a fragment of that same altered protein, displayed on HLA-A2, became a target for the patient’s T cells.
A mutation that weakened an internal brake on division simultaneously left a trace for external surveillance. A target arising from a mutation is called a neoantigen. The cell escaped one constraint and became more visible to another.
Two clues, two ways to give yourself away. MZ-2’s melanoma switched on something that should have stayed silent. A.V.’s melanoma broke something that should have worked. Both traces end up in the same display window, and the immune system can read both. If the Glasgow donor’s melanoma had even one such clue, there was something for the guards to see.
We can now assemble the chain of a targeted attack. A dendritic cell acquires tumor material and helps activate the appropriate T lymphocytes. Their descendants arrive in the tissue and inspect peptides on the surfaces of cells. On finding its target, a cytotoxic lymphocyte delivers a lethal strike.
But what if the tumor removes the display window itself: the MHC-I molecules? This is where the story of recognition took an unexpected turn.
A Line in a Dissertation
In the autumn of 1981, Klas Kärre was working on his dissertation at the Karolinska Institute. He was interested in natural killer cells, or NK cells. They could destroy certain tumor cells without prior immunization, but the rule by which they chose their targets remained unclear. What common features linked the very different cells they attacked?
Kärre later recalled struggling with a single sentence. He was trying to describe susceptible cells and noticed that it was much easier to state what resistant cells possessed. The question turned around: what feature allows a cell to avoid attack?
MHC-I already occupied a central place in ideas about recognition. Kärre proposed that its presence might serve as a signal holding an NK cell back from attacking. The disappearance of a familiar feature would then change a cell’s fate. To notice danger, it was not always necessary to find a new foreign molecule. Sometimes, losing a normal feature of the self could be enough.
The idea still had to be made to work in an experiment. A twenty-two-year-old student, Hans-Gustaf Ljunggren, joined Kärre. His summer project was to test whether NK cells could recognize tumor cells that had lost certain molecules belonging to their own organism.
The researchers took a mouse lymphoma resistant to NK cells and obtained variants with sharply reduced amounts of MHC-I. In the first stage of the experiment, the original and altered lymphoma cells multiplied at the same rate. Losing surface molecules did not take away the tumor’s ability to live and divide. In the second stage, after the lymphoma cells were injected into ordinary mice, the variants met different fates: the original lymphoma cells grew into a new tumor, while cells with low MHC-I died and did not establish new lesions. In the third stage, the researchers suppressed NK-cell activity. The previously rejected variants then began forming tumors again.
This principle became known as missing self. Normal MHC-I molecules send an inhibitory signal to an NK cell; as their numbers decline, that inhibition is lost. Today, we know that activating signals, including signs of cellular stress, also matter for NK-cell activation. But the direction of Kärre’s thinking has endured. The immune system can detect a disturbance through the disappearance of something that ought to be present.
The two branches of defense looked at the same surface in different ways. A T lymphocyte read the peptide on display; an NK cell took account of whether normal identification molecules remained. For a tumor lineage, these requirements made uncomfortable neighbors. Hiding from one means of detection could mean becoming more vulnerable to another.
Two Hundred Days
The mechanisms we have found let us picture the destruction of a dangerous clone: recognition, assembly of the response, arrival of lymphocytes, and cell death. But what was happening where no growing tumor could be seen?
The Glasgow donor’s story shows that cells with malignant potential could persist after sixteen years without clinical signs of melanoma. It raises a question: had the immune system been keeping them in check all that time?
Catherine Koebel and her colleagues modeled such a situation. They gave ordinary mice a small dose of the carcinogen 3-methylcholanthrene. Some animals developed growing sarcomas. The others remained under observation. Two hundred days passed, more than half a year. Some mice still had no detectable tumor; in others, a small, stable mass remained at the injection site.
On day 200, the researchers began giving one group a control immunoglobulin that did not affect immunity, and another group antibodies against lymphocytes. In effect, they switched off several important components of immune control. They did not give the carcinogen again. After this intervention, growing tumors appeared in seven of fifteen mice in the second group, compared with one of thirteen in the control group.
What happened forced a new reading of the preceding two hundred days. At least some of the animals had harbored a population capable of growing all along. Its presence became visible once the defenses were removed.
Cells taken from small, stable masses produced progressive tumors when transferred into immunodeficient mice. The long period of quiet had not made them safe. Within the masses themselves, researchers found fewer signs of cell division, more signs of cell death, and more T lymphocytes than in growing sarcomas. Together, these results supported a picture of dynamic equilibrium: the immune response was holding the tumor population’s growth in check.
After two hundred days of apparent quiet, another outcome of immune defense had emerged: it could contain a dangerous population without destroying it completely. This kind of work is easy to miss if total destruction of the enemy is the only thing that counts as victory.
For an evolving tumor, this meant that acquiring the ability to multiply was not enough. Its descendants also had to survive the pressure exerted by the surrounding organism. The lineage’s fate depended on whether it could produce more living cells than it lost. The immune system was part of that calculation.
Back to Glasgow
Now let us return to the donor. The kidney transplant replayed Koebel’s experiment in a human body, without the design or the controls. To allow the kidneys to take, the recipients were given drugs that suppressed their immune responses. The defenses were removed, much as antibodies had removed them on day 200, and melanoma grew within months.
In the second recipient, immunosuppression was withdrawn and interferon, which itself strengthens the immune response, was prescribed. After thirteen weeks, signs of rejection appeared. The kidney was removed, and extensive masses of dead melanoma cells were found inside it. Two years after the melanoma was detected, the man was alive and had no clinical signs of a tumor. When the guards returned, they began killing again.
A single case cannot prove that the immune system was what held the melanoma in check for sixteen years. But the laboratory findings fit that interpretation. Those sixteen quiet years were not empty. This defense had no witnesses because it was doing its job.
The Student’s Voice
On December 7, 2011, Michel Nussenzweig delivered the Nobel Lecture on dendritic cells. He was the same student who had helped Steinman show that a tiny fraction of spleen cells did the crucial work. Now he was telling the story of the discovery on his teacher’s behalf.
Ralph Steinman himself had died of pancreatic cancer three days before the Nobel Prize was announced. The disease had been diagnosed four years earlier. Steinman was treated with surgery, chemotherapy, and experimental immunotherapy based on dendritic cells that he had developed himself. We cannot say what role each part of the treatment played, but four years was a relatively long time for pancreatic cancer, especially then.
Alongside his story stand two others. Remember the two patients Alexander Knuth and Thierry Boon had worked on? MZ-2 and A.V. lived for more than thirty years without their disease returning. The researchers themselves linked this to the strong and diverse T-cell responses in both patients.
The body has defenses that keep working after the internal brakes of its cells have failed. These defenses receive information about what is inside cells, notice the loss of normal features, assemble a response, and destroy their targets. Sometimes, all their work leaves behind is the absence of a growing tumor.
