Viruses, Pandemics, and Immunity

by Arup K. Chakraborty

  An organ transplanted from one animal to another of the same species is usually destroyed in the host animal, and this was a barrier for organ transplantation in humans. In the 1930s and 1940s, George Snell, an immunologist working in Bar Harbor, Maine, on the isolated and beautiful Mt. Desert Island, was trying to understand whether transplant rejection had a genetic origin. His strategy was to transplant organs from one purebred mouse to another. While organs transplanted between mice of the same strain were not rejected, organs from a different mouse strain were. He then interbred these strains of mice to identify the genes responsible for rejection. These experiments required breeding many generations of mice and took many years to complete. Perhaps the isolation of Bar Harbor and the long winters allowed Snell to have the patience to carry out these experiments. He eventually found that transplanted organs were rejected because of differences in a single group of genes. In humans, these genes are called HLA genes, and they must be compatible to prevent transplant rejection. This is why it is standard procedure now to match the HLA types of organ transplant recipients and donors. It took a while, however, to figure out why differences in these genes mattered.

  The first clue as to the function of these genes was provided by the Australian immunologist Jacques Miller. The thymus is an organ located near the heart, and it was thought to be unimportant because removing the thymus of an adult mouse had no effect. At that time, the only known purpose of the thymus was in the context of sweetbreads, a delicacy in European cuisine that is prepared using the thymus of an animal, usually lamb. Miller found that he could prevent mice from rejecting transplanted skin by removing their thymus at birth. He also found that these mice were more prone to infections. Thus, he deduced that an important immune function must require the thymus, especially early in life.

  We now know that T cells undergo a number of maturation processes in the thymus before they start circulating through our body. The “T” in T cells refers to the thymus being the site where mature T cells are produced. Miller found that T cells kill the cells of an organ transplanted from a mouse with different HLA genes, and this destroys the organ. Mice with their thymus removed at birth were missing T cells, and so transplants were not rejected. Miller’s observation that these mice were also more prone to infections implied that T cells were an important part of our army of immune cells. Interestingly, shortly after puberty, the thymus begins to wither away, and thereafter we live largely with the T cells we have. By the way, this explains why all chefs are taught that sweetbreads are best prepared using the thymus from a young animal and that the adult animal’s thymus is small, fatty, and inedible.

  By the 1970s it was understood that T cells also kill virus-infected cells, but not free virus particles. But how they identify infected cells and thus know what to kill, and whether HLA genes had anything to do with this, was unknown. In a relatively short period of time various studies came together to provide us with our current understanding of how this works.

  Hugh McDevitt and Michael Sela, working at the National Institute of Medical Research in England in the 1960s, were studying immune responses in mice injected with a specific protein. Using some of the mouse strains developed by Snell, they found that the ability to mount a strong or weak immune response to a specific infecting substance depended on a mouse’s HLA genes.

  Expanding on the work of McDevitt and Sela, Rolf Zinkernagel and Peter Doherty working in Australia discovered another link between HLA genes and the T cell response. They were studying the ability of T cells to kill cells infected with a particular virus. They collected T cells that were able to kill virus-infected cells from a mouse. They then tested the ability of these T cells to kill virus-infected cells from mice with different HLA genes. They found that T cells could kill infected cells only when the infected cell came from a mouse that shared the same HLA genes. That is, mice could kill their own infected cells but not those from mice with different HLA genes. This was a huge surprise. The result suggested that to recognize a foreign invader (the virus), the T cells needed to first detect that its own cell, and not that of another with different HLA genes, was infected. But why was this the case?

  It was reasonable to assume that something on the surface of T cells was responsible for detecting infected cells, much as antibody-like B-cell receptors on the surface of B cells bind to the spike proteins of a virus. Soon, James Allison, John Kappler, Philippa Marrack, and Ellis Reinherz presented data from three independent studies that showed that indeed a protein on the surface of T cells was important for detecting infected cells. It was assumed that identification of this T-cell receptor (TCR) would explain why T cells from one mouse could kill virus-infected cells from another mouse only if they shared HLA genes. Because of this scientific importance, it was thought that the discoverer of this protein, and the gene that encoded it, would win a Nobel Prize. A mad race to find it began.

  The race was ultimately won by two groups of scientists who used a novel approach: Mark Davis and Steve Hedrick, working together at the National Institutes of Health, and Tak Mak, at the University of Toronto. Their experiments were cleverly designed to identify the genes that only a T cell possesses. One of these genes would correspond to the T cell receptor. These experiments had to be conducted with precision—Davis has likened them to the precision he had to exhibit as a fencer in college. On a Sunday in 1983, Hedrick was on the way to the zoo with his family and stopped in the lab to check on the experiments. The results he saw made him realize that they had discovered the identity of the TCR. He proceeded to the zoo and a bit later called Davis to tell him that something special had happened. They did not know that, at the same time, Mak had similar results. The two papers describing the TCR were published together in 1984. These results also showed that the TCR gene was different in each T cell, and the reason was that, just as we described for the BCR gene, it was made up of bits of DNA that were randomly shuffled and joined. So, along with our supply of B cells, we also have an equally diverse repertoire of T cells as well.

  Peering at the TCR protein and gene, immunologists were hoping to find the answer to the Zinkernagel–Doherty puzzle. But because the T cell receptor looks very similar to the antibody molecule, no new insights emerged regarding why T cells need to recognize self to recognize foreign. No one has been awarded a Nobel Prize for the discovery of the TCR.

  Other studies provided the answer. While working with Brigitte Askonas at the National Institute of Medical Research in England, Emil Unanue, a Cuban émigré to the United States, made a surprising observation. Adding a drug, chloroquine, to target cells prevented T cells from detecting them. Unanue was subsequently offered a faculty position at Harvard, where he followed up on this observation.

  Our cells have a mechanism to destroy used proteins. Used proteins are first chopped into fragments called peptides. These peptides are then further degraded into amino acids. Unanue’s experiments showed that the peptides created during the processing of used proteins bind to the proteins encoded by the HLA genes. He then found that these HLA-bound peptides are sent to the cell surface, and they are the molecular flags of infection that T cells recognize. It turns out that one of many things chloroquine does is block the ability of cells to break down used proteins. So, cells treated with this drug could not be detected by T cells. When Unanue presented his results for the first time in 1984 at a scientific conference, he was greeted with derision, with one immunologist comparing the ideas to cells displaying their own feces on their surface.

  Shortly after, in the mid-1980s, Pamela Bjorkman, a graduate student working toward her PhD in the laboratory of Donald Wiley at Harvard University, provided further evidence supporting Unanue’s findings. Wiley’s scientific specialty was structural biology. He studied the functions of proteins by coaxing them to form crystals that can then be imaged using X-rays to reveal what they look like. Bjorkman decided that her PhD research would focus on crystallizing and imaging one of the HLA proteins. After challenging effort
s, she finally succeeded. The structure of the HLA molecule that Bjorkman and Wiley reported shows that it has a groove, and that in the groove lies a peptide, exactly as predicted by Unanue.

  These findings, along with a few other facts, provided a solution to the Zinkernagel–Doherty paradox. Each one of us has between 6 and 12 different types of HLA proteins that are displayed on the surface of almost all our cells. These proteins are the most variable in the human population. Only identical twins and some siblings have exactly the same set of HLA proteins. Each HLA protein binds distinct subsets of peptides. To see why it is important to have so many different variants of HLA proteins in the human population, consider the following extreme scenario. If we all had exactly the same HLA protein, and one virus emerged whose protein fragments did not bind to its groove, none of us would be able mount a T-cell response to this microbe. Thus, this virus would pose a threat to the entire human species because none of us would be able to kill cells infected by it. Having many variant HLA proteins in the human population likely evolved as a bet-hedging strategy that prevents such an eventuality.

  One more fact needs to be added to close the loop on understanding how T cells work. The thymus is like a school for baby T cells. Only some of the baby T cells that enter the thymus pass the requisite tests to graduate. T cells are trained to detect their own HLA proteins, and not bind to their own HLA-bound peptides too strongly. Thus, the T cells that exit the thymus have TCRs that bind with modest affinity to peptides derived from one’s own proteins bound to one’s own HLA proteins. T cells that bind too strongly or weakly are eliminated. When a mature T cell binds strongly to an HLA-bound peptide, it infers that this peptide is of foreign origin and not derived from the organism’s own proteins. So, the cell displaying this HLA-bound peptide must be infected and should be killed. In Zinkernagel and Doherty’s experiments, T cells in one mouse could not kill virus-infected cells derived from another mouse because these T cells were not trained to bind to and detect the HLA proteins of the second mouse.

  T cells play a critical role in directing the immune response against viruses. When the TCR on a T cell binds strongly to an HLA-bound viral peptide displayed on a phagocytic cell that has eaten the virus, the T cell gets activated. Activated T cells begin to multiply, and the progeny go into tissues seeking infected cells. In tissues, if a T cell encounters a cell displaying the same HLA-bound viral protein fragment that originally activated it, the T cell can kill the infected cell. After the infection is cleared, some of these T cells live on as memory T cells, which can be quickly reactivated upon reinfection. It turns out that in addition to these killer T cells, there is another subtype of T cells. These T cells have many functions important for combating viral infections. For example, they secrete chemicals called cytokines, which have functions we will discuss in the next section.

  Innate Immunity

  As we recounted, the twentieth century was an age of great discoveries about our adaptive immune system. These discoveries taught us how our immune system does amazing things like mount virus-specific responses and establish memory of past infections that allows it to usually swat away viruses that reinfect us. The phagocytic cells that Metchnikoff had discovered were also found to be important for eating up all types of viruses present in tissues, transporting them to lymph nodes, and displaying HLA-bound viral protein fragments that activate T cells. But as the twentieth century progressed, it became clear that B cells, antibodies, T cells, and phagocytic cells could not be the whole story.

  It takes about 5–10 days for the adaptive immune system to identify the rare B cells and T cells specific for a particular virus and mobilize them for active duty. But early in the infection, the virus multiplies and infects new cells. By the time the adaptive immune system is mobilized to engage the enemy, the virus will likely have already won the battle. As we usually win battles with viruses, something else must be happening during the early stages of infection when time is of the essence. Controlling the infection early requires another part of the immune system that is ready to go right away upon infection, and as we mentioned early on in this chapter, this part of the immune system is called the innate immune system.

  As immunologists started to consider how the immune system reacts immediately after infection with any microbe, Charles Janeway, at Yale, started talking about the immunologists’ “dirty little secret.” What immunologists did not talk about was that if you injected a foreign protein or chemical into an animal, nothing happened. To induce an immune response, they had to mix in mysterious substances like dead bacteria, oils, or some other noxious material. For decades it was also known that for a vaccine to elicit an adaptive immune response that protects a person from a particular infection, something similar had to be added to get the immune system going. Janeway and others reasoned that there must be more to innate immunity than just phagocytosis. They proposed that cells of the innate immune system must have receptors that could recognize some features present in all bacteria, fungi, and viruses. Detection of these features would tell the immune system to mobilize.

  Evidence for such a system of cells came from studies in insects in the 1990s. A French biologist, Jules Hoffmann, was a specialist in studying grasshoppers and fruit flies. These organisms do not have T cells and B cells, and he wondered why they did not die of infections. He repeated Metchnikoff’s experiments and began to think about how cells of the innate immune system are alerted to go to the site of infection. To try and understand this, he mutated flies and looked for ones that died of overwhelming infections because of faulty immune systems. His studies showed that a receptor identified earlier by Nobel Prize–winning scientist Christiane Nüsslein-Volhard, in a different context, was also important for preventing infections. Nüsslein-Volhard studied genes that were important for proper formation of the fly body. Flies with mutations in one of these genes had bizarre and unusual shapes. Seeing these unusual flies for the first time under a microscope, she is said to have exclaimed “Toll!” (“Cool!” in German) and named the gene “Toll.” The idea that a gene implicated in controlling body shape would also function to mediate immunity in flies seemed hard to believe, but Hoffmann and his group proved that Toll did just that.

  Around the same time, Bruce Beutler, working at the University of Texas Southwestern Medical School in Dallas, was studying severe bacterial infections. As a graduate student in New York, he helped to identify a substance called tumor necrosis factor (TNF) that is produced upon overwhelming bacterial infection, resulting in a condition called septic shock. For decades, it was known that a component of bacteria called lipopolysaccharide (LPS) was a powerful stimulator of TNF. When he started his own laboratory in Dallas, Beutler decided to search for the receptor for LPS and discovered that it was a human version of the Toll protein Hoffmann was studying. Beutler and Hoffmann would win Nobel Prizes for their work in 2011.

  Ruslan Medzhitov, at Yale, and others have identified a large family of Toll-like receptors, and how they function, in mice and humans. Each of these receptors binds to different components that are specific to bacteria, fungi, and viruses. These discoveries showed that these receptors not only recognize microbial pathogens but also tell the body which type of microbe has invaded. The receptors that respond directly to bacteria, fungi, and viruses goes far beyond the Toll receptor family, and currently over 50 such receptors have been identified. This innate immune system is present in many species in nature, and functioned to protect living organisms long before vertebrates (like us) acquired an adaptive immune system.

  When innate immune cell receptors bind to a component of a microbe, the cells respond by producing hormones called cytokines (which T cells also produce). Cytokines signal to the body that a dangerous microbe is present and create an environment that is inhospitable for the invader. The effects of cytokines include increasing body temperature, inducing the liver to produce massive amounts of antimicrobial proteins, changing our metabolism so we can survive without much nutrition, a
nd mobilizing immune cells. Fever helps because most microbes do not replicate well at higher temperatures. The antimicrobial proteins from the liver bind to and damage the infectious agent, and the metabolic changes cytokines induce (that make sick people lose appetite) aim to starve microbes. There are also cytokines called interferons that tell cells to turn on genes that specialize in blocking viral replication. It is the cytokines produced by innate immune cells that make you feel sick when a virus infects you.

  The activated innate immune system is a blunt weapon that slows virus growth and spread, which allows us to survive until the adaptive immune system can take over. In some cases, the innate immune system is able to eradicate the invading virus by itself. The activation of innate immune cells is also required to release the functions of the adaptive immune system. This is why immunologists had to add noxious chemicals and bacteria to mimic a real infection when they wanted to elicit a T-cell or B-cell response.

  Over a hundred years ago, the humoralists won the day. Today, we know that Metchnikoff’s phagocytes are central to the immune system. The importance of the cells of the innate immune system for protecting us from infectious diseases is made vivid by the fact that humans who lack an important phagocyte, neutrophils, do not survive beyond a few weeks without major therapeutic interventions. Metchnikoff’s Nobel Prize in 1908 turns out to be prescient and highly deserved.