Immunity unravelled

The Prize for Medicine has gone to three scientists who found out the key mechanisms underlying the activation of the immune system.

TO defend themselves against infection by external microorganisms such as bacteria, viruses, fungi and parasites, living beings possess layers of defence with increasing specificity. The first layer is the physical block such as skin and external secretions that prevents microorganisms from entering the body. If these physical barriers are breached, the body's immune system provides an immediate non-specific innate response. This triggers inflammation one of the initial biological responses to infection or injury that can counter their attack and destroy invading pathogens. The innate immune system is present in all plants and animals. If the innate response is unable to counter the invading organism, the second layer of the immune system, called the adaptive or acquired immune system, takes over. Adaptive immunity produces antibodies that fight the infection and killer cells that destroy infected cells. The adaptive immune system is, however, known to be present only in jawed vertebrates.

Unlike the innate response, where the response is rapid on exposure to the pathogen, adaptive response is slower; there is a time lag between exposure and the maximal response that the system can mount (Figure 1). The second stage of immune response finally leads to the pathogens being cleared from the body. But, more significantly, while innate immunity does not retain any memory of the pathogen, adaptive immunity develops an immunological memory that enables the immune system to respond faster and mount stronger responses when the same pathogen causes a subsequent infection. While these two levels of the immune system provide good protection against infections, if the activation threshold is too low, or if endogenous molecules can activate the system, it may result in inflammatory diseases or autoimmune disorders.

This year's Nobel Prize for Physiology or Medicine has gone to three scientists who unravelled the key mechanisms underlying the activation of the immune system, both innate and adaptive. One half of the prize has gone to Jules Hoffman and Bruce Beutler for their discoveries concerning the activation of innate immunity and the other half to Ralph Steinman for his discovery of the dendritic cell and its role in adaptive immunity. By discovering how the innate and adaptive stages of immune response are triggered, the three Nobel laureates have provided new insights into disease mechanisms. Their work has opened up avenues for the development of preventive vaccines as well as therapy against various infections, cancer, inflammatory diseases and even autoimmune disorders.

Hoffman, 70, served last at the University of Strasbourg, France, between 1974 and 2009. It was here that he obtained his doctorate in 1969 and also did his award-winning research. Beutler, 54, has been at the Scripps Research Institute, La Jolla, U.S., since 2000 though his Nobel work was carried out at the University of Texas in Dallas in the late 1990s. However, Steinman is no longer alive to receive the award. In the 110-year history of the Nobel Prize, this will be the first time that the award is being given posthumously. The prize was announced on October 3 but Steinman had passed away on September 30.

In a press release issued after the announcement of the prize, the Nobel Foundation said that the information on Steinman's death had reached the Nobel Assembly at Karolinska Institutet via the president of the Rockefeller University only at 2-30 p.m. (European Time) on October 3, while the Nobel Assembly had announced the Prize at 11-30 a.m. According to Alexis Steinman, her father had joked only the previous week with his family about hanging on until the prize announcement. Steinman had apparently said: I know I have got to hold out for that. They don't give it to you if you have passed away. I got to hold out for that.

The events that have occurred, the press release stated, are unique and, to the best of our knowledge, are unprecedented in the history of the Nobel Prize. According to the statutes of the Nobel Foundation, work produced by a person since deceased shall not be given an award. However, the statutes specify that if a person has been awarded a prize and has died before receiving it, the prize may be presented. An interpretation of the purpose of this rule leads to the conclusion that Ralph Steinman shall be awarded the 2011 Nobel Prize in Physiology or Medicine. The purpose of the above-mentioned rule is to make it clear that the Nobel Prize shall not deliberately be awarded posthumously. However, the decision to award the Nobel Prize to Ralph Steinman was made in good faith, based on the assumption that the Nobel Laureate was alive. This was true though not at the time of the decision only a day or so previously. The Nobel Foundation thus believes that what has occurred is more reminiscent of the example in the statutes concerning a person who has been named as a Nobel Laureate and has died before the actual Nobel Prize Award Ceremony. Steinman, 68, had been at the Rockefeller University, where he had carried out the path-breaking research, since 1970.

Watchdog cells

The time line of scientific discoveries that has led to our present understanding of the immune system is not in the same order in which the two stages of the immune system work. It was the mechanism involved in the adaptive response that came to be discovered first. In 1973, Steinman, working with Zanvil A. Cohen, discovered a new cell type that he termed dendritic cells (DCs), which he hypothesised to be important in the immune system. DCs are tree-like cells that were first discovered in Germany in 1869 by Paul Langerhans, who thought that they were part of the nervous system. These cells are found at the interfaces between the body and the environment, like the epidermal layer of the skin, and in lymphoid or immune organs. In addition, DCs line the surfaces of the airway and the intestine. There they function as gatekeepers that sample substances from the environment.

Interestingly, in a video interview last year, Steinman had said that he had not been exposed to biology until he passed out from school in Quebec and joined McGill University. There he took a biology course to see what it was all about and then he was smitten. He said in the interview: I wanted to understand how the immune response begins and how this immune system is driven into action. This took him to the Rockefeller University. [The] most distinctive feature of the cells that we had discovered, he added, [is that it] had many processes. They are constantly probing the environment looking for all the challenges that the immune system has to deal with and when they see the challenge they have to take it into the body and teach the immune system what to do. [T]here was one system that people had discovered before us on how immune responses were generated in a test tube. But something was missing for the immune response to work in the test tube. So we looked at the immune cells with the microscope and there we spotted something that nobody had ever seen or taken note of before these strange probing dendritic cells.

By the late 1960s, it was already established that a kind of white blood cells (lymphocytes), the T-cells, and certain accessory cells, whose identity and function were unknown, played a role in cell-mediated immune response in mammals. Steinman and Cohen were studying spleen cells to understand the induction of immune responses in a major lymphoid organ of the mouse when they encountered a population of cells with unusual shapes and movements that had not been seen before. Because they had unusual tree-like or dendritic processes, Steinman named them dendritic cells. Steinman and Cohen then went on to study whether DCs were involved in activating T-cells. They were able to establish that in cell culture experiments, the presence of DCs led to a strong response of T-cells to pathogenic substances. Initially, these results were met with scepticism, but later work by Steinman established that DCs had the unique capacity to activate T-cells. DCs thus constitute a critical, and previously missing, link in the adaptive and innate branches of the immune system.

As the body's watchdogs, DCs seek out foreign invading pathogenic substances such as bacteria, viruses and toxins. One of the functions of DCs is to capture the antigens molecules characteristic of the invading pathogens and convert them to smaller pieces and display the antigenic fragments on their cell surfaces to be recognised by the T-cells to mount an immune response. DCs then travel to immune or lymphoid tissues and seek out regions rich in T-cells. In technical terms, DCs convert the antigens to peptides within the cell's cytoplasm, and these peptides then bind to products of the major histocompatibility complex (MHC) a region of the genome important for immunity and autoimmunity and then exit to the cell surface to present the antigens to the T-cells. This then signals the T-cells to mount an immune response to counter the attack, in particular by enabling white blood cells called B-cells to make antibodies to neutralise the infection and killer T-cells to destroy the infected cells (Figure 2).

But the key question that still remained was how does the system know in the beginning that there is an infection? Clinicians have known for long that patients have different levels of innate immunity. For instance, if patients do not have neutrophils white blood cells that migrate into the bloodstream during the beginning phase of infection they are at grave risk of infection of all kinds. This is nothing but innate immunity. But how is this initial innate immune response triggered? What is the mechanism that alerts us? It is obvious that the immune system has a genetically programmed capacity to recognise certain molecules of microbial origin and trigger some specialised sensor or receptor molecules. But what are these receptors?

Study of fruit flies

In 1996, Hoffman at the University of Strasbourg, and his co-workers, while investigating how Drosophila (fruit flies) fought infections, found that flies that had mutation in the gene called Toll could not mount an effective immune response when challenged with bacterial or fungal infection. Actually, Hoffman and colleagues were studying the general problem of how insects fought infections and had identified several antimicrobial peptides that the system produced to do this. The investigation culminated in identifying the important role played by the Toll gene in the promoter sequence for the antimicrobial peptide genes. The finding indicated that the molecular product of the Toll gene was involved in sensing pathogenic organisms, and Toll activation was necessary to activate the innate immune response.

In 1998, Beutler, then working at the University of Texas, found that certain mutant mice, the model system that he was investigating, were resistant to high doses of a bacterial product called lipopolysaccharide (LPS), which was otherwise known to cause life-threatening septic shock by overstimulating the immune system. Beutler and his associates determined that the mutation was in a gene that was very similar to the Toll gene in the fruit fly. The product of this Toll-like gene, called the Toll-Like receptor (TLR) molecule, turned out to be the receptor that was responsible for binding to LPS (receptor molecules reside on the surface of cells and signal the cells into some specific action.) When the TLR binds to LPS, signals are activated, triggering inflammation (Figure 2). But when the dose is excessive, it causes septic shock. The findings of Hoffman and Beutler showed that mammals and fruit flies make use of similar molecules to activate innate immunity against infections.

Of course, said Beutler in his post-prize announcement interview, we know that some things go very far back and are preserved even to the Cambrian times. And, nonetheless, it still was a surprise to me at the time that everything was so similar in the fly as in the mammal. When we made our discovery, which was a [of] couple years after Jules made his, I had only a very dim awareness of the situation in flies. [He had] showed [that] a fly was overwhelmed with fungal infection if it had mutation in Toll. Because it made perfect sense that in the mouse the same sort of situation applied, and there was overwhelming gram-negative sepsis if you had a mutation in Toll-like receptor 4 [TLR4]. So, I saw right away the parallelism.

But this finding was the culmination of research that had begun in the 1980s when Beutler had identified tumour necrosis factor (TNF) as an inflammatory protein. I realised quite early on, he said in an interview, that TNF was one of the major executors of endotoxic shock. And, it was strongly induced by LPS, and therefore, we always took it as a marker of the LPS response. It was the endpoint that we followed, just as Professor Hoffmann followed the production of antimicrobial peptides. It took a very long time to find the Toll-like receptor for a molecule because we didn't use a genetic approach for quite a while. We used conventional approaches: cross-immunising mutant mice with wild type mice and looking proteomically' to try to find a difference between the two strains. And all of that was fruitless. It took a very long time before we were able to start positionally cloning. (Positional cloning is reverse of the traditional approach where you find an area of interest in the genome by genetic screening and then find out what that region does functionally with the proteins that the region makes.)

The discoveries of these sensor molecules triggered an explosion of research in innate immunity. Over a dozen different TLRs have now been identified in humans and mice. Each one of them is programmed to recognise certain types of molecules common in microorganisms. While individuals with certain mutations in these molecules carry an increased risk of infection, other genetic variations in TLRs have been linked to increased risk of chronic inflammatory diseases.

As mentioned earlier, if innate immunity is breached, adaptive immunity comes into play. It is clear, therefore, that the innate immune response in some way contributes to the development of an appropriate adaptive immune response. But the exact mechanism of communication between the two stages remains unclear. Beutler and his co-workers in collaboration with David Nemazee's laboratory at the Scripps Institute have been able to show that TLR signalling is not required for effective antibody production, which has led them to conclude that a large number of other genes could be involved in supporting adaptive immune response.

As regards the potential applications of research on innate immunity, Beutler said, I think that the most hopeful [applications] lie in the realm of inflammatory and autoimmune disease because I believe now, as I believed long ago, that inflammation is something that evolved to cope with infection. And when we speak of sterile inflammatory diseases, like rheumatoid arthritis, and autoimmune diseases like lupus, probably some of the same pathways are utilised. And, it may very well be that by blocking TLR signalling we'll have very specific therapies for those kinds of diseases.

Steinman, too, saw similar potential from the ongoing work on DCs. Our most recent work is to take advantage of the science of dendritic cells, Steinman said in his interview. What we are trying to do is to use, to exploit, to harness what we have learnt about dendritic cells to make vaccines in a new way. Vaccines that are composed of very chemically defined substances and very safe, very specific and very incisive in terms of what they do. The dendritic cells help us understand the many diseases that involve the immune system. How does an autoimmune disease begin? How does allergy begin? What goes wrong? How can we fight cancer better. I think dendritic cells will bear upon the future of vaccine design, he said.

The basis for this lies in the new research at Steinman's lab. It has shown that DCs, which provide the important adaptive and are also probably linked to innate immunity, are also responsible for a seemingly opposite role in health called immune tolerance, which silences dangerous immune cells and prevents them from attacking innocuous materials in the body or the body's own tissues. In the absence of infection, DCs have been found to efficiently capture and process harmless self-' and environmental- antigens and silence the immune response; that is, induce immune tolerance. But during infection, it has been found that DCs undergo an intricate process of differentiation called maturation, which enable them to express additional receptors for responding effectively to microbial antigens and mount an immune response.

This work has led to a new understanding of the control of tolerance and immunity and has formed the basis of a new field of study in immunology: the role of DCs in immune regulation and their potential to help discover new vaccines and therapies for autoimmune disorders such as lupus and multiple sclerosis. During allergy, autoimmunity and organ transplantation, DCs initiate unwanted immune responses that cause inflammatory diseases and autoimmune disorders. But DCs are also known to suppress these conditions. That is, depending upon the effectiveness in tolerating self-antigens, their effectiveness in orchestrating innate and adaptive immune responses, DCs, in a sense, define the immunological self. Thus they can lead to new strategies for vaccines.