An immune system is required in any host that evolves slowly relative to the pathogens that attack it. This immune system must somatically generate and regulate new specificities. A history of immunological models^ref :

• 1959 : original self-nonself (SNS) discrimination (SNSD) model. Its conceptual underpinnings have been traced to Elie Metchnikoff's turn-of-the-century work on the phagocyte (Tauber, A. I.. 1994. The Immune Self: Theory or Metaphor? Cambridge University Press, London). As is by now well known, their work established the idea that during the neonatal period the immune system can learn to tolerate Ags that are normally present in the self. This education process-usually referred to as "central tolerance"-is now said to occur (at least with respect to T cells) throughout life in the thymus, where developing T cells strongly reactive with self-Ags are eliminated^ref. Of course, not every possible "self"-Ag is believed to be present in the thymus (but see^ref). Consequently, there must also be "peripheral" mechanisms that prevent mature, circulating T cells from attacking nonthymic self-Ags (here I ignore B cell tolerance^ref, though in many respects it resembles T cell tolerance). Although the distinction between central and peripheral tolerance is generally accepted, it is not known what fraction of self-reactive cells is inactivated in the thymus as opposed to the periphery; nor is it known whether a breakdown in central tolerance would lead to autoimmune disease or to what extent peripheral mechanisms could compensate

Supporting evidences :
• Paul Ehrlich
• Niels Kaj Jerne (Jerne, N. K. (1955) The natural selection theory of antibody formation. Proceedings of the National Academy of Science, USA. 41, 849-857; Jerne, N. K. (1966) The natural selection theory of antibody formation: ten years later. In Phage and the Origins of Molecular Biology. Edited by Cairns, J., Stent, G. S. and Watson, J. D., pp. 301-312, Cold Spring Harbor Laboratory Press, New York)
• David W.Talmage^ref
• Sir Frank Macfarlane Burnet's clonal selection theory^ref (Burnet, F. M. (1959) The Clonal Selection Theory of Acquired Immunity. Cambridge University Press, Cambridge; Burnet, F. M. (1968) Changing Patterns, an Atypical Autobiography. W. Heinemann, Melbourne, Australia; Ada, G. L. (1989) The conception and birth of Burnet’s clonal selection theory. In Immunology 1930-1980: Essays on the History of Immunology. Edited by Mazumdar, P. H., pp. 34-44, Wall and Thomson, Toronto; Sexton, C. (1991) The Seeds of Time. The Life of Sir Macfarlane Burnet. Oxford University Press, Melbourne, Australia; Burnet, F. M. (1967) The impact of ideas on immunology. Cold Spring Harbor Symposium on Quantitative Biology 32, 1-8; Burnet, F. M. (1956) Enzyme, Antigen, and Virus: A Study of Macromolecular Pattern in Action. Cambridge University Press, Cambridge) : each lymphocytes expresses multiple copies of a single surface BcR and signaling through this surface antibody initiates the immune response. The self-reactive lymphocytes are deleted early in ontogeny (Joshua Lederberg. Science (1959) 129, 1649-1653) and hence the universe of antigens is split into 2 sets : self (set a) and nonself (set b). The same principle was later applied to TcR of T cells.
• in 1945 Ray D. Owen discovered that dizygotic cattle twins sharing common blood circulation were mutually tolerant to each other's blood cells even in adulthood and displayed red cell chimerism--mosaicism as he called it--in adult life. Owen interpreted this extraordinary finding in terms of the much earlier discovery by F. R. Lillie that the placentae of cattle dizygotic twins undergo anastomosis early in fetal life, and he speculated that this would have permitted blood cells and their precursors to move from one twin to the other. Owen's discovery came out of the blue and it was ignored by immunologists until F. M. Burnet and F. Fenner highlighted it 4 years later in their influential monograph The Production of Antibodies, in which they predicted the existence of tolerance as a general phenomenon and developed their notion of "self-markers" to explain why the body does not react against self. Though it was Medawar's group that showed conclusively in 1953 that tolerance can be experimentally induced in fetal mice and in chick embryos, their entry into this field came from a totally different direction, an attempt to distinguish between mono- and dizygotic cattle twins by the exchange of skin grafts. This led to the seemingly paradoxic result that grafts exchanged between dizygotic twins were accepted (1951) and it was not until their cattle experiments had been virtually completed that they became aware of Owen's earlier discovery. Following the work of Billingham, Brent, and Medawar, and of Hasek, tolerance became incorporated into general immunologic theory and it helped to explain the fact that mammals do not normally suffer from injurious autoimmune manifestations. Ray Owen's discovery therefore has a secure place in the history of immunology^ref.
• RE Billingham, L Brent, and Peter Brian Medawar^ref found in 1953 that adult mice would accept foreign skin grafts if they had been injected as babies with cells from the donors. It started with a 1944 letter from a cattle breeder in Maryland to the University of Wisconsin immunogenetics laboratory, reporting a curious pair of twin calves, unusual in having different fathers. Ray Owen, a postdoctoral fellow in the laboratory and already interested in blood groups of cattle twins, thought they would provide an interesting opportunity for blood group analysis, so blood samples were sent to him. In the 30s and 40s, the genetics of blood cell antigens was an active field for investigation. It was then a popular view among geneticists that antigens, because of their simple inheritance, might be immediate gene products. For this reason, they might provide an insight into the nature of that maddeningly elusive entity, the gene. In pursuit of this possibility, new blood types were actively sought in various species, including Homo sapiens and Bos taurus. By the early 1940s, 40 different antigenic specificities had been identified in cattle. The world leader in cattle blood groups was the immunogenetics laboratory at the University of Wisconsin, founded by L. J. Cole and M. R. Irwin^ref. There was sometimes uncertainty about paternity in cattle, and when valuable animals were involved, that could be an important economic issue. Breeders and breed associations welcomed blood groups as a foolproof way of identifying sires. The immunogenetics laboratory provided valuable information and the breed associations provided financial support, vitally important in those pre-NIH/NSF days. It was a win-win situation. The events that led to the letter involved a Guernsey cow with twin calves. She had been properly mated to a Guernsey bull, but shortly afterward a lustful Hereford escaped from a neighboring area and got into the act. The color patterns of the calves showed clearly that the twins had different fathers. Blood analysis revealed that the cow carried (among many others) antigen G. The Guernsey bull had antigens S and X₂, while the Hereford bull had R and I’. The big surprise came with the calves. They had identical blood groups. This could not be explained by their being identical twins, for they were of different sexes to say nothing of having different fathers. Furthermore, each twin had antigens from the mother and from both sires, G S X₂ R I’. Why should nonidentical twins be identical for these blood groups (and for several others)? How could a calf inherit blood groups from both fathers? Ray Owen soon did a differential hemolysis, destroying cells of certain genotypes, and thereby demonstrated that each twin indeed had 2 kinds of red blood cells. One cell type was G S X₂ and the other was R I’, which made genetic sense. Ray was familiar with the peculiar uterine anatomy of cattle, which facilitates cross-connections between the extra-embryonic blood vessels of the twins (Lillie, F. R., 1916 The theory of the freemartin. Science 43: 611-613). These anastomoses provide a ready opportunity for exchange of blood between the 2 embryos. Ray, with his rural background, had long known about “freemartins.” These are frequently found when a female calf is born twin to a male. Such a female develops into a sterile, intersex-like adult, totally useless to breeders and dairy farmers. Long before, Lillie had demonstrated the union of circulatory systems of twin cattle embryos and postulated that hormones from the male suppressed the normal sexual development of his sister. The blood group admixture showed that more than hormones were exchanged. The study was soon extended to a large number of twins, and most of the time they were found to share identical blood groups (Owen, R. D., 1945 Immunogenetic consequences of vascular anastomoses between bovine twins. Science 102: 400-401). There were no regular proportions of the 2 types of cells, but whatever the proportion, it was similar in both twins. Thus, the vessels must be broadly connected so that the blood cells of the twins are thoroughly mixed. Are embryonic germ cells exchanged? Possibly yes, but one twin sired 20 progeny yet failed to transmit those antigens that he had gotten from his co-twin. Thus, the mixing of blood cells did not, at least in this case, extend to any mixing of germ cells. Ray's most spectacular example was a set of cattle quintuplets born on a farm in Nebraska (Owen, R. D., H. P. Davis and R. F. Morgan, 1946 Quintuplet calves and erythrocyte mosaicism. J. Hered. 37: 291-297). 4 calves were male, 1 was female, and all had identical blood groups. Each quint had 3 identifiable kinds of blood cells representing at least 3 genotypes; very likely there were 5. An immediate practical consequence of this work was that freemartins could be identified as very young calves. If opposite-sexed twins showed mixed blood types, the consequence of fused vessels (which occurred about 90% of the time), the female calf could be predicted to develop into a freemartin. The breeder could sell this one for veal and save the costs of feeding a calf that would turn out to be sterile. For many years the immunogenetics laboratory at Wisconsin offered a valuable freemartin-identifying service to cattle raisers. Another feature of the blood mixtures was quickly noted. The chimerism persisted far beyond the maximum life of blood cells. Therefore, what had been exchanged between the twins included blood cell precursors, not just the blood cells themselves. In fact, the antigenic phenotype persisted throughout the animals' lives. The blood admixture challenged a fundamental immunological tenet. Ordinarily, transfusion of blood from one individual to another leads to a specific, often severe transfusion reaction. Yet, somehow, each twin had survived and thrived, despite a massive transfusion of incompatible cells from the co-twin. Why should this embryonic exchange be exempt from the regular rules of blood transfusion? Ray wrote a longer paper discussing this question and foreseeing the possibility of what was later to be called immune tolerance. Unfortunately, the paper was rejected and only a much shorter one was published (Owen, R. D., 1945 Immunogenetic consequences of vascular anastomoses between bovine twins. Science 102: 400-401). It included only an explanation of the exchange, with no discussion of possible immunological implications. Alas, no copy of the first, unpublished paper can be located. It would be a great find for historians. A few years later, another serendipitous discovery was made, this time on the other side of the Atlantic. The late Hugh Donald, who did research on animal breeding in Edinburgh, was looking for identical twin calves. A genetically identical twin provides the perfect control for many kinds of experiments. The statistical gain to be gotten from reducing the between-twin variance was well understood, and identical twin calves were eagerly sought by researchers. The problem was, and is, that identical twins are rare in cattle. Also, especially in pure breeds with uniform color, it is often quite difficult to distinguish the 2 types of twins in newborn calves. In their search, Donald and his colleagues (1951) had made a rare find: identical quadruplets, identified as identical by exhaustive phenotypic analysis. I am sure the experimenters wished for many more; it would have been a statistical bonanza. At the 1948 International Genetics Congress in Stockholm, Donald encountered by chance Peter Medawar, who at the time was working on tissue transplants in mice. Medawar-over a cocktail, it is said-was certain that skin grafting would be an easy, certain way to distinguish between identical and fraternal twins. So he and his colleagues began an extensive grafting experiment in cattle. To their amazement, skin grafts were accepted by almost all the twin pairs, including those of the opposite sex. On still another continent, F. Macfarlane Burnet and Frank Fenner in Australia were developing the concept of self and non-self in antigen recognition (Burnet, F. M., and F. Fenner, 1949 The Production of Antibodies. Macmillan, London). Their book included a reference to Owen's work. According to Medawar, he read this, and the solution to the mystery was quickly apparent and soon published (Anderson, D., R. E. Billingham, G. H. Lampkin and P. B. Medawar, 1951 The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity 5: 379-397.; Billingham, R. E., G. H. Lampkin, P. B. Medawar and H. L. Williams, 1952 Tolerance to homografts, twin diagnosis, and the freemartin condition in cattle. Heredity 6: 201-212. Billingham, R. E., L. Brent and P. B. Medawar, 1953 Actively acquired tolerance of foreign cells. Nature 172: 603-606). Another version is that the connection with Owen's work was first noticed by Donald. Whatever the exact sequence of recognition, the explanation for the graft acceptance was immediately clear, and the new science of immune tolerance was born. Still a third party was involved at about the same moment, this time in Czechoslovakia. Milan Hasek was a follower of Lysenko and Michurin^ref. Impressed by the graft-hybrid results in plants claimed by Lysenko, Hasek decided that double-yolked eggs and parabiotic twins in chickens and between chickens and ducks would be an elegant way to demonstrate such effects in animals. The vascular connections seemed an excellent avenue for Lamarckian inheritance. He clearly showed the exchange of blood cells and the failure of antibody production. The paper, published in 1953, was written in Russian and the interpretation was in accord with Soviet orthodoxy. In 1955 Hasek met Medawar and Brent, who told him of their interpretation in terms of immune tolerance. Hasek’s views changed and he later adopted Mendelism. He became a leading figure, heading a large, productive laboratory in Prague. Thanks to his leadership, Prague became a world center in immunology. This didn’t last, however, for under the 1968 Soviet putsch in Czechoslovakia he became persona non grata. He was deposed, his laboratory and assistants were taken away, his co-workers were dispersed, and his subsequent personal life was a series of crises. He died in 1984. Remember that the cattle twin work was done in the dark ages of immunology. It was still believed that antibodies were molded by protein-folding on an antigen template. Shortly after, Burnet and others formulated the clonal selection hypothesis, and the research target changed from antigen to antibody^ref. This was a much more fruitful direction, and the field of immunology was poised to explode; the immune tolerance discovery helped light the fuse. In 1960 the Nobel Prize in Physiology or Medicine was awarded to Burnet and Medawar for the discovery of acquired immunological tolerance. Medawar assigned much of the credit to his colleagues, Brent and Bullingham. Some have suggested that Hasek should have shared the prize. Medawar, however, thought of Owen, who after all was there first. In a letter to Ray, Medawar said that he should have been included in the award, a statement that does honor to both men. Others-not including Ray-have also noted his absence from the Nobel Prize list and have wondered why. Yet, how much better this is than to have received the award and have people wonder why, as has been suggestedin some instances. Medawar got into the field of transplants while working with severely burned patients during World War II. He realized that skin grafts from other areas of the same person were permanently accepted, while those (homografts) from another person were eventually rejected (although they might persist long enough to be clinically useful). But, significantly, he found that a second homograft from the same donor was rejected very quickly. This, to him, was strong evidence for the immune nature of graft rejection. He went on to study mice and was involved with this work when the cattle twin question arose. Medawar was a man of many parts, a twentieth century renaissance man. He was an opera buff (as is Ray Owen). He studied mathematical logic and mastered the symbols needed to read the Russell-Whitehead Principia. He did experiments on such diverse topics as allometry and diffusion, and a number of other subjects (small experiments, he called them). He was fascinated by the transformation of Amphioxus from a highly asymmetric larva into a symmetrical adult and published an article on the evolutionary implications. Medawar’s best known work, aside from immunology, is on the evolution of senescence. He wrote two semi-popular essays on the subject (reprinted in Medawar, P. B., 1957 The Uniqueness of the Individual. Basic Books, New York) that have been widely heralded as the beginning of modern evolutionary theories of aging (e.g., Stearns, S. C., 1992 The Evolution of life histories. Oxford University, p. 200). Noting that post-reproductive (or post-progeny rearing) selection against deleterious mutations is weak at most, he suggested the accumulation of such mutations as one explanation. A second explanation, now called antagonistic pleiotropy, notes that selection can increase mutations that are favorable at early ages even if they are deleterious later. Although many other geneticists had similar ideas, Medawar set them forth explicitly. He used Fisher’s (1930) reproductive value as a measure of age-specific selection intensity. This intuitively appealing idea has been largely replaced by other measures due to Hamilton (1966)^ref, which can more readily be interpreted in terms of gene frequency change or probability of fixation (Charlesworth, B., 1994 Evolution in Age-Structured Populations, 2nd ed. Cambridge University Press, Cambridge, pp. 197ff). But Medawar was clearly on the right track. The relative importance of the 2 processes is still not clear and is being actively researched^ref (Rose, M., 1991 The Evolutionaly Biology of Aging. Oxford University Press, Oxford). Like his friends Julian Huxley and J. B. S. Haldane, Medawar enjoyed popular writing. He was a fluent writer of gracefully worded, easily understood essays, and many have been republished in book form. An example is a series of Sunday evening lectures broadcast by the BBC in 1959 and published under the title The Future of Man (Medawar, P. B., 1959 The Future of Man. Methuen and Co., London). His breadth of knowledge is impressive (as is the high level of programming of the BBC). As he said, “A human biologist must be  demographer, geneticist, anthropologist, historian, psychologist and sociologist all in one;” he came close. His mastery of English prose shows on every page. The book led to one minor disagreement. This was with H. J. Muller, who thought that Medawar overemphasized the importance of overdominant loci for quantitative traits and was therefore too pessimistic about the effectiveness of selection. Late in life, Medawar suffered a stroke that slowed his physical activity, but not his restless, wide-ranging mind. He continued to read, work (but not with his hands), and dictate essays and books. He published a charming autobiography with the intriguing title, Memoir of a Thinking Radish (Oxford University Press, Oxford. 1986). Death came in 1987 at age 72. Ray Owen was born the same year as Medawar (1915) and grew up on a Wisconsin dairy farm. For 8 grades, he attended a 2-room school. Then and through high school, he did his farm chores each day before and after classes. Followingraduation from Carroll College, he began graduate studies at Wisconsin with L. J. Cole and worked mainly with birds. His thesis was on the sterility of species hybrids. The observation of germ-cell migration alerted him to the possibility of  such events as were later observed in the twin calves. One of Ray's early papers-a favorite among his friends-describes “naked” pigeons (Cole, L. J., and R D. Owen, 1944 Naked pigeons. J. Hered. 35: 1-7). These birds, because of a recessive mutant gene, are completely featherless. The paper, largely written by Ray, was published in the Journal of Heredity. In those days science was less competitive and publications less crowded, and the editor, R. C. Cook, encouraged humorous, informal, clever writing (and contributed some himself). Here are some choice passages; those who know Ray will recognize the style. “Pigeon courtship, with its strutting, cooing and puffing out of feathers is an interesting performance. When there are no feathers to puff or to clothe the performer, it becomes a ludicrously macabre travesty. . . Although their wings are almost useless organs, these birds seem unable to learn to regard them as such. Placed on a table, they will hopefully take off into space, beating their wings vigorously, as though confident of a controlled landing which, however, ends in a ‘crash’. . . They are also active and aggressive lovers. Inadequate attire produces no inferiority complex in them; they strut and coo, puff and bow as if arrayed in the finest of raiment.” Alas, matings required artificial insemination and the fertility was low. Keeping the mutant gene by mating heterozygotes proved too laborious, and the strain was lost. “The perpetuation of the strain is so tedious that it will be a long time before the housewife can buy her squabs gene-plucked.” Ray’s immunogenetic work was done as a postdoctoral fellow. In 1947 he left Wisconsin for Caltech. The venue changed from cattle barns to rodent labs. Although the emphasis was always on immunogenetics, the organisms were strikingly varied. He and his students studied, in addition to rats and mice, viruses, ciliates, goldfish, birds, and humans. Always a popular teacher, he devoted considerable time to it. Along with Adrias SRB, he wrote a pathbreaking textbook (SRB, A. M, and R. D. Owen, 1952 General Genetics. W. H. Freeman, San Francisco) that started a trend in presenting genetics as an active, evolving subject. It quickly became a best seller. RAY’s later record shows a diminished number of scientific papers with his name attached, and there is a reason. In the 1960s, he decided no longer to permit his name to appear on papers by his students when they had done most of the laboratory work. But he continued to suggest problems and to offer assistance and guidance. His helpfulness to students, his and others’, is a Caltech legend. Ray is never too busy to help with a problem, be it scientific or personal. Nor is he too busy to accept a difficult administrative task, and this became a large part of his life. At Caltech he has become the students’ friend and advocate, and in many ways contributed to the conscience of the institution. Having recently passed his eightieth birthday anniversary, RAY has been the recipient of well-justified praise by former Caltech students and scientific colleagues. In the summer of 1996 a symposium in his honor was held at the University of Wisconsin. It was an exciting event, marred only by the death a few days earlier of George Snell, another pioneering transplant geneticist. The symposium was an intellectual feast. The latest theories and observations were on display. The contrast between what was known in 1946 and the state of the science in 1996 is amazing. It seems especially so to those, like me, who have viewed the subject with continued interest, but always from the outside. What a difference a halfcentury makes!^ref
Problems : this model does not explain ...
• since T cells respond to short peptides and crossreact on non-identical but similar peptides, "ignorant" T cells should often be activated by cross-reactive environmental antigens and autoimmunity should be a frequent and irrevocable event, rather than a rare occurrance
• what happens when the peptides change during puberty (new hormones), metamorphosis, pregnancy (new milk proteins, new foetal proteins), and aging
• why do we fail to make immune responses to vaccines composed of inert foreign proteins unless we add adjuvants
• why do we fail to reject tumors, even when many clearly express tumor-specific antigens (TSA)
• neonates are immunocompetent : for 5 decades, immunologists have thought that during embryonic development and early life, the immune system learns to distinguish ``self'' antigens, which are found on the body's own tissues, from ``nonself'' antigens, such as invading pathogens. This idea sprang partly from experiments in which Peter Medawar showed that fetal mice can become tolerant to transplanted tissues from immunologically different donors, presumably because the neonates' immune systems accept them as self, just as they accept the animals' own tissues. But 3 papers in 1996 by Fuchs et al, Ridge et al. and Forsthuber et al. have undermined the experimental basis of the self-nonself theory by showing that immunity can be induced in newborn mice under the right conditions. And conversely, tolerance can be induced in adults. The new work may not only have clinical implications, such as effective vaccines for infants or better success rates for organ transplantation, but it also opens the door to a new theory of how the immune system is activated: instead of distinguishing self from nonself, it may spring into action when an antigen is associated with harm (see below the danger theory)^ref. The classic system for induction of neonatal tolerance to protein antigens was reexamined in mice and the presumably tolerogenic protocol was found to trigger a vigorous T_h2 immune response. Thus, neonatal "tolerization'' induces immune deviation, not tolerance in the immunological sense. Neonates are not immune privileged but generate T_h2 or T_h1 responses, depending on the mode of immunization^ref. The susceptibility of neonates to virus-induced disease is thought to reflect, in part, the immaturity of their immune systems. However, inoculation of newborn mice with low doses of Cas-Br-M murine leukemia virus induced a protective CTL response. The inability of neonates to develop a CTL response to high doses of virus was not the result of immunological immaturity but correlated with the induction of a nonprotective T_h2 response. Thus, the initial viral dose is critical in the development of protective immunity in newborns^ref. Reexamination of the classic neonatal tolerance experiments of Billingham, Brent, and Medawar showed that tolerance is not an intrinsic property of the newborn immune system, but that the nature of the APC determines whether the outcome is neonatal tolerance or immunization^ref. Critics^ref admit that the fetus of many species may express immunological competence to many different antigens at different stages of gestation and even beyond birth^ref (J. B. Solomon, Foetal and Neonatal Immunology (North-Holland, Amsterdam, 1971)), and the mouse has long been known to slowly expand its immunological repertoire during the neonatal period^{ref1, ref2}. Neonatal tolerance has fascinated the immunologic community for 50 years, resulting in apparently conflicting publications that can be broadly divided into 3 groups which state that neonatal antigen exposure causes :
• clonal deletion
• suppression
• an immune response.
The prevalent view has been that the neonate is immune privileged. The 3 reports may have resolved this controversy and demonstrate why neonatally induced immunity has been perceived either as clonal deletion or suppression. For example, memory cells lose lymph node homing receptors and lose their ability to migrate to lymph nodes. Hence, when lymph node responses are studied, as has been the case in the past, the lack of antigen reactive cells seemed to suggest that these cells were clonally deleted, which substantiated the clonal deletion model for neonatal tolerance. But the memory cells were simply redistributed in the organism. Furthermore, neonatally induced T_h2 immunity results in apparent suppression when T_h1 immunity is measured. The previous data, which seemed to conflict, reflect a single mechanism: induction of T_h2 immunity. Clearly, the currently favored clonal deletion model is insufficient to fully explain self-tolerance. These findings substantiated the active T cell tolerance idea, showing that what was thought to be "suppressor cell''-mediated self-tolerance actually translates into T_h2 cell-mediated effects. Roughly 2 millennia before Copernicus, Aristarchus proposed a heliocentric model to explain the motion of the planets. Why was it ignored? Thomas Kuhn's suggestion was that, at that time, there might have been no general dissatisfaction with the reigning paradigm (Earth at the center) and therefore no reason to abandon it (T. S. Kuhn, The Structure of Scientific Revolutions (Univ. of Chicago Press, Chicago, IL, 1970)). By the time Copernicus suggested his version, the motion of planets could no longer be easily explained by the view that the Earth was central, and the intellectual community was ready for a "new'' idea. During that period of almost 20 centuries, there had been many findings that did not fit with the ruling paradigm, but most of them went unrecognized, examples of the "`retrorecognition'' phenomenon^ref (A. Lightman and O. Gingerich, Science 255, 690 (1992)), whereby clear anomalies in a paradigm are recognized only after a new conceptual framework has been set forth to replace the defective one. Roughly 100 years ago, Paul Ehrlich thought that the immune system's primary function was to detect and protect against noxious pathogens^ref (Ehrlich, P. (1900) On immunity with special reference to cell life. Proceedings of the Royal Society of London 66, 424-448; 12. Ehrlich, P. (1906) Collected Studies on Immunity. J. Wiley & Sons, London). It seems that this view was replaced by Burnet's self-nonself model because the latter was better able to explain 2 new phenomena, the ability to raise immune responses to nonnoxious substances^{ref1, ref2} and the discovery of neonatal tolerance^ref. Over the ensuing years, both of these findings were extensively examined and, in both cases, some clear anomalies appeared, many of which were ignored. For example, a variety of nonpathogenic substances can indeed induce immune responses, but, as Charles Janeway has emphasized, they almost never do in the absence of immunological adjuvants, which contain bacterial products^ref. Thus, the mere presence of "foreignness'' is not enough. Some essence of pathogenicity is also required. The picture with neonatal tolerance has also been clouded considerably since the original pioneering experiments. There have been scattered examples in which neonatal immunity rather than tolerance was seen^{ref1, ref2} : altogether, these studies showed that neonates were able to make many different kinds of responses. They cleared viruses, generated graft-versus-host disease, and made T_h1 and T_h2 responses; and a few experts in the field began to understand that neonatal tolerance was more complicated than had first been envisioned. Yet many immunologists were unaware of the complications, and recent textbooks continue to describe neonatal tolerance in terms of the immaturity of the neonatal T cells. Perhaps this was another case of retrorecognition. The findings simply didn't fit with a paradigm that had found its strongest early support in the original neonatal tolerance experiments, and there was no alternative model to fit them into. If youthful immune systems were able to respond to a variety of antigens, making a variety of responses, how could self be distinguished from nonself? Although some scientists attempted to deal with the problem by changing the temporal model to a spatial one (moving tolerance into the thymus, where the T cells, rather than the individual, are immature), these models could not easily account for tolerance to tissue-specific peripheral antigens, and no self-nonself model can account for new antigens that might appear throughout life. Looking back, a historian of science might wonder what would have happened if Peter Medawar's group had gotten a different result. Although Burnet's clonal selection would certainly have prevailed as the modern operating model, what would have happened to self versus nonself? Perhaps clonal selection would have served as a mechanism to generate specific responses to dangerous pathogens and to allow for antigen-specific memory. Although the results, by themselves, do not disprove the self-nonself model (this was better done by an earlier paper in which it was shown that unprimed adult female mice responded to the male antigen, H-Y, when it was presented by dendritic cells but became tolerant when it was presented by resting or activated B cells. The inevitable conclusion from this study was that their responses were dictated by the cell presenting the antigen rather than the "foreignness'' of the antigen itself^ref), they do undermine one of its experimental underpinnings and are more easily placed in the context of the "danger'' model^ref, which suggests that the immune system is primarily concerned with detecting and protecting against "dangerous'' pathogens and that tolerance is a continuous process regulated throughout life by each bodily tissue. The susceptibility of newborn mice to a virus was not the consequence of a "slowly expanding immunological repertoire'' but an example of immune deviation^{ref1, ref2} driven by the relatively high dose of virus encountered by the neonatal immune system. The development of T_h1 or T_h2 responses is determined by the dose of virus inoculated in newborn mice and influences the development of protective immunity. The 3 reports on immunological tolerance provide intellectual support for the clinical observation of down-regulation of cellular immune responses in patients receiving allogeneic blood transfusions^ref. This is particularly well documented in organ transplantation, surgery, and cancer. Recent evidence from both animal and human studies suggests that the high doses of alloantigen involved in clinical transfusions induce a T_h2 immune response, and the expected down regulation of the T_h1 response^{ref1, ref2}. The trauma of surgery alone causes T_h2 responses, compatible with Matzinger's "danger'' theory. Allogeneic transfusions also have been successfully used to treat 2 disease processes that likely represent overactive T_h1 immunity in adults : repetitive spontaneous abortion^ref and rheumatoid arthritis^ref. These observations in humans support the point made by the authors that manipulation of immunity in adults may be more feasible than previously believed on theoretical grounds^ref.
The above understanding of tolerance emphasizes the importance of the Ag-specific signal received through the TCR, a signal often called "signal one." In the simplest scenario, the tolerance mechanisms that eliminate "self"-reactive cells ensure that signal one can only originate from a "foreign" Ag, i.e., signal one is a self-nonself discriminator. However, immunologists are increasingly framing questions of tolerance in terms of a two-signal model of lymphocyte activation^{ref1, ref2, ref3, ref4}. Signal 2, or "costimulation," is a more generalized signal that is required to ensure self-tolerance in cases where, for some reason, central tolerance fails and a self-reactive T cell is not inactivated before functional maturity. The idea is that the second signal is required to properly activate T cells, but will only be provided during genuine infections and not to autoreactive T cells in healthy individuals. The central concept of "self" is ill-defined. Indeed, "self" has been variously understood as 1) the sum of genome-encoded Ags, 2) the body, 3) the total set of MHC/peptides, or 4) simply the subset of MHC/peptides expressed on APCs.
• 1969, 1^st modification (2 signal model for B cells / associative recognition model : Bretscher and Cohn, then updated and expanded over the years by Langman and Cohn^ref) : realizing that autoimmunity would be rare if immunity required the activation of 2 cells recognizing different specificities on the same antigen, they proposed that the B cells would die when they see antigen via BcR stimulation (signal 1) unless rescued by help (signal 2) from helper T lymphocytes.

Supporting evidences : autoimmunity is a rare occurrance
Problems : this model does not explain ...
• why alloreactivity is often stronger than xenoreactivity
• chicken lymphocytes respond more strongly to other chicken cells than to quail cells
• human T cells respond better to cells from another human than to mouse cells^ref
• what happens when the peptides change during puberty (new hormones), metamorphosis, pregnancy (new milk proteins, new foetal proteins), and aging
• why do we fail to make immune responses to vaccines composed of inert foreign proteins unless we add adjuvants
• why do we fail to reject tumors, even when many clearly express tumor-specific antigens (TSA)
• 1975, 2^nd modification (2 signal model for helper T cells : Lafferty and Cunningham^ref) : T helper cells die when they see antigen via TcR stimulation (signal 1) unless rescued by species-specific co-stimulation (signal 2) from accessory cell (now known as the antigen-presenting cell (APC)). Signal 1 alone would lead to tolerance of the T cell^ref.

Supporting evidences :
• alloreactivity is often stronger than xenoreactivity
• autoimmunity exists as responses are initiated by APCs, which are not antigen specific (they capture all sorts of self and foreign substances), making this model apparently unfitted into a self-nonself mode.
Problems : this model does not explain ...
• what happens when the peptides change during puberty (new hormones), metamorphosis, pregnancy (new milk proteins, new foetal proteins), and aging
• why do we fail to make immune responses to vaccines composed of inert foreign proteins unless we add adjuvants
• why do we fail to reject tumors, even when many clearly express tumor-specific antigens (TSA)
• 1989, 3^rd modification (infectious nonself (INS) / stranger model, Charles A.Janeway^{ref1, ref2}) : even APCs have their own form of self/nonself discrimination. APCs are not constitutively active and do not co-stimulate unless activated via binding of nonself pathogen-associated molecular patterns (PAMPs) to cell surface pattern recognition receptors (PRRs) for evolutionarily distant infectious non-self (signal 0), i.e. the immune system has a phylogenetic memory of infectious organisms. As PAMPs are not produced by healthy host cells, the universe of antigens is split into 2 sets : noninfectious self (set a) and infectious nonself (set f). It tends to ignore noninfectious nonself (set b - f). Thus, there are now 2 different types of nonself recognition : a genetically encoded set of PRRs assigned to the cells and molecules of the innate immune system and a somatically generatede set of receptors expressed by the cells of the adaptive immune system.

Supporting evidences :
• we fail to reject tumors even when many clearly express tumor-specific antigens (TSA)
• we fail to make immune responses to vaccines composed of inert foreign proteins unless we add adjuvants
• peptides can change during puberty (new hormones), metamorphosis, pregnancy (new milk proteins, new foetal proteins), and aging without triggering immune responses
Problems : this model does not explain ...
• why oral administration of antigen sometimes lead to vaccination (as in the oral polio vaccine (OPV)) and sometimes to tolerance
• why is the hamster (Cricetinae) cheek pouch (a place in which hamsters carry nuts, with all of their concomitant fungi and bacteria), an immunologically priviliged site
• what is the difference that leads to gene therapy (no immune response) versus DNA vaccination
• why viruses that do not generate dsRNA stimulate immunity
• how nonbacterial adjuvants, such as alum, work
• why transplants are rejected and why livers are mose easily transplanted than other organs
• why tumors are sometimes spontaneously rejected
• why do so many people have autoreactive T and B cells without any sign of autoimmunity while others get autoimmune diseases
• 1994, 4^th modification (alarm / danger model, Polly Matzinger^{ref1, ref2, ref3, ref4}) : an evolutionarily useful immune system should concentrate on those things that are dangerous, rather than on those  that are simply foreign. There are 3 reasons for this shift in viewpoint :
• there is no need to make a response to everything is foreign. Eg there is no need to eliminate ...
• a lot of harmless foreign material in the air we breath and the food we eat
• a virus that enters a cell, makes a few copies of itself and leaves without doing any damage (we might even want to welcome such viruses for the genes that they could bring us)
• the commensal bacteria in our guts that provide us with vitamin K
• the foetuses that make the next generation
• peripheral tissue-specific antigens that are not ectopically expressed in the thymus or bone marrow exist^ref
• bodies (self) do change through puberty (new hormones), metamorphosis, pregnancy (new milk proteins, new foetal proteins) and aging : have been all these new antigens previously ectopically expressed in thymus or bone marrow ? Although the distinction between central and peripheral tolerance is generally accepted, it is not known what fraction of self-reactive cells is inactivated in the thymus as opposed to the periphery; nor is it known whether a breakdown in central tolerance would lead to autoimmune disease or to what extent peripheral mechanisms could compensate.
The universe of antigens is split into 2 sets : those associated with dangerous entities or harmless ones, defining as dangerous anything that induces stress or necrotic (nonapopotic) cell death. APCs are not constitutively active and do not co-stimulate unless activated by endogenous cellular alarm signals (signal 0) sent from surrounding distressed cells :
• sudden loss of connection to a cell to which it was connected
• release of a substance elaborated or modified exclusively by stressed cells :
• MICA or MICB proteins
• monosodium urate (MSU) crystals increase the expression of co-stimulatory molecules by bone marrow-derived DCs, while soluble uric acid is unable to support DC maturation in vitro : when co-injected with antigen in vivo, MSU don't affect the uptake of antigen by DCs but significantly enhance the generation of responses from CD8⁺ T cells. Eliminating uric acid in vivo inhibits the immune response to antigens associated with injured cells, but not to antigens presented by activated DCs^ref. Increased uric acid production is observed to be a general characteristic of cells undergoing death : uric acid has been reported to precipitate in vivo and during cell death the levels of uric acid produced locally could increase sufficiently to cause this endogenous metabolite to precipitate and promote DC maturation and activation. This is the basis for the pathogenesis of gout. Significantly, uric acid can derive from the breakdown of ATP-itself a cell component released by necrotic cells-by phosphodiesterases and other enzymes, such as xanthine oxidase that can be found in extracellular fluids^ref. MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active IL-1b and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1b activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1b receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases^ref.
• IFN-a synthetized by virus-infected cells
• secreted heat-shock proteins (HSPs) HSP90 and gp96^{ref1, ref2} : molecular chaperones were proposed to be danger signals, because they are normally retained within healthy cells but can be released during unregulated and necrotic cell death^{ref1, ref2, ref3}. However, the mechanisms by which chaperones released from stressed or dying cell-elicited danger signals are not well understood. Chaperones released from necrotic tumor cells are reported to be immunostimulatory^{ref1, ref2, ref3, ref4}; however, recent evidence indicates that the stimulatory activity may be independent of peptides with which the chaperones may be associated^{ref1, ref2}. Chaperones have been reported to elicit a variety of responses, primarily in and from immunoregulatory cells, including cytokine release, APC activation and DC maturation, up-regulation of cell surface co-stimulatory molecules, and stimulation of autoimmune responses^{ref1, ref2, ref3, ref4, ref5, ref6}.
• tumor cells subjected to a nonlethal heat shock stress are unable to form tumors in syngenic mice, whereas they do so in athymic nude mice. Moreover, heat-shocked MethA immunity is tumor specific. Enhancement of T-cell-mediated immunogenicity correlates with the expression of the inducible HSP70 but not the constitutive HSC70. These observations have a bearing on the proposed functional role of Hsp-peptide association in antigen processing and presentation by MHC class I molecules under normal and stressful conditions^ref.
• in situ vaccination : HSP70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. A hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. The HSP70-peptide complexes from the tumor were purified after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity : in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. This hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity^ref.
• tumor-derived HSP90-peptide complexes (HSP90-PC) have strong immunogenicity and the DC sensitized thereby effectively induces the proliferation of CTL^ref.
Receptors :
• TLRs and CD14 have been posited to transduce chaperone-mediated signaling^{ref1, ref2, ref3, ref4, ref5}. However, recent reports^{ref1, ref2, ref3, ref4} indicate that some of the effects observed may have resulted from endotoxin contamination.
• CD91 / LDL-related protein 1 (LRP1) was originally reported to be the unique endocytic receptor for a variety of chaperones including CRT, gp96, hsp70, and hsp90^{ref1, ref2}. CD91, although not classified as a scavenger receptor, shares many traits of the scavenger receptor superfamily and was originally proposed as the sole APC endocytic receptor for a multitude of chaperones^{ref1, ref2}. However, subsequent studies identified a CD91-independent pathway for chaperone uptake^ref followed by the identification of canonical scavenger receptors as functioning in chaperone trafficking :
• LOX-1 was identified as an endocytic receptor for hsp70 but not gp96
• scavenger receptor class-A (SRA) was identified as an endocytic receptor for gp96 and CRT^{ref1, ref2}. Both of these scavenger receptors trafficked chaperone-associated peptides into the APC antigen presentation pathway(s)^{ref1, ref2, ref3, ref4}
But .... :
• the ability of colon cancer cells to elicit a T-cell-dependent immune response was suggested to depend on their sensitivity to death, either by apoptosis or by necrosis^{ref1, ref2}. Expression of antiapoptotic proteins such as Bcl-2 and Hsp27 could decrease colon cancer cell immunogenicity in syngeneic animals, thus increasing their tumorigenicity^{ref1, ref2, ref3}. On the other hand, selective depletion of HSP70 could sensitize a poorly immunogenic and strongly tumorigenic rat colon cancer cell line to death and facilitate induction of an immunogenic response in syngeneic animals^ref. Apoptosis is a conserved, energy-dependent programmed cell death pathway used to eliminate cells in response to stress as well as excessive cells in developing multicellular organisms. One of the central molecules in apoptotic pathways is cytochrome c, which upon release from the intermembrane space of the mitochondria in response to a variety of apoptotic stimuli activates the caspase cascade in the cytosol^{ref1, ref2}. In the mitochondria, cytochrome c is the only water-soluble component of the electron transfer chain and participates in the reduction of oxygen by cytochrome c oxidase, the terminal and putative rate-limiting step of electron transfer^ref. Once in the cytosol and in the presence of ATP, cytochrome c binds the adaptor molecule apoptosis protease activating factor 1 (Apaf-1) with a high affinity and triggers its oligomerization to form the apoptosome complex in which caspase-9 is recruited and activated^{ref1, ref2, ref3, ref4}. Activated caspase-9 in turn cleaves and activates caspase-3, caspase-6, and caspase-7, which function as downstream effectors of the cell death program^ref. The components of this pathway have been conserved throughout evolution of metazoan organisms^ref. Murine embryos devoid of cytochrome c demonstrate profound developmental delay and do not survive beyond day 10.5 post coitum. Furthermore, cell lines derived from early embryos of the cytochrome c-deficient genotype have reduced proliferation and viability and need special media to support ex vivo culture^ref. Decreased expression of cytochrome c after transfection with an antisense construct in tumor cells sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli, and facilitates in vivo "necrotic" colon cancer cell death and the induction of a specific immune response in rats^ref
• soluble heparan sulfate (an extracellular breakdown product of hyaluronan^ref made when vessels are damaged) binds to TLR4^{ref1, ref2}
• murine b-defensin-2 binds to TLR4^ref
• surfactant protein-A binds to TLR4^ref
• damaged DNA : it has become increasingly evident that alloreactivity of donor immune cells against host leukemic tumor cells plays a role in controlling the patient’s malignancy after allogeneic BMT3^{ref1, ref2, ref3}. Early GVL responses are probably mediated by direct donor T-cell allorecognition, whereas delayed responses leading to long-term tumor regression may be due to the indirect pathway, where donor-derived APCs, DCs in particular, cross-present host leukemic cell alloantigens to syngeneic (donor-derived) T cells^{ref1, ref2, ref3, ref4}. To facilitate GVL responses, reduced-intensity conditioning (RIC) chemotherapy regimens pre-BMT have been adopted to reduce early transplant-related toxicity while achieving donor hematopoietic and immune cell engraftment^ref. The 2-7-month time lag of tumor regression after BMT supports the importance of this GVL effect^ref. Clinical protocols optimize RIC with a purine analogue plus antithymocyte globulin, high-dose CTX, or the alkylating agent melphalan^{ref1, ref2}. The requirement for both RIC and GVL to mediate long-term tumor regression raises important questions about the interaction between immune cells and tumor cells dying after exposure to DNA-damaging agents^{ref1, ref2, ref3, ref4}. Dying tumor cells may provide DCs with a plentiful source of tumor antigens in combination with activating danger signals, i.e., "natural adjuvants," that may enable DCs to initiate antitumor T-cell responses and thus maintain tumor immune surveillance. There are several different types of potential DC-tumor cell interactions. The hypothesis that DCs mediate both T-cell immunity and the diametric opposite, i.e., T-cell tolerance, can be linked to the dynamic maturation of DCs. Immature DCs exposed to anti-inflammatory cytokines such as IL-10 and TGF-ß remain immature and may tolerize T cells to presented antigen^ref. Conversely, upon receipt of appropriate inflammatory stimuli, DCs mature with up-regulation of surface MHC [particularly MHC class II^ref] and T-cell costimulatory ligands, enhanced IL-12 secretion^{ref1, ref2}, and potent T-cell activation. With respect to cell-derived stimuli, DC maturation is perhaps best considered in terms of the danger hypothesis^{ref1, ref2}, whereby dying cells release endogenous mediators of DC activation such as HSPs^{ref1, ref2, ref3}, ROIs^ref, sCD40L, and IL-1ß^ref. Under physiological thresholds of in vivo cell death, cells dying by apoptosis are cleared rapidly by macrophages, whereas monocytes down-regulate secretion of TNF-a, IL-1ß, and IL-2 and increase IL-10, creating an anti-inflammatory milieu^{ref1, ref2}. If exposure of DCs to apoptotic cells is prevented, and DCs are bathed in anti-inflammatory cytokines, they will remain immature^ref, which will promote peripheral tolerance to self-antigens and avert autoreactivity^ref. However, should massive cell death overwhelm macrophage clearance, as in early postchemotherapy or viral infection^ref, apoptotic cells may progress to secondary necrosis characterized by nuclear pyknosis and cell membrane degradation^ref with spillage of intracellular contents into the extracellular milieu. In vivo, this inflammatory environment may enhance DC function and generate antitumor CTLs. This model is supported by reports of synergy between DC vaccination and tumor irradiation^ref or chemotherapy^ref. Likewise, IL-12 and chemotherapy synergize in promoting tumor regression^ref. However, questions about the relative role of tumor cells as a source of danger signals have not been addressed. Tumor cells dying via both apoptotic and necrotic mechanisms have been shown to be a plentiful source of antigens for processing and presentation by DCs to mediate T-cell immunity or tolerance. Nevertheless, controversy remains with respect to the immunomodulatory effects that dying tumor cells have on DCs after tumor antigen uptake. These conflicting findings may point to a delicate balance of DC activation that is biased in response to variations in the microenvironmental milieu. Biasing the response toward immunity may be reflected by the in vivo observations that direct anticancer therapy in combination with allogeneic BMT are necessary to drive powerful, and potentially curative, GVL responses. Although direct allorecognition of host tumor tissue by donor leukocytes is thought to play a prominent role in acute tumor regression^ref (after tumor debulking), the cross-presentation of allogeneic tumor peptides by donor DCs to mediate autologous GVL T-cell responses probably plays a key role in long-term rejection^ref. It has been suggested that DC vaccine strategies using allogeneic tumor-loaded DCs as an adjuvant to boost and maintain antitumor activity may be a powerful immunotherapeutic option^{ref1, ref2, ref3}. Different mechanisms of cell death may provide danger stimuli of different strengths to DCs developing from myeloid precursors. The effect of exposure of day 1 DCs to melphalan-treated tumor cells was to accelerate their acquisition of T-cell-stimulatory capacity with phenotypic up-regulation, and cytokine secretion. This reached a peak at day 5 of culture (4 days of exposure) and was associated with enhanced expression of HLA-DR, CD80, and CD86. The functional changes were duplicated using chlorambucil, a drug with a similar mechanism of action to melphalan, which supports a differential effect of alkylating agents compared with purine analogues, atypical alkylators, or primary necrosis by simple freeze-thaw. Pretreatment of whole cells with proteinase K did not affect CD86 up-regulation, thus excluding the role of protein mediators, whereas treatment of melphalan-killed tumor cells with DNase abolished their immunostimulatory activity. Both CD14 and CD1a were down-regulated, and DCs showed striking dendritic morphology, suggesting that monocytes respond to injured tumor cells with accelerated activation and maturation without deviation from DC lineage commitment. Taken together, these data suggest that DCs sense and respond to various types of tumor cell death via DNA damage in a differential manner. Typical immunotherapeutic strategies load DCs toward the end of their maturation process. DCs respond vigorously to injured tumor cells in an early stage of maturation (day 1). This is logical because immature DCs, and myeloid DC precursors for that matter, have the ability to efficiently take up antigen and cellular debris and thereby respond in a highly plastic manner by acquiring immunostimulatory or immunotolerizing qualities. The observations may help to explain the dichotomy reported in the literature about how DCs respond to dying tissue cells. Indeed, DC interaction with injured tumor cells is more complex than has been appreciated: alterations in the extracellular milieu of DCs may dictate the balance between potent antitumor immune responses and undesirable tolerogenic vaccinations. Furthermore, in the context of DC vaccine design whereby immature DCs are loaded with killed tumor cells (transfectomas)^ref before being matured with exogenous inflammatory stimuli (TNF-a, CD40L, and so forth), the judicious choice of how tumor cells are injured may obviate the need for the ex vivo maturation step. Clearly, both the nature of tumor cell death and the DC maturation states are important as variables in responses to dying cells. DCs respond to the DNA isolated from treated tumor cells by CD86 up-regulation, induction of enhanced T-cell proliferation, and secretion of IL-12. These DC responses to exogenously added damaged DNA were similar to responses to whole cells. However, DC responses to DNA were markedly lower than their responses to whole treated tumor cells, particularly because the amount of exogenous DNA was purified from 20-fold more tumor cells than the number used in the whole cell treatment studies. This may reflect the relatively higher efficiency with which DCs take up whole cells or a possible synergism between damaged DNA and other cell-derived proinflammatory mediators. The role of vertebrate DNA in danger signaling is controversial. In the murine system, Ishii et al.^ref reported that intact, unmodified dsDNA released from necrotic tissue cells had immunostimulatory properties on DCs, and fragmentation or strand separation abrogated these properties. Genomic DNA from normal cells had little effect on DCs in this human study, but the requirement for intact DNA is underscored by the finding that DNase treatment of both whole injured cells and their DNA abrogated the effects on DCs, and fragmented DNA had no immunostimulatory properties. Whereas the precise DNA modification caused by many antineoplastic cytotoxic agents has long been known, at present one can only speculate about the in vivo effects that DNA adducts or oxidized DNA have on DCs and the immune system as a whole. However, in this context, it is noteworthy that there are isolated clinical reports of autoimmune side effects concomitant to antineoplastic therapy (pemphigus^ref or multiorgan ^ref), which could represent in vivo DNA stimulation of immune cells. It is feasible that, in a manner similar to the response to CpG oligodeoxynucleotide or viral dsRNA via pattern recognition, DNA modification by induction of ROI^ref oxidation, alkylation, or cross-linking may enhance its immunogenicity by exposing otherwise cryptic immunostimulatory sites in DNA sequences to which the corresponding DC receptors may bind. This resembles TLR ligation by autologous DNA sequences that may be critical in the pathogenesis of systemic lupus erythematosus^ref. The implications of the finding that modified DNA may act as a danger signal extend beyond tumor immune therapy strategies. In the cancer context, the findings are particularly striking, showing clearly that the type of cell death invoked may be a significant determinant of danger detected at the tumor site^ref. Tumor-free survival may be mediated by T-cell responses that are primed by activated DCs during the initial antitumor treatment. This may theoretically account, at least in part, for the long-term effectiveness of one chemotherapy regimen over another. Therefore a better understanding of the molecular details concerning various forms of stress-induced death and their effects on dynamic, i.e., time-dependent DC immunobiology is needed. An improved understanding of the nature of tumor-elicited danger signals may lead to the discovery of "natural" immunological adjuvants that mimic those signals or else generate the appropriate type of cancer cell damage that is necessary to elicit them^{ref1, ref2}; ^ref.
Increased immunogenicity explains synergy with necrosis-inducing treatments : while macrophage exposure to necrotized tumor cells causes pronounced stimulation of macrophage antitumor activity, exposure of macrophage to apoptotic tumor cells in contrast results in impairment of macrophage-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms^ref. Apoptotic tumor cells can be either immunogenic or nonimmunogenic in vivo, depending on whether or not these cells are heat stressed before induction of apoptosis. Stressed apoptotic cells express HSPs on their plasma membranes and DCs are capable of distinguishing them from nonstressed apoptotic cells. When purified HSP70 or chaperone-rich cell lysate (CRCL) from syngeneic normal tissue is used as an adjuvant with nonimmunogenic apoptotic tumor cells in vaccination, potent antitumor immunity can be generated. This antitumor immunity is mediated by T cells because antitumor effects are not observed in either SCID or T cell-depleted mice. Vaccination of mice with apoptotic tumor cells mixed with liver-derived CRCL as adjuvant were capable of enhancing the production of T_h1 cytokines, inducing specific CTLs and eliciting long-lasting antitumor immunity. Stress proteins from autologous normal tissue components therefore can serve as danger signals to enhance the immunogenicity of apoptotic tumor cells and stimulate tumor-specific immunity^ref. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. AxV preferentially targets irradiated lymphoma cells to CD8⁺ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV-coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection^ref.
• sinergy with chemotherapy :
• intratumoral injections of bone marrow-derived immature naïve DCs into mice with advanced transplanted syngeneic B-cell lymphoma had no antitumor effect. Systemic chemotherapy alone resulted in only transient tumor regression. However, the intratumoral injection of DCs after chemotherapy led to complete, long-term tumor regression in the majority of treated mice. This DC-mediated antitumor effect was systemic, resulting in simultaneous elimination of the tumor at second uninjected sites. In addition, it resulted in long-term memory with resistance to tumor rechallenge. Both CD4⁺ and CD8+ T cells are necessary for the antitumor effect. Furthermore, tumors that occasionally recurred in mice with initial complete tumor regression could be retreated by the same combined chemoimmunotherapy approach. These results show that immunotherapy can succeed in the setting of advanced lymphoma if DCs are restored and loaded with tumor antigens in situ at a single tumor site^ref.
• CTLs sensitized with gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF)-treated colon cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs^ref.
• i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8⁺ T cell response eradicates systemically established B16 tumors. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and TRP-2 antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. Inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy^ref.
• myeloid suppressor (Gr-1⁺/CD11b⁺) cells accumulate in the spleens of tumor-bearing mice where they contribute to immunosuppression by inhibiting the function of CD8⁺ T cells and by promoting tumor angiogenesis. Elimination of these myeloid suppressor cells may thus significantly improve antitumor responses and enhance effects of cancer immunotherapy, although to date few practical options exist. The effect of the chemotherapy drug gemcitabine on the number of (Gr-1⁺/CD11b⁺) cells in the spleens of animals bearing large tumors derived from five cancer lines grown in both C57Bl/6 and BALB/c mice was analyzed. Suppressive activity of splenocytes from gemcitabine-treated and control animals was measured in NK cell lysis and Winn assays. The impact of myeloid suppressor cell activity was determined in an immunogene therapy model using an adenovirus expressing IFN-b. The chemotherapeutic drug gemcitabine, given at a dose similar to the equivalent dose used in patients, was able to dramatically and specifically reduce the number of myeloid suppressor cells found in the spleens of animals bearing large tumors with no significant reductions in CD4⁺ T cells, CD8⁺ T cells, NK cells, macrophages, or B cells. The loss of myeloid suppressor cells was accompanied by an increase in the antitumor activity of CD8⁺ T cells and activated NK cells. Combining gemcitabine with cytokine immunogene therapy using IFN-b markedly enhanced antitumor efficacy. These results suggest that gemcitabine may be a practical strategy for the reduction of myeloid suppressor cells and should be evaluated in conjunction with a variety of immunotherapy approaches^ref.
• synergy with radiotherapy : radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes
• radiation modulates MHC class I-mediated antitumor immunity by functionally affecting bone marrow-derived dendritic cells (DCs) Ag presentation pathways, with divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-127-35 that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-127-35 also protected mice against growth of B16-A2/Kb tumors in HLA-A2.1/Kb transgenic mice^ref.
• localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site : antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 x 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-g upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45⁺ cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-g and lysed tumor cell targets. Peptide-specific IFN-g responses were directed against both the class I and class II MHC-restricted OVA peptides OVA_257-264 and OVA_323-339, respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2180-188. Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site^ref.
• UV irradiation appears to be an effective modality for altering tumor cell immunobiological properties, including increased tumor cell sensitivity to T-cell and/or natural cell-mediated immunity to the nonimmunogenic MHC^- MCA102 fibrosarcoma. In parallel, the effect of short wavelength UVC light on the sensitivity of tumor cells to natural cell-mediated cytotoxicity and TNF was also investigated. MCA102 fibrosarcoma cells were irradiated in vitro twice with UVC light (610 and 457 J/m²). Surviving cells were expanded and maintained in vitro as the MCA102UV subline. UV treatment changed tumor cell morphology and increased their in vitro rate of proliferation. However, after inoculation of 1 x 10⁵ to 2 x 10⁶ MCA102UV cells into C57BL/6 mice, growth of these cells was completely prevented. Lyt2.2 and not L3T4 lymphocytes were responsible for the rejection of these tumor cells. To determine the minimal and optimal dose of UV irradiation capable of increasing tumor cell immunogenicity, MCA102 cells were irradiated once or twice with different doses (76 to 610 J/m²) of UV light. After a single dose of UV treatment, tumor growth in C57BL/6 mice was inhibited, particularly with lines irradiated at the highest doses (610 or 457 J/m²). After a second round of irradiation, tumor cells became more immunogenic, and the level of tumor growth inhibition increased with higher doses of UV irradiation. Thus, cells irradiated twice with 610 and 457 J/m² became rejectable in all immunocompetent C57BL/6 mice. The increase in tumor cell immunogenicity induced by UV light was not associated with the appearance of MHC I H-2 antigens. In parallel with the induction of tumor cell immunogenicity, UV irradiation made tumor cells more sensitive to NK cell-mediated cytotoxicity and to lysis by TNF. An increase in sensitivity to natural cell-mediated cytotoxicity and TNF was observed after single or double doses (152 to 610 J/m²) of UV irradiation. The cells that showed the highest levels of immunogenicity were found also to be most sensitive to lysis by TNF. MCA102UV cells cultured in the presence of increased doses of recombinant TNF became resistant to its cytotoxicity without losing their immunogenicity, suggesting that immunogenicity and TNF sensitivity are 2 independent UV-induced properties^{ref1, ref2}.
• mice bearing s.c. D5 melanoma or MCA 205 sarcoma tumors were treated with intratumoral (i.t.) injections of bone marrow-derived unpulsed DCs in combination with local fractionated tumor irradiation. DC administration alone slightly inhibited D5 tumor growth and had no effect on MCA 205. RT alone caused a modest inhibition of both tumors. DC administration combined with RT inhibited D5 and MCA 205 tumor growth in an additive and synergistic manner, respectively. In both tumor models, RT intensified the antitumor efficacy of DC administration independent of apoptosis or necrosis within the tumor mass. Combination treatment of i.t. DCs + RT was superior to s.c. injections of tumor lysate-pulsed DCs + IL-2 in inhibiting D5 tumor growth and prolonging survival of mice. Splenocytes from mice treated with i.t. DCs + RT contained significantly more tumor-specific, IFN-g-secreting T cells compared with control groups. Moreover, adoptive transfer of these splenocytes mediated significant tumor regression in mice bearing established pulmonary metastases. Combined treatment followed by resection of residual s.c. tumor conferred protective immunity against a subsequent i.v. tumor challenge. Furthermore, i.t. DC + RT treatment of s.c. tumor in mice bearing concomitant pulmonary metastases resulted in a significant reduction of lung tumors. i.t. DC administration combined with RT induces a potent local and systemic antitumor response in tumor-bearing mice. This novel regimen may be beneficial in the treatment of human cancers^ref.
• synergy with oncolytic viruses
• synergy with suicide gene therapy : using HSVtk suicide gene therapy, macrophages can distinguish between tumor cells dying through classical apoptosis and tumor cells engineered to die through nonapoptotic mechanisms, resulting in secretion of either immunosuppressive cytokines (IL-10 and TGF-ß) or inflammatory cytokines (TNF-a or IL-1ß), respectively. Additionally HSP70 acts as one component of a bimodal alarm signal that activates macrophages in the presence of stressful, immunogenic tumor cell killing. These differential responses of macrophages can also be used to vaccinate mice against tumor challenge, using adoptive transfer, as well as to cure mice of established tumors^ref
• synergy with viral fusogenic membrane glycoproteins (FMGs) is a potent strategy for antitumor cytotoxic gene therapy in which tumor cells are fused into large multinucleated syncytia. K1735 cells, a poor allogeneic melanoma vaccine, can be made more effective for protection against B16 in vivo. To promote antigen release in an immunologically effective manner, tumor cells were transfected with VSV G glycoprotein, which kills cells through the formation, and degeneration, of large multinucleated syncytia. Vaccines consisting of a 1:1 mix of fusing allogeneic and autologous cells led to dramatic increases in survival of mice in both prophylactic and therapy models, dependent upon T cells, the mechanism of tumor-tumor cell fusion, and the nature of the fusion partner. Syncytia activate macrophages and fusogenic membrane glycoprotein-mediated cell killing very efficiently promotes cross-priming of iDCs with a model tumor antigen^ref. Syncytia are highly ordered structures over 24-48 h but then die through processes that, by multiple morphological and biochemical criteria, bear very little resemblance to classical apoptosis. Death of syncytia is associated with nuclear fusion and premature chromosome condensation as well as severe ATP depletion and autophagic degeneration, accompanied by release of vesicles reminiscent of exosomes (syncytiosomes). Dying syncytia produce significantly more syncytiosomes than normal cells or cells killed by irradiation, freeze thaw, or osmotic shock. These syncytiosomes also load DCs more effectively than exosomes from cells dying by other mechanisms. Finally, syncytiosomes from either autologous or allogeneic fusing melanoma cells lead to cross-presentation of a defined tumor antigen, gp100, by DCs to a gp100-specific CTL clone. Cross-presentation was significantly more efficient than that with exosomes from normal, irradiated, or HSVtk/ganciclovir-killed tumor cells^ref
• electrofusion (EF) process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation : EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells^ref.
• intraprostatic administration of live bacille Calmette-Guerin (BCG) in humans has been found to produce tumor necrosis; unfortunately, the number of severity of complications have made its clinical use prohibitive. Previous studies have shown that soluble and microparticulate components present in the supernatants obtained after centrifugation of a reconstituted BCG preparation exhibit similar immunogenicity to the one shown by live bacteria. The supernatants, however, are not associated with disseminated infection of the progressive regional tissue destruction observed with the use of viable vaccine. Experiments were conducted to determine the effect of intraprostatic injection of BCG and its supernatants. Adult dogs, after positive conversion to protein purified derivative (PPD), were randomly assigned to 3 groups. Under direct vision and with digital rectal control, intraprostatic injections of various agents were given as follows: group I, normal saline; group II, live BCG; group III, 200 mg of BCG supernatants. 2 months later the animals were sacrificed, and the prostates removed in toto and submitted for a thorough histological examination. Extensive but variable tissue necrosis was noted in groups II and III. No histological alterations were present in group I. The histological picture of the animals receiving BCG supernatants conclusively demonstrated circumscribed necrosis of the gland. Side effects and complications were present in animals receiving live BCG but conspicuously absent in the ones receiving supernatants^ref.
• the induction of cell death by high hydrostatic pressure (HHP) is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures = 150-250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures > 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells^ref.
• synergy with thermal therapy :
• transpupillary thermotherapy (TTT) may not only have a direct destructive effect on the primary tumour, but may also influence the immunogenicity of uveal melanoma cells, induce infiltration of macrophages into the tumour, and induce apoptosis^ref.
• radiofrequency ablation (RFA) of the liver produces necrosis of the hepatocytes. Increased HSP70 expression enhances immunogenicity of cells in the zone of transition that can have therapeutic implications for the treatment of liver^ref.
• heat immunotherapy : dendritic cells (DCs) are potent APCs that play important roles in regulating immune responses in cancer. Immunotherapy using these immunocytes has become an accepted therapeutic modality. Hyperthermia using magnetic nanoparticles induces antitumor immunity, which could be activated by adjuvant including cytokines. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs were injected into a mouse EL4 T-lymphoma nodule in C57BL/6 mice, which were subjected to AMF for 30 min. The temperature at the surface of the tumor reached 45 degrees C and was maintained by controlling the magnetic field intensity. Hyperthermia treatment was repeated twice with 24 h intervals. After hyperthermia, immature DCs were directly injected into the EL4 nodule. As a result, complete regression of tumors in 75% (6/8) of the mice was observed, while the percentage of complete regression of tumors was 12.5% (1/8) in the case of mice treated by hyperthermia alone^ref.
Dendritic cells (DCs) primed with tumor antigens can effectively mediatethe regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-g release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-g production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in 2 of 3 cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in 2 analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge^ref.
In the absence of phagocytosis, apoptotic cells may undergo secondary necrosis, a process associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflammatory conditions^ref.
• release of a normal intracellular molecule : the maturation of APCs is also necessary for immune responses that occur in the absence of pathogens^ref. However, the adjuvant signals involved are less characterized. Tissue damage per se activates immune responses^{ref1, ref2, ref3, ref4}. Injured tissue evokes acute but generally transient immune responses against "self" constituents^ref :
• autoimmunity represents a caveat to the use of DCs as adjuvant for human vaccines. DCs from normal BALB/c mice or from mice prone to autoimmunity (NZB x NZW) F₁ were allowed to phagocytose apoptotic thymocytes and vaccinated syngeneic animals. All mice developed anti-nuclear and anti-dsDNA Abs. Autoantibodies in normal mice were transient, without clinical or histological features of autoimmunity or tissue involvement. In contrast, autoimmunity was maintained in susceptible mice, which underwent renal failure and precociously died. The data suggest that DC vaccination consistently triggers autoimmune responses. However, clinical autoimmunity develops in susceptible subjects only^ref.
Antigens from dying cells are preferentially recognized in vivo^ref, and dying cells release adjuvant factors that amplify and sustain T-cell-dependent immune responses, in situ and at a distance^ref.
• endogenous ligands of TLRs^ref :
• RNA (dsRNA regions of mRNA bind to TLR3)
• DNA
• chromatin-IgG complexes bind to TLR9
• mouse and human NKG2D ligands are upregulated in non-tumour cell lines by genotoxic stress and stalled DNA replication, conditions known to activate a major DNA damage checkpoint pathway initiated by ATM (ataxia telangiectasia, mutated) or ATR (ATM- and Rad3-related) protein kinases. Ligand upregulation was prevented by pharmacological or genetic inhibition of ATR, ATM or Chk1 (a downstream transducer kinase in the pathway). Furthermore, constitutive ligand expression by a tumour cell line was inhibited by targeting short interfering RNA to ATM, suggesting that ligand expression in established tumour cells, which often harbour genomic irregularities, may be due to chronic activation of the DNA damage response pathway. Thus, the DNA damage response, previously shown to arrest the cell cycle and enhance DNA repair functions, or to trigger apoptosis, may also participate in alerting the immune system to the presence of potentially dangerous cells^ref.
• cytoplasmic heat-shock proteins (HSPs) bind to TLR2^ref
• high-mobility group B1 protein (HMGB1) is an abundant and conserved nuclear constituent loosely bound to chromatin, and a mediator of inflammation in the extracellular environment^{ref1, ref2}. Extracellular HMGB1 is responsible for the inflammatory response to cell necrosis, as shown in a model of acute liver toxicity^ref. Receptor for advanced glycation end products (RAGE) is a surface receptor for HMGB1 on dendritic cells^ref, but TLR2 and TLR4 may be involved, too^ref. RAGE activation results in NF-kB translocation^ref, and the NF-kB pathway is responsible for most events elicited by necrotic cells^ref. Administration of HMGB1 to normal animals causes inflammatory responses, including fever, weight loss and anorexia, acute lung injury, epithelial barrier dysfunction, arthritis, and death. Anti-HMGB1 treatment, with antibodies or specific antagonists, rescues mice from lethal endotoxemia or sepsis and ameliorates the severity of collagen-induced arthritis and endotoxin-induced lung injury^ref. HMGB1 is released into the extracellular milieu by :
• damaged and necrotic cells (passive release)^ref; in contrast, the chromatin of apoptotic cells sequesters HMGB1 : human monocyte-derived DCs matured when challenged with necrotic HeLa cells. Cell constituents released into the supernatant were sufficient to cause DC maturation. In contrast, DCs challenged with low numbers of early apoptotic cells or their supernatant did not mature. The addition of HMGB1-specific antibodies or of the HMGB1 inhibitory fragment box A^ref to the supernatants of necrotic HeLa cells abrogated DC maturation. Fibroblasts from wild-type or Hmgb1^-/- mice were necrotized; supernatants from wild-type cells contained HMGB1, whereas those from Hmgb1^-/- cells did not. Only supernatants containing HMGB1 activated the DCs, as shown by the upregulation of membrane markers (HLA-DR, CD40, CD83, CD80 and CD86) and the induction of TNF-a secretion; the insoluble fraction from wild-type or -/- necrotic cells was not active. The addition of recombinant HMGB1 to the supernatant of necrotic -/- fibroblasts only partly reconstituted the effect of the endogenous counterpart, suggesting a possible role for intracellular regulatory activities. Necrotic wild-type cell supernatants complemented with anti-HMGB1 antibodies and necrotic -/- cell supernatants had similar effects on the DC activation state. In living polymorphonuclear neutrophils (PMNs), HMGB1 is contained in cytoplasmic vesicles as well as in the nucleus. Necrotic PMNs do not release HMGB1; the protein is tightly associated with an insoluble fraction and fails to be released by treatment with ionic and nonionic detergents. Necrotic PMN supernatants, which therefore do not contain HMGB1, did not trigger DC maturation. C57BL/6 mice injected with apoptotic RMA lymphoma syngeneic to C57BL/6 mice are poorly immunogenic at protecting from challenge with a large number of living lymphoma cells 14 days after vaccination^ref: all mice subsequently challenged with living lymphoma cells developed the tumour within 10 days. C57BL/6 mice injected with apoptotic lymphoma cells together with the supernatants of necrotic fibroblasts, either wild type or Hmgb1^-/-. The mice that had received wild-type necrotic cell supernatants rejected the tumour; 80 days later, they also rejected a further challenge with living lymphoma cells, indicating that the response was long-lasting. Supernatants of necrotic Hmgb1^-/- fibroblasts were less efficient in eliciting a protective immune response: 75% of the mice developed lymphomas and eventually died. The blockade of HMGB1 with antibodies reduced (but did not abolish completely) the protection elicited by necrotic -/- cell supernatants by 50%. These results indicate that HMGB1 released by necrotic cells is a potent adjuvant in vivo and that other intracellular components contribute to the adjuvant activity of necrotic cell supernatants. Candidates include heat-shock proteins^ref and uric acid^ref. Subcutaneous coinjection of ovalbumin (OVA) with purified recombinant HMGB1 elicited a striking increase in OVA-specific IgG antibody titres, a well-characterized feature of endogenous adjuvants^ref. Similar effects on OVA immunogenicity were obtained in wild-type C57BL/6 mice, wild-type LPS-responsive C3H/HeN mice and in C3H/HeJ mice, which are LPS-resistant due to a mutation in TLR4. This result indicates that contamination with bacterial endotoxin is not involved. Recombinant HMGB1 obtained from prokaryotic or eukaryotic cells exerted similar effects in vivo. HMGB1 per se did not induce the production of OVA-specific IgG antibodies. At the concentration used, HMGB1 was consistently less potent than complete Freund's adjuvant (CFA), considered the gold standard against which all adjuvants are measured. This is consistent with the general message conveyed by extracellular HMGB1-namely, that tissue damage has occurred^ref. Whereas the blockade of extracellular HMGB1 might represent a suitable therapeutic target for the treatment of sepsis^ref and rheumatoid arthritis^ref, the local administration of recombinant HMGB1 may enhance the immune responses against cancer^ref.
• activated dendritic cells (monocytes and macrophages on activation by LPS, TNF-a or IL-1b^{ref1, ref2}) : this actively secreted HMGB1 is necessary for the up-regulation of CD80, CD83, and CD86 surface markers of human DCs and for IL-12 production. The HMGB1 secreted by DCs is also required for the clonal expansion, survival, and functional polarization of naive T cells^ref. In myeloid cells, HMGB1 is secreted through specific organelles, the secretory lysosomes^ref, and is extensively acetylated in order to reach them from the nucleus^ref. Thus, HMGB1 secreted actively by inflammatory cells is molecularly different from HMGB1 released passively by necrotic cells. There is no reason to believe that the secreted protein cannot activate immune responses, but this should be verified formally when adequate amounts of acetylated HMGB1 become available.
Necrotic HMGB1^-/- cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants. In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines^ref.
• heme released from hemeproteins has been shown to promote the formation of oxygen radicals, playing a role as a catalyst in the oxidation of lipids^ref, proteins^ref, and DNA^ref. In addition, free heme can promptly bind to and oxidize low-density lipoprotein (oxLDL), acting as a biologically relevant lipoprotein oxidant^ref. Hemoglobin (probably because of the release of free heme and heme iron) may contribute to the acute renal failure often seen after episodes of intravascular hemolysis^ref. In fact, it has been proposed that heme could be considered one causative agent in organ failure after ischemia-reperfusion because heme-oxygenase is induced in heart and kidney^ref. In normal conditions, diverse species produce avid heme-binding plasma proteins, such as hemopexin, that efficiently remove most of the heme produced intravascularly^ref, thus preventing nonspecific cellular heme uptake and heme-catalyzed oxidation reactions. However, pathologic situations of increased hemolysis can lead to very high levels of free heme, as in malaria^ref, sickle cell disease^ref (>20 µM^ref), HELLP (hemolysis, elevated liver levels, and low platelet count) syndrome^ref, or regions with high erythrocyte shear forces or turbulent blood flow^ref. Very little work has been done to assess the consequences of the interaction of free heme with intact cells. It has been demonstrated that free heme is promptly incorporated into endothelial cells in vitro and that this association potentiates the oxidative damage induced by chemical agents^ref. It is interesting to note that patients suffering from sickle cell disease often exhibit a low-grade chronic inflammation. This state correlates with the enhanced expression of adhesion molecules on activated endothelial cells, leukocytes, and reticulocytes^{ref1, ref2, ref3}, but the specific agent that triggers this process remains unknown. Leukocyte migration into tissues is the hallmark of all types of inflammatory responses. Polymorphonuclear neutrophils (PMNs) are blood-borne inflammatory cells with oxidative and proteolytic potential that are usually the first cells involved in pathogen recognition, playing an essential role in the host defense against invading microorganisms. The inflammatory process can be amplified by the neutrophils themselves through the production of arachidonic acid-derived bioactive lipids such as LTB₄, cytokines (ie, IFN-g, IL-1, TNF-a), or chemokines (IL-8, GRO-a). On exposure to the proper stimulus, PMNs also release a variety of reactive oxygen species (ROS), including superoxide anion (O₂^.-), hydrogen peroxide (H₂O₂), hydroxyl radical (^.OH), and hypochlorous acid (HOCl). The generation of O₂^.- by PMNs occurs during phagocytosis or upon stimulation by soluble agonists, such as phorbol ester, chemotactic peptides, and calcium ionophore. Binding of several agonists to their specific receptors has been shown to trigger a signal-transduction cascade that ultimately activates NADPH oxidase. Several pieces of evidence point to PKC activation as an essential step for the initial activation of NADPH oxidase and, consequently, to the production of ROS^{ref1, ref2, ref3}. Heme can modulate several neutrophil-related responses either by a PKC-dependent pathway (such as migration, actin cytoskeleton reorganization, and ROS production) or by a PKC-independent mechanism (IL-8 expression). Heme can act as a physiologically relevant proinflammatory signaling molecule that may be associated with the development of inflammation in hemolytic diseases. Hemorrhagic episodes and severe hemolysis are often associated with tissue injury that can trigger the inflammatory response characterized by intense leukocyte infiltration. The demonstration that hemopexin levels are decreased in patients with a variety of hematologic disorders suggests the importance of other plasma constituents able to bind heme during hemolytic episodes. Although albumin is capable of drawing hemin away from red cell membranes, the detection of heme-albumin complexes portends an extremely poor prognosis for patients with life-threatening hemorrhagic shock^ref. In experiments with rats, intrathoracic injection of hemin induced an acute inflammatory reaction, characterized by edema formation and intense accumulation of neutrophils in the pleural cavities. The stimulation of neutrophil migration in vivo and in vitro reported suggests that free heme has the potential to serve as an endogenous chemoattractant. Association of hemin with human albumin did not reduce its propensity to promote neutrophil migration. This is consistent with the fact that binding of heme to albumin^ref does not prevent heme uptake, and therefore, after dissociation, heme will eventually diffuse into circulating and endothelial cells. A primary event in the inflammatory response is the recruitment of neutrophils into sites of inflammation. In vivo or in vitro, leukocyte migration induced by different chemoattractants is promptly stimulated by phorbol esters, indicating that PKC stimulation can lead to neutrophil chemotaxis. Heme was shown to be a potent activator of PKC in an insect cell model^ref. Because of its highly hydrophobic nature and considering that at neutral pH, the 2 lateral carboxyl groups of hemin are dissociated, a possible explanation for the PKC activator effect might be interaction of hemin carboxyl groups with the phospholipid-binding regulatory domains of the enzyme. However, an indirect effect, such as oxidation of regulatory and/or binding domains, should not be discarded because it has been shown that PKC can be activated by superoxide anion, redox cycling quinones, and micromolar levels of periodate^{ref1, ref2}. Because hemin was able to activate PKC in PMNs, a possible direct implication is the modulation of PKC-related gene expression. Expression of proinflammatory and immunoregulatory cytokines rapidly increases in the lung after hemorrhage^ref, but the specific agent responsible for the onset of these proinflammatory responses remains unknown. Hemin can boost the expression of a₂-macroglobulin after systemic inflammation induced by LPS, indicating a possible role for heme in the expression of positive acute-phase proteins (APP)^ref. Hemin could act as a signaling molecule involved in the triggering of inflammatory processes often associated with hemolysis^ref.
• thioredoxin (Trx)^ref is a 12-kD, ubiquitous intracellular enzyme with a conserved CXXC active site that forms a disulfide in the oxidized form or a dithiol in the reduced form^{ref1, ref2}. Trx catalyzes dithiol-disulfide oxidoreductions, and, together with Trx reductase 1 and 2 and NADP, is a general protein oxidoreductase and a hydrogen donor for ribonucleotide reductase essential for DNA synthesis^ref. In addition, it has intracellular antioxidant activity and, when overexpressed, protects against oxidative stress^ref. Trx is secreted by monocytes, lymphocytes, and other normal or neoplastic cells in infection and inflammation by a leaderless pathway, a property it shares with IL1-b^ref. Secreted Trx exhibits several cytokine-like activities^ref. In particular, Trx secreted by HTLV-1-transformed T cells was originally described as adult T cell leukemia-derived factor and acts as an inducer of the IL-2R^{ref1, ref2}. Other activities include activation of eosinophil functions, such as cytotoxicity and migration^{ref1, ref2}, and potentiation of TNF production^ref. Clinically, increased Trx levels have been reported in HIV disease^ref and in the synovial fluid of patients with rheumatoid arthritis^ref. Trx is a unique chemoattractant for neutrophils, monocytes, and T cells with a potency comparable to other known chemokines. Trx contributes significantly to the chemotactic activity released in HTLV-1-infected cells, that have long been known to secrete Trx^ref. In particular, the HTLV-1-transformed MT4 cell line spontaneously releases chemotactic activity for monocytes that is not mediated by TNF, IL-8, or MCP-1^ref. A major monocyte chemotactic factor was purified from MT4-conditioned medium and identified as MIP-1/LD78^ref : it was showed to be active on monocytes but only weakly chemotactic for PMNs^ref. An anti-Trx antibody significantly inhibits the chemotactic activity of MT4 supernatants, an observation that might explain why the known chemokines produced by these cells could not account for all of their chemotactic activity. As far as the mechanism of the effect of Trx is concerned, we obtained convincing evidences that Trx does not act through a chemokine receptor. First of all, Trx does not induce an increase of intracellular Ca²⁺, which is observed with all chemokines, whose receptors are coupled to G proteins. Furthermore, chemotactic activity of Trx is not inhibited by PTX. Although chemotactic receptors have been shown to be able to couple with both PTX-sensitive and PTX-insensitive GTP binding proteins^{ref1, ref2}, only bg subunits associated with Ga_i are responsible for chemotactic receptor-mediated cell migration^ref. The finding that Trx chemotactic response is PTX insensitive, along with the lack of effect of Trx on intracellular Ca²⁺, strongly argue against its direct interaction with a 7TM domain chemotactic receptor. In contrast, Trx may initiate signal transduction for chemotaxis by oxidizing and cross-linking appropriate cell surface molecules. Trx is mainly known as a protein disulfide-reducing enzyme; however, it can also act as protein disulfide isomerase in the formation of disulfides during protein folding^ref, particularly when in an oxidative environment^ref. Thus it is possible that, in an extracellular environment, Trx acts by oxidizing thiols of one or more membrane proteins or catalyzing isomerization reactions. However, since Trx's chemotactic activity is G protein independent, this putative oxidized receptor is likely to differ from any of the known chemokine receptors. In fact, the Trx target could behave like a "sensor", in a fashion similar to the hemoprotein, which acts as the oxygen sensor in the induction of erythropoietin^ref. Several lines of evidence suggest that the chemotactic activity of Trx is due to its enzymatic action on cell surface protein substrates. First of all, a C32S/C35S mutant of Trx where the cysteines of CXXC active site have been mutated, and that has lost enzyme activity^ref, was not chemotactic. Furthermore, glutaredoxin, which differs from Trx in substrate specificity, has no chemotactic activity. In fact, although Trx catalyzes the oxidation/reduction of a wide range of inter- or intra-protein disulfides, the preferred substrates of glutaredoxin are mixed disulfides between proteins and glutathione, and the substrate specificity for disulfides is very different^{ref1, ref2, ref3}. A further support to this hypothesis is the chemotactic activity of Trx from Spirulina. Since algal Trx has only little (~20%) similarity with human Trx^ref, but has the same conserved CGPC active site human Trx, we think it is more likely that Trx may act through its enzymatic activity rather than by binding to a membrane receptor. Consistent with this, various investigators have been unable to identify specific binding sites for Trx on the cell membrane of various cell types^ref. The local release of Trx is likely to be important, in concert with other chemokines, in recruiting cells during infection and inflammation. Consistent with this hypothesis, Trx is secreted by IFN-g- or endotoxin-stimulated macrophages or activated T lymphocytes^{ref1, ref2} and has been measured locally in arthritic patients^ref. Trx is also an acute-phase protein in that its production by the liver is increased in rats injected with LPS^ref. In addition, Trx may also be a major chemoattractant in diseases associated with oxidative stress, such as ischemia/reperfusion, since Trx production is induced by oxidants^refand Trx is elevated locally in cerebral ischemia or brain injury^{ref1, ref2} and open-heart surgery^ref. Thus, according to this hypothesis, Trx might function as a signal of oxidative stress that amplifies the cellular response at a site of inflammation.  second hypothesis on the significance of Trx in disease stems from the observation that Trx levels were found to be elevated (>30 ng/ml plasma) in a proportion of HIV- infected subjects with CD4 T cell counts <200/µl blood^ref. Nearly all of the high-Trx subjects died within 18 mo of the Trx measurement, although none had active opportunistic infections or other signs of debilitating disease at the time of measurement. Deaths in an otherwise similar group of HIV-infected subjects with normal Trx levels were minimal during the same period^ref. This survival difference may be directly traceable to the chemoattractant activity of the Trx present in circulation. Intravenous injection of IL-8 has been shown to inhibit PMN accumulation in response to local injection of IL-8^ref. Furthermore, leukocyte migration is impaired in transgenic mice overexpressing human IL-8 or MCP-1 in circulation^{ref1, ref2}. High levels of circulating Trx may similarly decrease the ability of leukocytes to migrate efficiently to a site of infection, either by counteracting the gradient of local chemoattractants or downregulating the chemokine receptors by desensitization (or both). Consistent with this, PMNs and monocytes from AIDS patients have been shown to be deficient in their ability to migrate^{ref1, ref2}. Thus, by potentially interfering with chemotaxis, elevated serum Trx levels in HIV patients could contribute to augmented susceptibility to bacterial or viral infections and hence constitute a serious threat to survival in the ensuing months. The possibility that a chemoattractant acts through its enzymatic activity, rather than through classical receptor binding, opens the way to new strategies aimed at inhibiting its action, using enzyme inhibitors rather than receptor antagonists or antibodies^ref
• cytoplasmic lectins : galectin 3^{ref1, ref2, ref3}
• displacement of a molecule normally not found in a given compartment :
• extravascular fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation, binds to TLR4^ref
• phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P₂X₇. Changes in PS distribution were shown to modulate several membrane activities: Ca²⁺ and Na⁺ uptake through the P₂X₇ cation channel itself; P₂X₇-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance^ref.
Cells that are healthy may send calming signals to local APCs, while cells that die by apoptosis send "eat me" signals to a neighbouring scavenger that should not induce the expression of costimulation..
Dangerous entities may be self (set c) such as mutations that lead to stress or inappropriate cell death or inefficient scavenging; or nonself, such as pathogens (set e), environmental toxins (set d), and such. Set f would contain fetuses and evolutionarily distant nonself organisms that have PAMPs, but that are not dangerous (e.g. symbiotic organisms, well-adapted viruses).

Anyway the infectious nonself (INS) model and the danger model share common ground as mitochondria are essentially intracellular bacteria, so the same mechanisms could potentially be employed both to detect pathogens and to detect host cell lysis. In self-nonself models, T cells must be trained to tell the difference between these foreign and self antigens. Instead in an immune system poised for danger, the T cells need only to distinguish APCs from everything else. To build such a T cell population, we need 3 basic "laws of lymphotics" and 1 minor exception :
• resting T cells need 2 signals to be activated :
• signal A from TCR binding to MHC/peptide
• signal B (co-stimulation) from an APC
T cells die if they receive signal A without signal B (leading to tolerance) and become activated if they receive both.
• resting T cells can receive signal B only from APCs : dendritic cells (DCs) serve as APCs for both virgin and experienced T cells, and B cells can re-stimulate experienced but not virgin T cells. Though other tissues might express surface MHC/peptide complexes (signal A), they do not express the appropriate co-stimulatory signals (signal B) needed by T cells.
• exception : during negative selection thymocytes  would be unable to receive signal B from any cell, including professional APCs. Any thymocyte recognizing the normal surface MHC/antigen profile of a DC would thus be eliminated by the receipt of signal A without signal B.
• the activated effector stage only lasts for a certain period of time. During this time, T cells do not require signal B and can be triggered to function (eg. help B-cells or kill targets) by signal A alone (ie even by cells other than APCs). After a while, they either die or return to a resting state from which they can only be drawn again by the appropriate combination of signal A + signal B.
Among the virgin T cells circulating through the lymph node draining the infected site, some are specific for danger peptides displayed by the activated APC and others, having had no opportunity to become tolerant of non-thymic tissue antigens, will recognize peptides from the extra-thymic tissue : but because such autoantigen(s) is everywhere they will distribute all over the body. Each will kill a few tissue cells and then drain to a local lymph node : unless that node is draining an infected site, the autoreactive killers will not be reactivated. They will exit the node as resting memory cells and recirculate from blood to tissues : encountering autoantigen again on normal tissue cells, and thus receiving signal A without signal B, they will die, as will do any autoantigen-specific cell returning to the original infected area after the infection is cleared. "Normal" autoimmunity is self-limiting as killer cells induce apoptosis in their targets : although the ⁵¹Cr release assay gives the impression that targets die by membrane disruption, DNA fragmentation actually occurs hours before the membranes disintegrate^ref. In vivo, these apoptotic cells are probably scavenged long before their membranes fall apart. Consequently death induced by killer cells does not signal local APCs to perpetuate the immune response.Thus, although both autoreactive and danger-specific killers follow the same laws, their fates differ because their antigens are critically dissimilar : autoantigens are continuously expressed by healthy cells incapable of delivering signal B. By sheer numbers and persistence, they ensure that circulating autoreactive cells should regularly be deleted. Of course the efficiency of deletion will depend on the size of an organ and the rate at which lymphocytes circulate through.
The danger model differs from the associative recognition model solely with respect to the origin of effector T helpers (eT_h). More properly, the danger model should be juxtaposed to the Ag-independent model for the origin of eT_h cells (i.e., the primer question). The introduction of danger in no way challenges the need to make a self-nonself discrimination, nor, for that matter, does it challenge the need to define self as those Ags encountered in the absence of eT_h and that persist, whereas nonself are those Ags encountered in the presence of eT_h and that are transient^ref.
Supporting evidences :
• neither lytic (necrotic) cell death nor production of stress proteins occur in ...
• metamorphoses
• puberty (many new hormones are produced)
• pregnancy
• new milk proteins from lactating breast
• new foetal antigens : fetal cell migration is not universal but occurs in only a fraction of pregnancies, the mother is not continuously exposed to circulating fetal cells and, in fact, has the capacity to eliminate them without eliminating the fetus^ref
• ageing
• infections from ...
• useful commensal organisms
• harmless opportunistic organisms
With such an immune system we are not sterile static entities and we live in harmony with our internal and external environment, becoming a habitat
• tumors^ref (cells die by apoptosis). Further as tumor grows it induces deletion of tumor specific T cells^ref. This view fits with the finding that :
• T cell clones isolated from such patients can kill melanoma cells taken from HLA matched patients that have not rejected their tumors, showing that it is not lack of antigen expression that prevents tumor rejection
• temporaneous induction of active tumor immunity :
• when it becomes repeatedly infected, stressed or otherwise necrotic, small tumors spontaneously regress thanks to any tumor specific T cell clone that had not yet been deleted^ref. In some cases, the T cells might also destroy normal cells of the same type expressing similar antigens (e.g. melanocytes leading to the vitiligo occasionally found with spontaneous melanoma regression).
• an injection of heavily irradiated tumor cells transfected with GM-CSF can evoke protection against the untransfected tumor. This treatment has 2 important features :
• though radiation death is usually apoptotic, an injection of large numbers of dying cells may well overload local scavenging capacities, allowing some of the tumour cells to disintegrate before they can be cleared, and thus signalling local APCs.
• GM-CSF enhances the well being of DCs in vitro and may do the same in vivo.
The combination of dying cells and APC-enhancing cytokine would thus provoke activation of tumor specific T cells that could now kill a certain number of living, untransfected tumor cells.
• activation by any crossreactive antigen should be enough : the immunizations could merely be injections of disrupted tumor cells in adjuvant. The injections might consist of tumor cells that had been infected with a lytic virus or an inducible death gene, with or without the addition of APC-enhancing cytokines. One could paint visible surface tumors (repeatedly) with noxious substances, calculated to kill off enough tumor cells to activate local APCs. It might help to add carrier determinants to activate helper cells, or to inject professional APCs fed with the tumor cells (or their antigens, if known).
Though a single immunization would initiate immunity, the response would soon die down for lack of repeated stimulus : the killers would rest down and, in the absence of more danger signals, would not be re-activated, explaining why the protection generated by the transfected tumor can be overcome with a large enough challenge of normal tumor cells. Even if some of the tumor were destroyed by killers activated during the first immunization, this apoptotic death would not maintain the response. The tumor meanwhile, like any other tissue expressing signal A without signal B, would induce deletion of tumor-specific memory cells as they rested down. To be effective, therefore, immunizations should be repeated until the last vestiges of tumor are gone. In addition such immunizations should be done early, while the tumor is small and has not had time to delete a large number of tumor specific T killers, or alternatively they should be held off for a while after removal of the main tumor mass in order to give the thymus time to repopulate the periphery with new tumor specific T cell populations.
• temporaneous induction of adoptive tumor immunity : if TILs are removed from the tumor, activated in vitro and re-injected along with a source of IL-2, they may well return to the tumor and engage in a round of destruction. If the tumor burden is small, this may be enough to destroy it. However, if there are too many tumor cells for the injected T cells to destroy in the first round, the T cells will go through their natural cycle of resting down and waiting to be re-stimulated. Without a source of activated APCs, they will remain in the resting state, now to be tolerized by recognition of tumor antigens in the absence of signal B.
No signal 0 is delivered => no activation of local APCs => hence no immune response occurs.
• lytic cell death occurs in ...
• tissue transplants : they cannot be performed without surgical and/or ischemic damage
• the lower extent of damage can explain why long-term living unrelated renal donation (LURD) allograft survival rates are lower than those for HLA-identical but equivalent to those of haploidentical and better than those of cadaveric kidney transplantations^ref
• graft-versus host disease (GvHD) is less severe ...
• ... in gnotobiotic animals undergoing allogeneic bone marrow transplantation (BMT)^{ref1, ref2}
• ... in recipients that have had gentle rather than harsh preconditioning treatment : synergistic damage by increased total body irradiation (TBI) and allogeneic donor cells permits increased translocation of lipopolysacharide (LPS) into the systemic circulation, which triggers excess TNF-a from macrophages priming the GVH reaction ^ref
• organs with a larger number of parenchymal cells, and those that have minimal blood-tissue barriers (like the liver^ref) are more tolerized after allogeneic transplantation whereas small and/or well barricaded organs (brain, ...) induce deletion at a much slower rate. The process begins when APCs in the liver become activated by the surgical trauma, home to the local (recipient) lymph node and activate recipient's donor-specific T cells which then migrate to the newly transplanted organ and begin killing all donor cells, including hepatocytes, endothelial cells and APCs. After a while, all the bone marrow-derived APCs will have died, but the regenerating liver lasts longer, continues to offer signal A without signal B and, rather quickly because of its size, deletes all the relevant T cells. Should any HSCs remain, they can now take up residence, creating the chimerism that is often (but not always) seen in recipients of liver grafts. In immunologically mature hosts, chimerism resulted in immunity and stronger graft rejection. In immature hosts, it resulted in tolerance to the chimeric T cells, but not to graft antigens not expressed by the chimeric cells^ref.
• an apparent exception to the idea that tissues induce tolerance to themselves comes from transplant recipients who suddenly stop taking cyclosporin A and reject their grafts, even though they may have had them for over 20 years! According to the model, these patients should be tolerant. The problem here is not the model but the drug : since CsA blocks signal A not signal B, the first law of lymphotics cannot operate and no deletion can occur. Although this effectively blindfolds the lymphocytes, the drugs must be given for life. This is nicely illustrated by an experiment in which rats were given allogeneic livers under a 2 week course of CsA and, at various times later, were immunized with donor-type skin :
• skin grafts given 0-4 weeks after CsA withdrawal were rapidly rejected and also stimulated rejection of the livers
• skin grafts given 8 weeks after CsA withdrawal were rapidly rejected but livers were not
The authors determined that there were 3 stages of reactivity :
• during CsA treatment, alloreactive T cells were unable either to respond or be tolerized.
• "transitional" stage : when the drug is withdrawn, the T cells were slowly tolerized unless activated by a new source of APCs. This stage is delayed by the CsA treatment
• the animal had become solidly tolerant of the graft
Tissues cannot induce tolerance to themselves in the presence of a signal A blocker. This may also be why irradiated rodents whose immune systems are allowed to regenerate under the cover of CsA suffer autoimmune symptoms when the drug is discontinued. The solution, of course is to find drugs that block signal B without obstructing signal A, and some steps have already been made in this direction.
• soluble CTLA-4 ...
• evoked long lasting tolerance of xenogeneic islet grafts : the grafted tissue stayed in place, continually tolerizing for itself by expressing its antigens in the absence of signal B
• inhibited antibody responses to KLH and SRBC though not permanently : that's why neither KLH nor SRBC persist for long in mice and, in their case, a temporary ablation could not generate long lasting tolerance
• antibodies to adhesion molecules run the risk of inhibiting signal A, in which case, like CsA, they may stop rejection but nevertheless not allow for the induction of tolerance
An optimal combination might be to give blockers of signal B along with a source of donor's HSCs in addition to the grafted organ : in this case, the organ itself should tolerize any mature T cells and the stem cells should generate the necessary chimerism to induce tolerance of newly maturing T cells in the thymus, a protocol occasionally mimicked by liver grafts, accounting for their success.
• extracorporeal shock wave lithotripsy (ESWL) causes IgA antibody against Tamm-Horsfall glycoprotein (THP) / uromucoid to be significantly higher (P < 0.05) in patients 30 days after treatment^ref
• fine-needle aspiration biopsy (FNAB) of the thyroid causes appearance of IgM against thyroid hormones (THAb) in 4.2% and against thyroglobulin (TgAb) in 7% (however, in only a fraction of patients the immune response is followed by a IgG secondary one, long lasting in only a minority of patients)^ref
• endolymphatic sac surgery in an opposite ear felt to be afflicted with Meniere's disease causes autoimmune hearing loss in a healthy ear^ref
• lymphocytes respond to myelin proteins after spinal cord injury (SCI) and may contribute to post-traumatic secondary degeneration^ref. Nerve crush during Wallerian degeneration (WD) of the rat optic nerves (ON) induces expression of IL-18 mRNA and protein^ref
• ionizing radiations
• radiation-induced thyroid diseases include benign thyroid nodules, hypothyroidism and autoimmune thyroiditis, with or without thyroid insufficiency, as observed in populations after environmental exposure to radioisotopes of iodine and in the survivors of atomic bomb explosions^ref
• administration of radiation has been utilized in vitro and in vivo to create an inflammatory setting, via induction of apoptosis, necrosis, cell surface molecules, and secretory molecules. Caspase-mediated cellular apoptosis is induced by radiation through multiple signaling pathways. Radiation upregulates expression of immunomodulatory surface molecules (MHC, costimulatory molecules, adhesion molecules, death receptors, heat shock proteins) and secretory molecules (cytokines, inflammatory mediators) in tumor, stromal, and vascular endothelial cells. Results of animal studies indicate possible radiation-mediated modulation of tumor antigen-specific immunity. Experimental data could indicate that the radiation-induced danger microenvironment engenders a DC-mediated antigen-specific immune response. Further enhancement of radiation-mediated inflammation and cell death can be achieved via administration of radiosensitizing pharmaceuticals. Ionizing radiation could be optimized for use with cancer vaccines and generate tumor antigen-specific cellular immunity^ref.
• irritant contact dermatitis is the clinical result of sufficient inflammation arising from release of pro-inflammatory cytokines from skin cells (principally keratinocytes) in response to (usually) chemical stimuli. Different clinical forms may arise. The 3 main pathophysiological changes seen are skin barrier disruption, epidermal cellular changes and cytokine release. An important role of irritancy in allergic contact dermatitis (ACD) comes from earlier animal and human studies. Evidence is outlined which is consistent with a "danger model" of ACD rather than one based on a traditional SNS immune model. In such a model an antigenic signal will produce sensitization only in the presence of a danger signal; in the absence of a danger signal tolerance will occur. The danger signal in ACD is cytokine release from nonimmune skin cells (principally keratinocytes) and both the antigenic and "danger" signals arises from the hapten^ref.
• eosinophils are able to recognize and be activated by danger signals released from damaged epithelial cells, which may be of importance in understanding the role of eosinophils in the various inflammatory conditions in which they are involved. Necrotic intestinal cells (HT29) induced chemotaxis in human eosinophils. EPO release was elicited in eosinophils stimulated with necrotic cells derived from all cell lines, as well as from viable genital (HeLa) and respiratory (HEp-2) cells. Release of ECP was only seen in eosinophils incubated with necrotic intestinal or genital cells, not viable ones. Both necrotic intestinal and genital cells elicited FGF-2 secretion from eosinophils. TGF-b₁ was released from eosinophils exposed to viable and necrotic HT29 cells^ref.
• platelets can undergo gradient-driven migration, or chemotaxis, toward supernatants from cells injured by repeated freeze/thaws, UV light, or ischemia/reperfusion. Platelet-derived CD154 induces immature dendritic cell maturation with upregulation of costimulatory molecules and IL-12p40 production. Overall, these results provide a mechanism for platelet activation of APC facilitating the induction of adaptive immunity in environments of cell injury^ref.
• processes leading to some autoimmune diseases :
• mutations in genes governing the normal physiological death and clearance processes, in the absence of any foreigness
• environmental agents that cause cellular stress or death
• pathogens : viral antiapoptotic proteins could protect infected cells from apoptosis induced by CTLs, alter antigen cross-presentation and the priming of the immune response, or modulate the expression of danger signals from sites of infection.
• toxins :
• coculture of immature DCs with tumor cells treated with the alkylating agents melphalan and chlorambucil leads to enhanced autologous and allogeneic T-cell activation, up-regulation of surface expression of MHC and costimulatory molecules, and increased IL-12 secretion. Exposure of the same DCs to tumor cells killed by cytarabine or by freeze-thaw (primary necrosis) resulted in significantly less T-cell proliferation and IL-12 production. Exposure of DCs to DNA purified from tumor cells treated with alkylating agents also increased their T-cell-stimulating capacity, expression of CD86, and IL-12 secretion, supporting the hypothesis that the activating effects of tumor cells are linked to the nature of the DNA damage^ref
• traumas :
• autoimmune vitiligo after vaccination with melanoma-melanocyte differentiation antigens is a consequence of secondary trauma to the skin : it occurs after i.d. injection but not after i.m. injection^ref
• women seem to be more susceptible than men to certain autoimmune diseases
• Rh disease of the newborn is much more often a problem rising in the second pregnancy than in the first
Although it has long been thought that the effector class of an immune response is tailored to the targeted pathogen (e.g. virus, intra- or extra-cellular bacterium, or worm), it is actually tailored to the tissue in which the response occur. Different tissues seem to have different means of determining the effector class of a response. For example, the class of response that occurs most often in the skin, type IV hypersensitivity, is characterized by swelling, redness, an influx of macrophages, and the production of TNF-a and IFN-g. Unlike skin, however both the gut and the eye can be destroyed by DTH responses, and the most common response in these organs is the production of IgA, an antibody found at high levels in tears, saliva, and gut secretions. To ensure that IgA is made, and TNF-a and IFN-g are not, the cells of the anterior chamber of the eye produce VIP and TGF-b₁, 2 cytokines that are also made by the gut and that promote a switch to IgA and suppress the DTH response. Even innate lymphocytes appear to be useful self-reactive cells involved in local immunity.
Problems^ref : critics argue that when explaining why T cells generally respond only to infected and not normal tissue, danger theorists tend to downplay the idea that the thymus selects self-restricted T cells that can respond specifically to "foreign" peptides presented on the surface of infected tissue : but how danger theorists propose to assess the relative importance of signal 1 vs signal 2 ? Further some PAMPs that are not, by definition, strictly "dangerous" (such as bacterial DNA, or mimics of viral RNA such as poly(I:C)) are potent immunostimulatory compounds. However, in some cases the immune system may be tuned neither to danger nor strangers. Zinkernagel, for instance, argues that it is the repetitive pattern of Ag (as found for example on viral coats) or Ag localization kinetics that govern immunogenicity^ref. In the case of NK cells, which express inhibitory receptors that recognize MHC class I, cytotoxicity is enhanced when a target cell down-regulates self-MHC due to viral infection or transformation^ref. And so on. The moral is that if the diversity of immune detectors is to be appreciated, it becomes necessary to speak not of a single "danger" signal, but of multiple "danger + stranger + other" signals. But if we&rsquo;re going to appreciate the plurality of immunogenic signals, why not discuss the signals directly, and dispense with the monolithic and misleading label of "danger"? . Experiments explicitly designed to test the danger theory have been proposed^ref, and in some cases conducted, with results that apparently either confirm^ref or elegantly refute the danger theory : in the latter case skin and cardiac allografts (i.e. neo-self peripheral Ags) in immunodeficient Rag1^-/- mice without evidence of inflammation are vigorously rejected by transferred T cells or when recipients are reconstituted with T cells at a physiologic rate by nude bone graft transplantation. These results provide new insights into the role of inflammation or "danger" in the initiation of T cell-dependent immune responses. These findings also have profound implications in organ transplantation and suggest that in the absence of central deletional tolerance, peripheral tolerance mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger^ref.
The popularity of the idea that great science proceeds by the revolutionary overthrow of reigning paradigms is due to Thomas Kuhn, a historian and philosopher of science (Kuhn, T. S.. 1970. The Structure of Scientific Revolutions University of Chicago Press, Chicago). Some danger theorists have even gone so far as to compare the danger theory to the Copernican Revolution of the 16th century, in which a heliocentric view of the solar system came to replace the ancient geocentric view. Danger theorists even cite Kuhn in a defense of their radicalism^ref (Fuchs, E. J., J. P. Ridge, P. Matzinger. 1996. Response to A. M. Silverstein. Science 272:1406) (the irony is that one of Kuhn&rsquo;s most infamous suggestions was that new paradigms are not necessarily superior to their predecessors). As quoted above, one prominent danger theorist appears to think that the immune system should be viewed as having a primary driving force^ref. This belief chimes with Kuhn&rsquo;s notion that disciplines are governed by "incommensurable" paradigms and forces danger theorists to choose between self and danger.
• 2004, 5^th modification (damage-associated molecular patterns (DAMPs), Polly Matzinger and Seung-Yong Seong). As there is evidence for both the INS and danger models^{ref1, ref2}, one can ask whether there might be a way to bring them together. 3 main features of the innate immune system and its receptors can be considered :
• many immunostimulatory PAMPs, such as lipopolysaccharide (LPS), lipoarabinomanan and peptidoglycan, are truly microorganism specific and cannot be found in any multicellular organism, from insects to vertebrates^{ref1, ref2}. There is also a growing list of host-derived immunostimulators, such as hyaluronan^ref, heat-shock proteins (HSPs)^ref, surfactant proteins^ref, IFN-a^ref,uric acid^ref, fibronectin^ref, b-defensin 2^ref and cardiolipin^ref, many of which signal through the same receptors as those used by PAMPs. Because these endogenous immunostimulators are difficult to fit into self-non-self (SNS)-discrimination models, there has been a tendency to ignore them or to attribute their activity to trace LPS contamination^{ref1, ref2}.
• the innate immune system is not only the first line of defence against pathogens, it is also involved in several normal physiological processes, such as tissue remodelling after damage^ref and during development^ref, transport of blood lipids^{ref1, ref2} and scavenging of apoptotic cells^ref

Immune component		defence	functions other than defence
humoral factors	complement and  surfactant proteins	bacterial clearanceref1, ref2, ref3, ref4	clearance of apoptotic cells and necrotic cellsref1, ref2, ref3, ref4, ref5, ref6, ref7
	MBP	bacterial clearanceref1, ref2, ref3	clearance of apoptotic cells, necrotic cells and lipidsref1, ref2, ref3
	LBP, CD14 and BPI	shuttle of PAMPs to TLRsref1, ref2, ref3, ref4	lipid transfer and mammary-gland involutionref1, ref2
	apolipoproteins and phospholipid transfer protein (PLTP)	neutralization of LPS and bacterial clearanceref1, ref2, ref3, ref4, ref5	lipid transferref1, ref2, ref3, ref4
membrane receptors	Toll	recognition of PAMPsref1, ref2, ref3, ref4	dorsoventral patterningref
	Toll-like receptors (TLRs)	recognition of PAMPsref1, ref2	recognition of endogenous ligandsref1, ref2, ref3
	scavenger receptors	recognition of PAMPsref1, ref2	clearance of apoptotic cells and lipid uptakeref1, ref2, ref3, ref4, ref5, ref6

• dual functions of scavenger receptors :

ligands		scavenger receptor A	scavenger receptor B
exogenous ligands evoking defence mechanisms	lipoteichoic acid, Gram-negative and -positive bacteriaref1, ref2, ref3, ref4	+	N.D.
	LPSref1, ref2, ref3, ref4, ref5	+	+
	Plasmodium-infected RBCsref	N.D.	+
endogenous ligands	amyloid b-proteinref and extracellular matrixref	+	N.D.
	acetylated or oxidized LDLs (oxLDLs)ref1, ref2, ref3, ref4,, oxidized phospholipidsref, cholesterolref1, ref2, ref3, apoptotic cellsref1, ref2, ref3 and maleylated BSAref	+	+
	oxidized HDLref1, ref2, apoE-phospholipid particlesref, long-chain fatty acidsref, anionic phospholipidsref,  modified RBCsref1, ref2 and phosphatidylserineref	N.D.	+

• dual functions of humoral factors for innate immunity :

humoral factors		exogenous ligands	endogenous ligands
collectin-family proteins	surfactant protein A	lipomannanref, lipoarabinomannan (LAM)ref and Pseudomonas aeruginosaref	apoptotic cellsref1, ref2, phosphatidylinositol,  phosphatidylglycerol, phosphatidylserine, cardiolipin, lipoglycans, galactoceramide, sphingophospholipid, glycosphingolipids and dipalmitoyl phosphatidylcholineref1, ref2, ref3
	surfactant protein D	LPSfrom Helicobacter pylori, Escherichia coli and Klebsiella pneumoniaeref1, ref2, ref3	apoptotic cellsref
	MBP	Staphylococcus aureus, Cryptococcus neoformans, and Trypanosoma cruziref1, ref2, ref3, ref4	apoptotic cellsref1, ref2, phosphatidylinositol, phosphatidylglycerol and phosphatidylserineref1, ref2
lipid-transport proteins (LTPs)	BPI	LPSref	phosphatidylcholineref
	LBP	Escherichia coli and Bacillus subtilisref1, ref2	phosphatidylinositol, phosphatidylcholine and  phosphatidylethanolamineref1, ref2, ref3
	PLTP	LPSref1, ref2	phospholipidsref
	CD14	LPS, phosphatidylglycerol, lipoteichoic acid and Bacillus subtilisref1, ref2	phosphatidylinositol, phosphatidylethanolamineref
	apolipoproteins	LPS, Klebsiella pneumoniae and Listeria monocytogenesref1, ref2, ref3	cholesterol and triglycerideref and amyloid proteinsref
complement	C1q	liposomeref; streptococcal lipoteichoic acidref; cell envelope of Escherichia coliref1, ref2; LPS from Escherichia coli and Helicobacter pyloriref1, ref2; outer membrane region of HIV-1 gp41ref; surface lipoprotein of Mycoplasma fermentansref; hydrophobic peptide of HIV-1 gp160ref and outer membrane proteins of Klebsiella pneumoniaeref	cellular and subcellular membranes obtained from the heart, liver and brain tissue from humans, baboons, cattle, rabbits and ratsref; substances released on brain contusions, without open injuryref; mitoplasts derived from human heart mitochondriaref; cardiolipinref; aminophospholipidsref; fragment of activated Hageman factorref; b-amyloid peptidesref and modified LDLref

• TLRs promiscuity : unlike most receptors, which bind only a few ligands, TLR2 and TLR4 interact with a large diversity of structures, and this promiscuity has puzzled those who work on them^{ref1, ref2}. As an example, ligands for TLR4 include both exogenous ligands, such as LPS, the fusion protein of RSV and bacterial fimbriae, and endogenous ligands, such as hyaluronan, b-defensin and HSP60. These ligands do not seem to have common structural motifs. Blue colouration indicates hydrophobicity^{ref1, ref2, ref3} :


Conventional chemical models of receptor-ligand binding evoke van der Waal's forces, hydrogen bonding, ionic interactions and hydrophobic interactions to stabilize configurationally fitted receptor-ligand pairs. Such models can explain the preferences of peptides for particular MHC grooves^ref and the specificity of antibodies to antigens^ref. It has been difficult, however, to find sequence or structural similarities among ligands that bind to the same TLR^ref.
These 3 features can be reconciled if we make one rather simple assumption, namely that many of the receptors that activate the innate immune system, whether to initiate immunity or to remodel tissues during development or repair, have evolved to recognize and react to the hydrophobic portions (hyppos) of molecules should these become suddenly exposed. Why hyppos? Hyppos are both useful and dangerous. The crucial feature of hydrophobicity is that water molecules prefer to associate with each other, rather than with hydrophobic structures. This drives hydrophobic molecules and patches of molecules to separate from water and aggregate with each other. Because life evolved in water, this aggregative tendency has both positive and negative consequences for living organisms.
• on the positive side, hydrophobicity is the main stabilizer of properly folded proteins and protein complexes. It is the force that drives phospholipids to form membranes, the 'glue' that keeps integral membrane molecules embedded and the main force that stabilizes the extracellular matrix. So, the tendency of hyppos to aggregate has important intracellular and extracellular physico-chemical functions in both unicellular and multicellular organisms.
• on the negative side, the same tendency to aggregate can cause problems. Depending on their structure and on the reactive mixture, molecules that contain exposed hyppos can either form symmetrical micelles, in which the hyppos are hidden, or unsymmetrical aggregates with exposed hyppos. The exposed hyppos will interact non-specifically/inappropriately with each other to form even bigger aggregates, and these aggregates are usually non-productive or even toxic. Sickle-cell anaemia, for example, illustrates the importance of the hydrophobic/hydrophilic balance, as a single mutation from a hydrophilic glutamine at position 6 of b-haemoglobin to a hydrophobic valine can completely disrupt the molecular structure and lead to the haemoglobin aggregation that results in the sickling of red-blood cells^ref.
Some of the structures that hydrophobic portions can form in water :

If the population of exposed hydrophobic portions (hyppos) is uniform, they can form ordered single-layer micelles as well as lamellar vesicles. If the population of hyppos is not uniform, they nevertheless aggregate, forming disordered micelles as well as complex aggregates, such as inverted hexagonal phase aggregates^ref. Aggregation is a particularly acute problem intracellularly, where macromolecules can exist at concentrations of 50-400 mg/ml. As a result of such high concentrations, hydrophobic molecules should aggregate even more rapidly in the cytoplasm than we would expect in dilute solutions^ref. To prevent rampant non-specific aggregation of cytoplasmic contents, the cell uses numerous components that bind, quench or otherwise deal with the hydrophobicity.
• Proteins undergoing synthesis are particularly vulnerable, as the hyppos that will later function as internal structural anchors are temporarily exposed. To prevent inappropriate aggregation, the cell uses ATP-dependent chaperones, such as members of the HSP70 and HSP40 families, to cover the exposed hyppos and ensure proper folding^ref. Under stress conditions, such as temperature changes (see cold shock and heat shock), even mature proteins can unfold and/or aggregate, and to tackle this, cells upregulate inducible HSPs that mediate catalytic solubilization and refolding of protein aggregates, thereby preventing aberrant protein aggregation^ref. Furthermore, when protein synthesis is greatly upregulated during viral infection, the production of HSPs is also upregulated. Although many investigators believe that the production of HSPs is the cell's response to infection, others suggest that the virus itself induces HSPs to chaperone its own nascent proteins under conditions of increased protein synthesis^ref. Extracytoplasmic protein misfolding can also lead to the exposure of hyppos, rendering the proteins potentially cytotoxic. Examples in which this has been shown to occur include prions, amyloid b-protein, amylin, calcitonin, serum amyloid and C-type natriuretic peptides. These proteins have hyppos that can become exposed and interact with lipid membranes, thereby inducing membrane damage and activation of innate immune responses that can then lead to chronic inflammation^ref.
• protein-associated hyppos are not the only potentially vulnerable hydrophobic molecules. Lipids and lipid derivatives are the main constituents of cell and organelle membranes. In multicellular organisms, lipids are absorbed from the gut, transported to peripheral cells, and stored in adipose cells through a complex process involving a variety of collectins^ref, scavenger receptors^ref and apolipoproteins^ref that handle normal and aberrant hydrophobic molecules in the blood and other extracellular spaces^ref. Apolipoproteins, for example, assist LDL in transporting lipids in the blood. If a lipid-LDL complex has any exposed hyppos, the apolipoproteins bind and cover them to prevent non-specific aggregation in the blood^ref. Intracellularly, there are ATP-binding multidrug transporters that transport lipids and other small hydrophobic molecules, such as taxol, from within cells^ref, and NOD (nucleotide-binding oligomerization domain) proteins, which use leucine-rich repeats (LRRs) to bind to phospholipid vesicles, indicating that they may have a role in the early detection of hyppos in the cytoplasm^ref1,ref2. Apolipoprotein E (apoE) is synthesized in the liver and in macrophages, and it has antiatherogenic properties that are mediated, at least in part, through the regulation of plasma cholesterol homeostasis. Previous data suggest that apoE also has antiinflammatory properties that may contribute to protection against atherosclerosis independent of its role in lipid metabolism. ApoE knockout and C57BL/6 mice were stimulated with low-dose LPS and other TLR agonists : the proinflammatory cytokines TNF-a, IL-6, IL-12, and IFN-g were upregulated to a significantly greater extent in apoE-deficient mice than in wild-type mice at both the mRNA and protein levels following administration of LPS. In contrast, hypercholesterolemic LDLR/APOBEC1^-/- mice had a similar cytokine response as wild-type mice, eliminating hypercholesterolemia as a cause for the exaggerated cytokine response. Importantly, reconstitution of apoE expression in the liver of apoE-deficient mice normalized the LPS-induced plasma protein levels of IL-12p40. Furthermore, there was selective upregulation of plasma IL-12 in apoE knockout mice by a TLR3 agonist, poly I:C, but not by other TLR agonists, CpG oligonucleotide or Toxoplasma gondii antigen. This implies that apoE selectively regulates TLR4- and TLR3-mediated signaling of IL-12 production. These results indicate that apoE modulates the T_h1-type immune response in vivo by modulating IL-12 production^ref
Overall, organisms go to a great deal of trouble to prevent the aggregation of hyppos, which are therefore rarely exposed under normal conditions. After injury, however, both prokaryotic and eukaryotic cells can release several sorts of hyppos, including membrane lipids, integral membrane molecules and nascent or denatured proteins. Exposure of hyppos also results from protein-misfolding diseases or modifications of the extracellular matrix by tissue enzymes, or pathogen-derived toxins. Sudden exposure of hyppos is therefore a sign of injury and disintegration, and hyppo-specific receptors are likely to have appeared early in evolution, to initiate intracellular and extracellular repair and remodelling. Some of these receptors would have functioned under normal conditions (as in protein synthesis or the transport of dietary lipids). Others would have operated during conditions of stress or injury (such as inducible HSPs), and some would have evolved to serve a dual function under both types of condition (such as CD14, scavenger receptors, constitutive HSPs and Toll). The modern immune system might have evolved from these primordial damage-detection systems, adapting some of the ancient hyppo receptors. Many receptors that trigger inflammation have ligands that are both endogenous (self) and exogenous (non-self); in many cases, it is the exposure of a hyppo that makes a particular endogenous or exogenous ligand a danger signal.
• exogenous hyppos : chemists creating polymers for implantation in humans have long known that hydrophobic polymers cause far more inflammation than non-hydrophobic polymers^ref, and this prompted us to look at the presently known ligands for TLRs, scavenger receptors, C1q and collectins. With rare exceptions, these immunostimulatory molecules are either entirely hydrophobic, or have large hydrophobic surfaces that can be exposed when injured, denatured or dysregulated. Incomplete Freund's adjuvant (IFA), for example, is simply mineral oil. LPS, peptidoglycan, lipoteichoic acid and many other immunostimulatory microbial products have large hydrophobic portions that are exposed when microorganisms are damaged, and bacterial proteins comprising structures such as flagellae have hydrophobic regions in their transmembrane domains or in buried portions between protofimbriae. In all cases tested so far, the immunostimulatory activities co-purify with the hyppos of these molecules^{ref1, ref2}. Viral fusion proteins, which are required for entry and exit from a cell, and for syncitium formation, have hydrophobic amino-acid regions that enhance membrane fusion. We would predict that the immunostimulatory activity of those that use the TLR pathways, such as the fusion protein of RSV^ref, would co-purify with the hyppos, rather than with the non-hyppos of the molecules.
• endogenous hyppos : uric acid, the most recently discovered endogenous immunostimulator^ref, forms hydrophobic crystals^ref. Defensins have large hydrophobic surfaces that bind membrane lipids^ref. HSPs have hydrophobic binding sites that are exposed in an ATP-dependent manner to allow binding to the hyppos of nascent or unfolded proteins^{ref1, ref2}. After ATP depletion (such as might occur if HSPs were released by damaged cells into ATP-poor environments), HSP polymers dissociate, resulting in increased hydrophobic exposure and more accessibility to digestion by proteases^{ref1, ref2}. Hyaluronan polymers, which serve as major components of vertebrate extracellular matrix and of bacterial capsules, seemed at first to be an exception, but a closer look shows that hyaluronan polymers are held together by evenly spaced hyppos that aggregate to help polymerize the molecules into sheets^ref. These hyppos are not exposed in the polymeric form of hyaluronan that lines blood vessels, but become exposed when the tissues are damaged. There has been controversy over the idea that endogenous molecules might be immunostimulatory. Some suggested that the immunostimulatory effects of HSPs might be due to contamination with LPS, because purified HSP preparations are sometimes no longer active^ref. However, one must be careful of the details here. The steps used in purification chromatography can remove many aggregates, and might easily remove the denatured/dysregulated and aggregated HSPs, leaving only the non-stimulatory water-soluble complexes that lack exposed hyppos. Similarly, polymyxin B, which inhibits LPS and is often used to rule out LPS contamination, not only binds to the lipid-A portion (the hyppo) of LPS^ref, but also to other lipid molecules^ref, and might therefore inhibit other immunostimulatory hyppos as well.
One might wonder, in fact, whether any molecule containing a hydrophobic core could potentially become immunostimulatory. There is some evidence in favour of this. Purified bovine serum albumin (BSA) became tolerogenic if it was injected into and then collected from the blood of a rabbit^ref. One of the results of such intravital passage would be the removal and/or quenching of degraded/denatured/aggregated molecules by blood-borne hyppo quenchers (see later). Aggregated/denatured immunoglobulin is immunogenic, whereas the soluble native monomers are not^ref. Humans make transient immune responses to the clotting factors VIII and IX after a bleeding episode^ref. These factors are cleaved during the clotting process, leading to the exposure of otherwise hidden hyppos, which are necessary for clotting^ref. Hydrophobicity might even be important in the immunostimulatory capacity of nucleic acids. Sequence analysis of RNA-binding peptides showed that a hydrophobic patch is highly conserved^ref. This indicates that hydrophobic domains on the supramolecular organization of RNA might be crucial in protein-RNA interactions, and indicates that hydrophobicity might be important in interactions of ssRNA and dsRNA with their respective receptors, TLR7 and TLR3^{ref1, ref2}. We would predict that the size of the exposed hyppos might matter, and suggest that any molecule containing hyppos that are large enough to cause aggregation after exposure should potentially be immunostimulatory when denatured.
Hyppo receptors have dual functions. There are many hyppo-binding molecules involved in covering hyppos during the synthesis, transport and degradation of lipids and other hydrophobic molecules. In the blood, for example, hyppos are masked, opsonized or cleared by humoral factors such as the collectin-family proteins, complement proteins, clotting proteins and lipid-transfer proteins (LTPs)^{ref1, ref2, ref3, ref4}, and many of these hyppo receptors also have a role as part of the innate immune system.
• LPS-binding protein (LBP), a member of the LTP family (which also includes phospholipid-transfer protein, cholesterol-ester-transfer protein and bactericidal/permeability-increasing protein), is a classic example of a dual functioning hyppo receptor. LBP facilitates the transfer of LPS monomers from LPS aggregates to CD14, thereby enhancing the sensitivity of CD14-TLR4 stimulation^{ref1, ref2}. But LBP is misnamed. It should have been called something like cell-associated membrane-lipid shuttle, because its main function is to shuttle insoluble lipid derivatives from cell membranes or aggregates to CD14 and high-density lipoprotein (HDL)^ref.
• CD14, which has been called a PRR for LPS^ref, is actually a receptor with three activities and could have been named HDL-associated lipid transporter, as it functions physiologically to facilitate the transfer of both endogenous and exogenous lipid-derivatives to HDL^ref, and also recognizes apoptotic cells^ref. Because apoptotic cells lose their asymmetric distribution of phospholipids, it has been suggested that macrophages might sense an increase in hydrophobicity at the apoptotic cell surface^ref. In this light, we should also mention that the hydrophobicity of bacteria correlates with their phagocytosis by macrophages, with the more hydrophobic bacteria being more readily phagocytosed^ref. And erythrocytes that have lost their asymmetric distribution of phospholipids across the plasma membrane bilayer become more hydrophobic and are also more readily phagocytosed by macrophages^ref. It is therefore possible that CD14 could be involved in the recognition of these changes in hydrophobicity^ref.
• C-reactive protein (CRP), the expression of which is upregulated by either tissue injury or infection, is another hyppo receptor that detects both endogenous and exogenous hyppos. It uses a hydrophobic pocket to recognize unesterified cholesterol or phosphorylcholine^ref - a universal prokaryotic and eukaryotic membrane molecule. CRP binds damaged membranes and can facilitate opsonophagocytosis of pathogens and of necrotic-cell debris^{ref1, ref2}.
• the scavenger receptors, which are important in the phagocytosis of non-opsonized molecules, bind endogenous molecules, such as cholesterol and phospholipids exposed on apoptotic cells^ref, as well as exogenous LPS and lipoteichoic acid^ref. Scavenger receptors contribute to host defence against bacteria by activation of NF-kB^ref and can also induce inflammation in response to endogenous hyppos, such as oxidized HDL, damaged red-blood cells, atherosclerotic plaques and b-amyloid fibrils^{ref1, ref2, ref3}, therefore they have a dual role in response to injury as well as to infection.
• even some of the TLRs, the classic PRRs, bind to endogenous molecules and mediate normal physiological functions. The original Toll receptor of Drosophila, for example, mediates dorsoventral patterning during larval development and also initiates immunity in adult flies^ref. In humans, atherosclerosis is linked to TLR polymorphism, indicating that endogenous lipid derivatives might also activate human TLR pathways^ref. In some cases, the normal function of a hyppo receptor can become a liability when that receptor is bound by a pathogen. Mammals can become ill or die from shock in response to pure LPS, and the horseshoe crab, Limulus polyphemus, solidifies completely^ref. Although CD14 is known as a PRR for LPS6, CD14-knockout mice are protected from this lethal response, clearing bacteria better than their normal littermates^ref.
• CD36 - a class B scavenger receptor - is a lipoprotein receptor that mediates platelet aggregation and adhesion after injury, and dendritic-cell recognition and uptake of apoptotic cells^ref. It is also involved in oxidized HDL-mediated or thrombospondin-1-mediated stimulation of cytokine production by human macrophages^ref. CD36 also binds (or is bound by) Plasmodium falciparum-parasitized erythrocytes, an interaction that leads to the inability of dendritic cells to stimulate T cells against the parasite^ref. Hyppo receptors are promiscuous. Because of the physical and chemical characteristics that drive hyppos in aqueous solutions to aggregate with each other non-specifically, hydrophobic interactions are far less dependent on exact molecular configuration than other types of chemical interaction. This principle is fundamental, for example, to algorithms that predict protein structure, in which a string of hydrophobic amino acids is interpreted to represent part of a protein's inner core or a transmembrane region, regardless of the exact amino-acid sequence. Hydrophobicity, per se, is therefore the main predictive feature^{ref1, ref2}.
Although immunologists tend to extol specificity, non-specific interactions are also useful, as a small number of receptors can deal with a large variety of ligands. A handful of chaperones, for example, protect a large variety of nascent proteins. A few hyppo-binding molecules transport a large variety of lipids in the blood and interstitial fluids. And a small number of hyppo-binding cell-surface receptors can initiate immune responses to a large variety of insults. This is what is seen with the TLRs and other receptors used by the innate immune system, such as scavenger receptors, surfactant proteins and C1q, which bind to many different pathogen-derived and host-derived hyppos. All of the molecules that signal through TLR4 have hydrophobic regions and that the TLR itself has a large hyppo made up of LRRs, with which it can bind to many different types of hyppo. Another example is the complement protein C1q, which binds an area that is exposed when IgM molecules change their conformation after antigen binding^ref. It also binds to liposomes, LPS, cardiolipin, -amyloid and many other host-derived molecules^ref.
How can these receptors bind hydrophobic molecules without exposing hyppos of their own and succumbing to self-aggregation? An example comes from the analysis of GroEL, an Escherichia coli HSP that preferentially binds immature proteins. GroEL uses a small, exposed hyppo 'finger' to initiate binding to nascent proteins. It then uses ATP-derived energy to serially open a large internal hydrophobic cavity that binds to the nascent protein^ref. We might predict that the hydrophobic LRRs of TLR4 might be similarly hidden on the concave b-face^ref, only to be exposed when binding is initiated. The exact nature of such two-step binding might confer some specificity to the otherwise non-specific hydrophobic binding, perhaps explaining why the TLRs and other hyppo receptors are promiscuous but not totally non-specific.
Not surprisingly, we find that hyppo-handling systems are an essential component of many innate immune systems. In plants, for example, the LTP that normally shuttles lipids between organelles and cells^ref is also upregulated by stresses that cause tissue damage, such as infection, drought, heat and cold, and confers plants with resistance to a broad range of virulent pathogens^ref. Other plant-resistance proteins also bind a great variety of endogenous and exogenous hyppos^ref. Insects use another LTP, apolipophorin III, as an immunity-inducing receptor^ref, and mutant apolipophorins that lose their lipid-binding capacity also lose their immune-inducing properties^ref. Even the original Toll receptor of Drosophila is a hyppo receptor, as it has a hydrophobic LRR (similar to that of TLR4) that it uses to bind its endogenous ligand; the hydrophobic carboxy-terminal end that is cleaved off the protein Spaetzle by a serine protease^ref. So, early detection of hyppos seems to be an evolutionarily conserved step for innate immunity.
As an aside, might recognition of stress-induced lipids also have been the first step towards adaptive immunity ? A hint that this might be so comes from recent studies on NK1.1⁺ T cells - an evolutionarily ancient set of innate T cells that recognize the ancient, stress-induced, MHC class I molecule CD1. Unlike T cells that are restricted by conventional MHC molecules, the NK1.1⁺ T cells bind endogenous lipids in the CD1 groove^ref. Similarly, the Vg9d2 human T cells are triggered by isopentenyl pyrophosphate^ref - an intermediate in cholesterol synthesis^ref.
Are all hyppos damage-associated molecular patterns (DAMPs) ? Any molecule that is not normally exposed can be a DAMP if it is revealed during, after, or because of injury or damage. Hyppos can therefore become DAMPs if exposed after damage or injury. IFN-a, however, is also a DAMP, as it is produced by infected cells. Exposed mitochondria could be DAMPs, along with numerous other intracellular molecules that are not normally found outside of cells. So, not all DAMPs are hyppos. Is the converse true? Are all hyppos, whether pro- or eukaryotic, DAMPs? And are all DAMPs immunostimulatory? Or do some of them simply initiate post-injury repair and/or remodelling? Most immunostimulatory exogenous or endogenous hyppos seem to be DAMPs. In healthy bacteria, the hyppos of bacterial flagellae and fimbriae are buried in the membrane, as is the lipid-A portion of LPS, leaving only the non-stimulatory polysaccharide exposed. In addition, the hydrophobic surfaces of lipoteichoic acid and peptidoglycan are hidden in aggregates^ref. Although flagellin might seem to be an exception, studies with TLR5, which binds to bacterial flagellin and activates host inflammatory responses^ref, showed that the 13 amino acids that form the relevant flagellar ligand for TLR5 normally participate in intermolecular interactions between flagellar protofilaments and are buried in the complete filament. It might be impossible for TLR5 to recognize the buried portions in undamaged flagellae, as monomeric flagellin, but not the complete filamentous molecule, is stimulatory. So, bacterial hyppos, similar to their eukaryotic counterparts, seem to be exposed only under conditions of stress, damage or injury. We might therefore predict that stressed/injured/dead pathogens should activate immunity better than healthy ones. Although this has not been well studied, there are a few examples. First, anti-tuberculosis therapy can initially lead to clinical or radiological worsening due to increased lung inflammation in patients with tuberculosis^ref. A rapid increase in the lymphocyte count, associated with a strongly positive tuberculin-skin-test result has been observed in these patients after treatment. Second, children with malaria often do not make antibodies to the particular parasite variant with which they are infected until after they become sick and are hospitalized and treated with anti-malarial drugs^ref. Together, these examples indicate that dead pathogens might evoke more prominent inflammation than live ones. Not all hyppos, however, are immunostimulatory and some are even inhibitory^ref. E. coli lipid IVa (an LPS derivative) and Rhodobacter sphaeroides lipid A (RSLA), for example, antagonize the effect of LPS on TLR4^ref, and some arachidonic acid derivatives also show anti-inflammatory effects. A search for the properties that might distinguish stimulatory from non-stimulatory lipids indicates that the crucial feature might be the size and molecular organization of their aggregates. When the concentration of hyppos reaches a critical point, they aggregate, however, not all aggregates are the same^{ref1, ref2, ref3}. For example, lipid-A analogues that have conical shapes (where the cross-section of the hydrophobic moiety is larger than the hydrophilic moiety) have a strong tendency to form inverted hexagonal structures with exposed hydrophobic surfaces, and are highly endotoxic^{ref1, ref2, ref3}. Lipid-A analogues that have a more cylindrical shape show a strong preference for lamellar structures that hide the hydrophobic tails, and are not immunostimulatory^ref. This indicates that the tendency of a molecule to allow hyppo exposure, rather than its hydrophobicity per se, might determine its immunological activity. In this light, it is interesting that polymyxin B, which inhibits the activity of LPS, induces a change in the organization of LPS aggregates such that they no longer form inverted cubes with hydrophobic surfaces, but instead form lamellar structures that have hydrophilic surfaces^ref. Another example is found in bronchial secretions. Although they contain many phospholipid and surfactant proteins, alveolar macrophages are not constitutively activated. As the name surfactant implies, these molecules easily form symmetrical micelles^ref, allowing them to capture and expel environmental hyppos from the bronchial linings without stimulation of the local APCs. Indeed, such lipids can incorporate and quench other hyppos to reduce inflammatory responses^ref. In brief, it seems to be the supramolecular organization of hyppos, and the capacity to be hidden or exposed, that determines their ability to activate immune responses. How do dual function hyppo receptors 'know' when to initiate inflammation? In some cases, a hyppo receptor must deal quietly with dietary lipids and other physiologically exposed hyppos. In other cases, it will bind to hyppos that are exposed because of injury and initiate repair mechanisms, and in others, it should induce the processes that result in immunity. How does it know when to do what? The short answer is, we don't know. There are, however, several possibilities.
• first, the concentration matters. In physiological conditions, humoral quenchers, such LDL, HDL, chylomicron, apolipoproteins and LTP^ref, maintain homeostasis by masking, transferring and opsonizing hyppos that are produced by dietary lipids and apoptotic cells. The LDLs and chylomicrons are then cleared by cellular uptake through specific receptors in the absence of cellular activation^{ref1, ref2}. If the concentration of hyppos rises above the level that is controllable by the quenching systems, such as might happen locally or systemically after injury or infection, the unquenched hyppos can activate the coagulation system and/or the complement cascade, as well as cell-surface hyppo receptors such as TLRs.
• second, stimulatory hyppos are sometimes accompanied by inhibitory molecules. For example, apoptotic cells can bind to CD14, which is known to aid in TLR4-mediated stimulation. However, apoptotic cells do not activate APCs or other inflammatory systems. The reason might be that phosphatidylserine, which is exposed on the outer leaflet of apoptotic cells, binds to its specific receptor, the phosphatidylserine receptor, which sends negative signals that inhibit local immune reactivity by suppressing the maturation of dendritic cells and/or the production of pro-inflammatory mediators^ref. The phosphatidylserine receptor is not alone. The receptors for arachidonic acid derivatives^ref, PPAR family^ref and various other orphan nuclear receptors^ref have also been shown to inhibit cytokine production and dendritic-cell migration. Activation and inhibition of T and B cells is the result of a balance of stimulatory and inhibitory signals, and APCs are likely to be under similar stimulatory and inhibitory controls following hyppo recognition.
To stimulate or be silent: 3 destinies of hydrophobic portions.
• a | At normal physiological concentrations, free hydrophobic portions (hyppos) are bound by hyppo quenchers, such as apolipoproteins, collectins and lipid-transfer protein (LTP), which transfer hyppos to hyppo carriers, such as LDL, HDL and chylomicron. These complexes are cleared by cellular uptake through specific receptors for the quenchers or carriers that do not send stimulatory signals.
• b | In pathological conditions, in which the concentration of free hyppos might overload the local scavenging capacity, unquenched hyppos can activate the coagulation system, complement systems and cell-surface hyppo receptors, such as the Toll-like receptors (TLRs). Signalling through TLRs activates antigen-presenting cells (APCs), which provide co-stimulatory signals and cytokines for T-cell activation.
• c | A combination of negative and positive signals might result in silence. For example, during normal programmed cell death, various hyppos might be exposed on apoptotic cells, however, the exposed phosphatidylserine binds to the phosphatidylserine receptor, which sends negative signals^ref and prevents the activation of APCs. TCR, T-cell receptor.

DAMPs among the PAMPs^ref. On the one hand, it is possible that some hyppos are truly bacterial PAMPs that are totally unrelated to the ancient signs of injury and distress. For example, exposure of hyppos on the surface of bacteria might help them to colonize solid surfaces or target tissues, thereby enhancing their survival chances^ref. Indeed, there is a family of hydrophobic small proteins known as hydrophobins which are secreted by filamentous fungi^{ref1, ref2, ref3, ref4, ref5,ref6, ref7} which react to interfaces between fungal cell walls and the air or between fungal cell walls and solid surfaces, promoting adhesion to animal cells. They have been shown to be important in many morphogenetic processes, including sporulation, fruit body development, and infection structure formation. Hydrophobins form hydrophobic surface layers by self-assembly of secreted protein monomers in response to the environment. This process results in amphipathic polymers of interwoven rodlets on surfaces of fungal aerial structures and hyphal aggregations. Hydrophobin self-assembly is also involved in attachment of hyphae to hydrophobic surfaces and this may act as a conformational cue for certain developmental processes. Although hydrophobins appear to be ubiquitous among fungal taxa, a second class of fungal protein with very different biochemical characteristics could fulfill a similar role. These proteins, called repellents, have been identified in only one fungal species so far, but clearly help to make aerial hyphae hydrophobic. The functional similarities between hydrophobins and repellents highlight the importance of aerial development to the fungal lifestyle.
On the other hand, if exposed hyppos are evolutionarily ancient signals of injury and destabilization in eukaryotic organisms, is it possible that they might have the same function in bacteria? Most of the bacterial molecules that are immunostimulatory are not normally exposed in healthy bacteria. Could the lipid-A hyppo of LPS act as an alarm signal for E. coli? Although bacteria are unicellular creatures, they do not normally live solitary lives. They form aggregates, colonies and multi-species establishments. They communicate using quorum sensors and make biofilms^ref. Might it be useful for individual bacteria, in a colony of relatives, to have advance warning that something is amiss? If the bacteria around them begin to die, for example, the survivors might want to do something different, for example, leave the area or, possibly, sporulate. Could the E. coli LPS molecules that are currently thought to be a sign of the 'enemy' therefore really be distress signals released by commensal bacteria that have taken a wrong turn out of the gut, lost their way and found themselves in the wrong tissue environment? Perhaps the ancient hyppo alarm signals form a common language of distress spoken by all living creatures.
The hyppos of bio-molecules are hidden from the aqueous environment, either by conformational folding, by insertion into membranes, or (for example, during lipid transport) by help of accessory molecules. Because exposed hyppos rapidly form non-productive or even toxic aggregates, organisms use a great deal of metabolic energy preventing such exposure and maintaining homeostasis. A sudden increase of exposed hyppos is therefore a universal sign of damage, and receptors that detect such alarm signals are likely to have evolved early in the history of life to activate repair and remodelling systems. It would have been an easy step for the innate immune system to adapt these damage-detection receptors to initiate immune responses. Many PAMPs and endogenous alarm signals belong to an ancient subfamily of universal DAMPs that consist of exposed hyppos. This view explains why most immunostimulators are hydrophobic or contain exposed hyppos, why TLRs and other hyppo receptors are so promiscuous in their binding specificity, and suggests that there might be a universal language of injury and repair shared by most, if not all, living organisms. Not all immunostimulators are hyppos. IFN-a, for example, is a primary immunostimulator with its own specific receptor that uses standard molecular interactions for its specificity (though, even here, hydrophobic patches seem to have a role^ref). What we are suggesting, however, is that the modern immune system remembers its history. If the early immune system 'piggybacked' onto or evolved from the ancient hyppo-detection system, then many (perhaps most) primary immunostimulators act through exposed hyppos. Today, a fully developed immune system might have added new variations to its repertoire of triggers, but the oily evolutionary footprint remains, revealing the kinship of all water born life forms.
Clinical implications of the hyppo hypothesis : as the innate immune system cannot determine the origin of hydrophobic portions (hyppos), both endogenous and exogenous hyppos (for example, environmental hyppos, hyppos in drugs, mutations in lipid-handling pathways or in protein folding) can lead to inflammation. A few of the diseases that might be hyppo based are described here.
• allergies : what makes an allergen an allergen? Many are hyppos, including hydrophobic chemicals, aromatic drugs, aromatic hydrocarbons in diesel exhaust, animal lipocalins, plant lipid-transport proteins, plant-pathogenesis-related proteins (hydrophobic plant proteins induced by infection, trauma or environmental stress), and seed storage proteins (which have large hydrophobic faces to prevent evaporation).
• atherosclerosis : increased lipid and/or cholesterol concentrations, if not adequately quenched/blocked/scavenged, could activate antigen-presenting cells (APCs) to present self antigens and stimulate autoimmunity and inflammation. Indeed in humans, increased cholesterol levels are linked with normocytic anaemia and autoimmune thyroiditis. Autoimmune-prone MRL/lpr mice spontaneously develop hyperlipidaemia as well as lupus, and are susceptible to diet-induced aortic cholesterol deposition. By contrast, Myd88-deficient mice are resistant, implicating a role for Toll-like receptors in atherosclerosis. Perhaps the inflammation of atherosclerosis is a direct consequence of non-specific APC activation by hyppos that are exposed on plaques?
• protein-misfolding diseases : Alzheimer's disease, amyloidosis and spongiform encephalopathy exhibit marked inflammation due to the inherent toxicity of the extracellular protein aggregates. This might be due to aberrant hyppo exposure on the aggregates. For example, melatonin binds hydrophobically to b-amyloid and inhibits its spontaneous aggregation. Furthermore, apolipoproteins, which normally quench blood lipids, can ameliorate inflammation in AD
• arthritis : exposed hyppos on degraded collagen in the articular space might initiate rheumatoid arthritis. Could hyppo-quenching agents counteract this effect? Indeed, the chemical agent bindarit, which inhibits denaturation, strongly reduces the development of adjuvant-induced arthritis (AIA), indicating that hyppos on denatured proteins in articular spaces might be important in the pathogenesis of autoimmune arthritis.
CD200, a potent immunoregulatory protein, is expressed on Langerhans cells (LCs) and keratinocytes (KCs) in mouse epidermis. CD200 expression is concentrated on KCs comprising the outer root sheath (ORS) of murine hair follicles (HF). Skin deficient in CD200 is highly susceptible to HF-associated inflammation and immune-mediated alopecia. In this concept review, the results of recent studies on CD200 and its inhibitory receptor, CD200R, are summarized and integrated to yield a model whereby CD200-CD200R interaction attenuates perifollicular inflammation, prevents HF-specific autoimmunity and may protect epidermal stem cells from autoimmune destruction. Further elucidation of the CD200-CD200R signaling pathway in cutaneous tissues may advance understanding of how immune homeostasis is established and maintained in the skin^ref.
However, it is unclear whether or how the inducible expression of co-stimulatory signals would distinguish between self antigens and microbial antigens when both are encountered by dendritic cells during infection. The efficiency of presenting antigens from phagocytosed cargo on MHC class II is dependent on the presence of TLR ligands within the cargo. Furthermore, the generation of peptide–MHC class II complexes is controlled by TLRs in a strictly phagosome-autonomous manner^ref.
Synthetic decapeptides (N=206) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (FAH) were used to analyze the specificities of the autoantibodies (autoAb) elicited towards this enzyme in mice infected with mouse hepatitis virus (MHV). These autoAb bound mainly to N- and C-terminal FAH peptides, the most reactive sequences being 1-50 and 390-420, respectively. Surprisingly, although FAH sequence 1-50 shares a high degree of homology with various MHV proteins, the C-terminal portion does not. Moreover, whereas the autoAb reacted with homologous peptides surrounding residues 70, 160 and 360, non-similar sequences around residues 130, 210, 240, 250, and 300 were also recognized, indicating that autoAb were not restricted to epitopes with sequence homologies. There was also a lack of correlation between the amount of anti-MHV or anti-FAH antibodies produced and the reactivity towards the peptides. Moreover, the spectrum of peptides recognized by the autoAb of a given mouse did not change significantly with time, which suggests that the MHV-elicited autoimmune response does not induce an epitope recognition spreading. Finally, anti-FAH Ab produced after immunization with rat liver FAH recognized essentially the same mouse FAH regions than autoAb from MHV-infected mice. Results indicated that the induction of the autoAb is not only related to molecular or structural mimicry, but rather supports the Danger model, in which any aggression, in this case the MHV infection, is susceptible to trigger the production of autoAb^ref.
Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas : senescent tumors became infiltrated by neutrophils, macrophages, and natural killer cells that eventually destroyed the tumor cells and the tumoral vasculature. This finding indicates an alternative mechanism to cell death that can also explain cancer regression^ref.

• leukemia (see also granulocytic leukemoid reactions and lymphocytic leukemoid reactions) :
• T-LGL leukemia : CR in an 82-year-old man. The patient died 2 years later of an unrelated condition, with no evidence of recurrence of T-LGL leukaemia^ref
• AML : since 1980 about 33 cases of SR in adult AML have been reported, 27 of which are summarized in the table below :

sex	age	diagnosis	time to remission (months)	duration of remission (months)	associated infection	transfusions	other conditions	karyotype	survival (months)	reference
M	34	AML	4	2	+	(?)		Normal	24+	Ruutu et al. (1982)
M	52	APL	0.5	7	+	+		ND	8	Raza et al. (1985)
M	28	APL	2	NR	+	+	?		100+	Enck (1985)
M	56	AML M1	2	34	+	+		Complex	56+	Ifrah et al. (1985)
M	73	AML M4	2	?	+	+ (?)		ND	3	Sanz et al. (1986)
F	21	AML M2	?	NR	+	+		ND	6+	Rajni et al. (1988)
F	53	Hypoplastic L	8	5	+	+		Normal	24	Kizaki et al. (1988)
F	54	AML M2	1	1	–	–		Normal	6+	Tsukada et al. (1989)
M	51	Hypoplastic L	1	4	+	+		Tetraploid	?	Maekawa et al. (1989)
F	28	APL	1	2	–	+	Termination of pregnancy	ND	6	A. de Mayolo et al. (1989)
M	70	AML	?	5	+	+	?		6	Nakamura et al. (1989)
M	69	AML M5a	1	3	–	–		ND	15+	Spadea et al. (1990)
F	47	AML M5b	1	NR	–	–		+8	12+	Okagawa et al. (1990)
F	74	AML M5	1	7	–	–		Complex	25	Paul et al. (1994)
F	49	AML M5a	0.5	6	–	(?)		ND	10	Musto et al. (1994)
M	48	AML M2	1–3	36	+	+		t(8;21), –y	38	Delmer et al. (1994)
F	41	AML M5	1–3	14	+	+		Normal	17	Delmer et al. (1994)
M	54	AML M2	1–3	3	+	+		Normal	71+	Delmer et al. (1994)
M	54	AML M2	?	1	+	?	G-CSF	+8	?	Hayatsu et al. (1994)
M	83	AML	1	1	+	+	G-CSF	ND	4	Mitterbauer et al. (1996)
M	64	AML M5b	1	NR	+	+	hydroxyurea 5.5 g	ND	14+	Mitterbauer et al. (1996)
F	62	AML	?	4	+	?		?	40	Gau et al. (1997)
F	60	sAML M1	2	2	+ (invasive pulmonary hyalohyphomycosis highly suggestive of aspergillosis)	+	G-CSF	Normal	26+. 2 months after the diagnosis of leukaemia, she achieved a spontaneous remission lasting 3 months, although neither cytostatic drugs nor corticoids were administered because of a septic condition. At the time of remission, a chronic HCV infection and a polyclonal hypergammaglobulinaemia were present, and the patient received G-CSF factor once	Tzankov et al. (2001)
M	31	AML M5a	1	2	+	–	biphenotypic	Normal	4+	Maywald et al. (2004)
?	?	?	?	?	interstitial pneumonia caused by Pneumocystis carinii	?	?	?	6	Fassas et al (1991)
?	?	?	?	?	?	?	?	?	?	Jimenez et al (1993)
		AML M2			severe pneumonia. High-dose methylprednisolone was administered, and the leukemic cells disappeared without chemotherapy, although dysplastic hematopoietic cells were observed transiently after the first therapy. After the disappearance of leukemic cells, FISH for AML1 splitting was negative, and RT- PCR results for quantitative chimeric AML1/ MTG8 mRNA were less than the detectable level, however, RT-PCR results for AML1/MTG8 mRNA remained positive. These findings suggest that the patient acquired morphological, cytogenetic, and possibly molecular genetic remission by the synergistic effects of severe infection and high-dose methylprednisoloneref.			t(8;21) (q22:q22). The patient tested positive by FISH for AML1 splitting and positive by RT-PCR for chimeric AML1/MTG8 mRNA, which indicated splitting of the MTG8 gene on chromosome 8 (q22) and the AMLI gene on chromosome 22 (q22)		Shimohakamada et al, 2001
2 cases										ref

The mechanisms of SR in adult AML are unknown. Severe systemic infections often precede SR^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11, ref12, ref13, ref14}. Although SRs are most often reported to be associated with bacterial infections, even fungal infections have coexisted with SR^{ref1, ref2}. Activation of the immune system as documented by an increased serum level of TNF-a, IFN-g, and IL-2^{ref1, ref2, ref3} as well as an increased number of NK cells and hypergammaglobulinemia^ref before and during SR have been implicated as involved mechanisms.
• the possible role of infections has led to therapeutic trials of bacterial extract inoculations, vaccination strategies, or immunotherapy using bovine gammaglobulin (BCG)^{ref1, ref2} with limited effect on prolonging remission or survival.
• promising effects of IL-2 have recently been shown especially in patients with refractory or relapsed acute myeloid leukemia with >5% and <30% bone marrow blasts who were not suitable for further chemotherapy^ref.
• transfusion of blood components before SR has been reported in association with SR^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11, ref12}.
• cytotoxic antibodies against leukemic cells as described by Dore et al.^ref or other soluble factors in donor serum such as cytokines may play a role.
Furthermore, mechanisms similar to those described as GVL effect in allogeneic transplanted patients^ref due to the transfused allogeneic lymphocytes have to be taken into consideration as underlying mechanism to induce SR in these patients. Nevertheless, most patients suffering from AML receive blood transfusions during therapy and SR has not been observed. In some patients termination of pregnancy preceded SR and relapse occurred in one patient with leukemic breast infiltration^ref. Additional presence of estrogen-binding sites and growth-stimulating effect of estradiol in human myelogenous cell lines could be demonstrated in vitro^ref. Therefore, hormonal changes have been highlighted to be a possible mechanism of SR. Nevertheless, androgens had no additional benefit when combined with conventional chemotherapy versus chemotherapy alone during maintenance therapy of patients with AML in complete remission^ref. In some cases neither infection nor blood transfusion, G-CSF or hormonal changes such as termination of pregnancy could be associated with SR^{ref1, ref2, ref3, ref4, ref5}, implying that these conditions are not sine qua non. In summary, spontaneous remission in AML is rare but well documented even in acute myeloid leukemias with a poor prognosis such as secondary AML or AML with biphenotypic markers of the blast cells^{ref1, ref2, ref3}. SR is mostly associated with severe infection or with transfusion of blood components. Either soluble factors such as TNF-a, IL-2, IFN-g, hypergammaglobulinemia, cross-reactive antibodies, direct cytotoxic antibodies in donor serum, and G-CSF as well as cellular factors such as an elevated number of NK cells or allogeneic lymphocytes with a GVL effect in blood transfusions are discussed to be involved in SR. Nevertheless, the exact mechanisms of SR still remain unclear. Though most remissions are of short duration, even long-term remissions and even complete cytogenetic remissions have been documented. Since standard therapeutic options in AML such as intensive chemotherapy and allogeneic bone marrow transplantation are associated with a high rate of therapy-related morbidity and mortality, better insights into the mechanisms of spontaneous remission could offer new therapeutic approaches in AML.
• primary cutaneous peripheral T-cell lymphoma, unspecified, that completely regressed after skin biopsy^ref
• lymphoid blast crisis in chronic myelogenous leukaemia : returned to chronic phase, without the use of cytostatic chemotherapy, following an episode of viral infection and blood transfusion^ref
• ALL :
• ALL-L3 in a 5-year old Turkish girl. Because of infection she was given antibiotics and a blood transfusion. Her bone marrow was in remission after 15 days without chemotherapy^ref
• of childhood following acute infectious disease^ref
• B-CLL :
• with virus infection in a Japanese patient^ref
• three patients following viral infection^ref
• HCL : remission occurred spontaneously or in association with Legionnaires or subsequent HIV infection. Currently it is 17 years since the diagnosis of hairy cell leukemia and 14 years since the infusion of HIV infected blood. Hairy cell leukemia has yet to return and HIV infection has not progressed to AIDS^ref
• NHLs :
• T-NHLs : gingival T cell non-Hodgkin's lymphoma that responded initially to chemotherapy, recurred at another site a year later, but regressed spontaneously after incisional biopsy^ref
• ATL :
• spontaneous remission without any anti-cancer therapy in a 57-year-old woman with adult T-cell leukemia (ATL) is reported. The patient was referred to our department because of persistent cough and appearance of abnormal lymphocytes in the peripheral blood, and she was diagnosed as having chronic ATL. Eight months later, she was re-admitted because of cystitis, watery diarrhea and worsening of respiratory symptoms with an increase of ATL cells (WBC 31,000/ml with 56% ATL cells). Acute exacerbation of ATL was diagnosed. Interestingly, antibiotic therapy for the pulmonary and urinary tract infections brought about spontaneous reduction of the ATL cell count. Spontaneous remission of ATL continued for one year without chemotherapy^ref
• spontaneous remission in a 50-year-old woman. The patient was referred to our hospital because of generalized lymphadenopathy and the appearance of abnormal lymphocytes with convoluted nuclei in the peripheral blood. She was diagnosed as ATL because of the characteristic morphological ATL cells, cell surface marker analysis and the presence of serum antibody to human T-cell lymphotropic virus type-I (HTLV-I). However during the following weeks before admission, her leukocyte count and ATL cells in the peripheral blood decreased in number even though she received no therapy. After admission, abnormal lymphocytes in the peripheral blood disappeared and lymphadenopathy decreased in size, LDH level was normalized. Spontaneous remission had continued ten months without chemotherapy, but she developed recurrence thereafter and died two years later after onset with progressive disease. Gene analysis study by Southern blot analysis showed monoclonal integration of HTLV-I proviral DNA at the same positions both before regression and after recurrence. In this case, the trigger of spontaneous remission was unclear^ref
•  a 79-year-old woman with spontaneous remission of acute type ATL. Spontaneous remission was preceded by surgical biopsy and pneumonia and lasted for 2 years until she died with pancreas cancer. Monoclonal integration of HTLV-I provirus DNA became undetectable after remission^ref
• spontaneous regression of pulmonary infiltration^ref
• B-NHLs :
• DLBCL : spontaneous regressions of EBV⁺ DLBCL with subsequent relapses three times. In the literature, there are several reports of aggressive non-Hodgkin's lymphoma cases that showed spontaneous regressions without relapse till the last observation. Over half of the cases were the extra-nodal type. The tendency toward regression of swollen lymph nodes detected by clinicians occurs within 2 weeks after biopsy^ref
• spontaneous remission of a histopathologically supported highly malignant B-cell Non-Hodgkin's lymphoma with primary manifestation in the oral cavity is presented. This regression, which has showed no signs of recurrence for more than 18 months, occurred following a diagnostic biopsy and without any therapeutic intervention^ref
• complete spontaneous remission after surgical excisional biopsy of a small noncleaved cell malignant lymphoma (non-Burkitt's lymphoblastic lymphoma) of the tonsil in a 12-year-old boy. The diagnosis was confirmed by immunohistologic and immunoglobulin gene analysis. Similar studies were performed on the cervical lymph node excised 2 weeks later when spontaneous remission had occurred. The patient has remained in good health for over 3 years^ref
• a 58 years-old woman was diagnosed with high grade immunoblastic lymphoma after excision of a single cervical lymph node, the remaining bilateral cervical, inguinal, and axillary adenopathy regressed completely without any cytotoxic treatments 22 days after biopsy. At the time of this writing, the patient has been free of disease for 24 months^ref
• nodal ALK positive anaplastic large cell lymphoma of the axilla^ref
• HIV-associated plasmablastic lymphoma in the oral cavity^ref

• Danger theory by Chris Forden

• The danger model: a renewed sense of self by Polly Matzinger in Reflections on self : immunity and beyond, Science Vol.296 12 April 2002
• Hydrophobicity : an ancient damage-associated molecular pattern that initiates innate immune responses by Seung-Yong Seong & Polly Matzinger, Nat Rev Immunol. 2004 Jun;4(6):469-78.

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