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