Immunity against solid tumors? (2001)

The immune system protects higher vertebrate hosts against lethal effects of infections with acute pathogenic infectious agents, because these infections kill hosts before procreation has been successful. In contrast, tumors—particularly solid peripheral tumors—in general are rare and only become more frequent at a relatively late age, when evolutionary pressure cannot exert its selective influence. From this point of view, the evolutionary role of the immune system in controlling tumors may be questioned. Nevertheless, general rules about when the immune system is most efficient against infections may help us to understand why solid peripheral tumors in particular often do not induce an immune response, or do so much too late to be of significant influence on the patient's survival. This short review attempts to summarize some of these general rules and explores the ways in which one may use this knowledge to improve immunity against solid tumors. Rolf Zinkernagel, distinguished speaker in the Joint International Journal of Cancer Meyenburg Stiftung Lectureship. His talk, on which this Mini Review is based, was presented at the Deutsches Krebsforschungszentrum on 30 October 2000. During the past few years, the reactivity of the immune system has been described by the signal 1 plus signal 2 hypothesis.1-5 This two-signal hypothesis proposes that T and B cells recognize antigen via specific antigen receptor as signal 1; this signal 1 alone is a negative signal and turns off the specific lymphocyte. The same T cell is, however, induced to respond, proliferate and differentiate to become an effector cell if a second signal is provided via B7, CD40, fas, interleukin-2 (IL-2) or several of the adhesion molecules, acting alone or in combination. Antigen-presenting cells (APCs) are given a key function in this concept because they possess the capacity to process antigens and present them from the outside via class II MHC molecules and from the inside via class I MHC molecules. However, more importantly, they not only express various second signals but can also migrate to lymphatic organs. Two-signal theories were first proposed to explain B-cell activation via specific linked T-helper signals.1 The proposal was extended to T cells, to include the possibility that second signals could also be provided in a bystander fashion rather than by direct contact.2 More recently, these two views were combined as follows:3-5 a signal 1 alone always turns off T or B cells. A signal 1 plus a signal 2 turns on T or B cells. For naive T or B cells, signal 2 can come exclusively from so-called professional APCs, i.e., dendritic cells, whereas memory T cells may receive the second signal in a bystander fashion. Any T cell—naive, activated or memory—can be made tolerant if exposed to signal 1 alone. These two-signal theories have been largely favored to explain both induction and absence of immune reactivity (i.e., immunological tolerance) against foreign or self-antigens, respectively, and have been widely used to explain tolerance to peripheral self-antigens or absence of responses to peripheral tumors. We have proposed an alternative view: immune reactivity is not regulated by negative signals, such as signal 1 alone, but rather by the dose of antigen, the time it is available and the localization of antigen, within or outside of organized lymphoid tissues.6-10 Accordingly, if antigen does not reach secondary lymphoid organs in minimally sufficient doses or during a sufficiently a long time period, it is immunologically ignored. Antigens that are always available and persist in the lymphoid system and in blood delete T cells. Antigen that reaches secondary lymphoid organs in a localized fashion in sufficient, but not excessive amounts, and for a sufficient time period, but does not persist, induces protective immune response. A balance of available specific precursor T or B cells relative to amount, duration and distribution of antigen is relevant for T-cell induction versus exhaustion or versus ignorance. If a foreign protein (bovine serum albumin, hen egg lysozyme, fowl gamma globulin) is injected subcutaneously in substantial amounts, usually neither T-cell nor antibody responses are induced. This correlates with the usually short half-life of foreign antigens because of rapid degradation. In contrast, if antigens are brought into the host, particularly via direct intravenous injection in a monomeric form (i.e., deaggregated) at sufficiently high concentrations and persisting for a long period, T-cell responses are usually deleted.11-16 This has been particularly well documented for antigens that are very close to self-antigens and only differ in a few amino acids (e.g., fowl gamma globulin and bovine serum albumin).15, 17 In contrast to these immunization protocols, adjuvants save antigen from immediate degradation and permit long-term release of antigen, fulfilling all three key conditions (localized, sufficient but not excessive dose, for sufficient time) for enhancing induction of a T-cell response.18 Antigen is retained in a depot locally that leaks antigen to the draining local lymph node; thus antigen half-life is extended, and uptake by macrophages or dendritic cells is improved. Taken together, these and many unsuccessful experiments attempting to induce either T- or B-cell responses indicate that it is generally difficult to induce an immune response. Dose, time period of antigen availability and localization and distribution of antigen are important, and the relevant thresholds are impressive. In fact, these factors together are probably the most important guarantors against autoimmunity. Acute cytopathic infectious agents usually induce T- and B-cells responses efficiently because they fulfill requirements optimally: infection of cells within lymphatic organs or efficient transport of antigen to such organs via dendritic cells or macrophages; also, infections are initially localized and usually only spread in a generalized fashion later. Foreign or self-antigens not reaching organized lymphoid organs are immunologically ignored. Mice expressing lymphocytic choriomeningitis virus-glycoprotein (LCMV-GP) exclusively in insulin-producing islet cells of the pancreas as a transgene did not immunologically react against this new self-antigen if they were left untreated.6 If, however, they were infected with LCMV, a prompt cytotoxic T-lymphocyte (CTL) response was induced that destroyed transgenic islet cells by a perforin-dependent mechanism.19 Interestingly, infection of the same mice with a vaccinia-recombinant virus expressing the GP of LCMV resulted in CTL priming but failed to induce diabetes.19 These experiments showed that induction of a CTL response alone is not sufficient to cause immunopathological autoimmune disease but that there is an important quantitative requirement, fulfilled by LCMV infection but not by vaccinia GPLCMV infection. The difference between the two infections is that about 100 to 1000 times more effector T cells are generated by LCMV infections compared with vaccinia GPLCMV.19 A sufficiently damaging autoimmune response may, however, also be induced by highly repetitive immunizations with low doses of antigen.20 Immunological ignorance of antigen outside secondary lymphoid organs is also the key in the understanding of a series of classical experiments. Immunization experiments against rhabdoviruses indicated that this virus infects neurons exclusively and is transported retroaxonally to the nucleus, eventually destroying the neuronal cell.21 During this time of retroaxonal flux, the virus and viral antigens remain confined to neurons and stay outside secondary lymphoid organs. In fact, no immune responses—in particular no antibodies—can be measured during this period. If active immunization with attenuated or inactivated rabies virus is attempted during this incubation period (with antigen formulations that enhance antigen reaching secondary lymphoid organs), a neutralizing antibody response and/or a T-cell response may be induced more rapidly, thereby overtaking virus kinetics and mediating protection by systemic antibody responses. (There are, however, unresolved questions about the accessibility of neuronal tissues.22) Similarly, papillomavirus infections of skin-epithelial cells are often ignored for long periods, resulting in the formation of tumors (warts: see ref. 23). Also, foreign organs or cells expressing several major or minor histocompatibility antigens are immunologically ignored if they stay outside secondary lymphoid organs. This has been documented by experiments done in the thirties with allogenic dog parathyroids24 and more recently with allogenic thyroid grafts.25 Such grafts were rejected if transplanted fresh but were not rejected if transplanted after 3 to 4 weeks of tissue culture, a procedure that depletes the organs of so-called passenger leukocytes (including APCs and macrophages), which have the potential to emigrate to local lymph nodes and induce a T-cell response sufficiently powerful to cause rejection. We have compared tumor-take in mice exposed to single-cell-suspensions with tumor-take after transplantation of small solid tumor pieces of a highly antigenic methyl-cholanthrene-induced sarcoma.26 Whereas single cells never caused a tumor to grow, even when up to 108 cells were injected subcutaneously, 2 × 106 cells in a small solid tumor piece transplanted subcutaneously usually caused a lethal tumor to develop. Detailed analysis showed that after injection of single cells, some tumor cells reached draining lymph nodes within the first day and promptly induced a CTL response; this did not occur in the mice given a small solid tumor piece. Recipients of the solid tumor pieces were, however, not immunologically tolerant or unresponsive, as the two-signal hypothesis would predict. In fact, these same mice were found to respond promptly to injection of additional single tumor cells at the time—or 14 days after—the solid tumor piece had been transplanted; after similar repeated injections, the tumor was rejected. This shows that the strictly peripheral tumors neither deleted nor energized the specific T cells but that such tumors were simply ignored. Exhaustive induction of T cells causes their deletion. A long series of experiments illustrates that persistence of antigen in the thymus—but also in secondary lymphoid organs plus or minus the periphery—causes deletion of specific T cells. This happens irrespective of whether the antigen is there before or at the time of birth, or later in adult life. This has been documented for lymphohemopoietic cell chimerism in twin calves,14, 27, 28 in mice and in humans29 exposed to sufficient numbers of allogenic cells before or at birth, which can persist in the host. More recently, the same phenomenon was shown with spleen cells expressing a so-called super-antigen30 and with a minor histocompatibility antigen (LCMV Gp33 transgenic C57Bl/6 mice). The latter study demonstrated that effector T cells were induced by skin transplants but were deleted by intravenous injection of >106 spleen cells; a strict correlation between persistence of antigen-expressing transgenic cells and T-cell deletion was demonstrated.31 Similar results have been obtained with viruses tending to establish persistent infections. Intravenous infection of mice before or at birth with the noncytopathic LCMV (or of adult mice with high doses of LCMV or with very rapidly replicating variants of the virus) generally results in mice carrying LCMV persistently often at low levels for life. In these mice, LCMV-specific T cells are absent, and mice remain healthy.11, 16, 32 This T-cell deletion by overwhelming induction of all available CTL precursors is independent of the presence of the thymus. This mechanism eliminates T cells reactive against either self- or foreign antigens persistently available at sufficiently high levels in blood and lymphoid tissues. If, however, neonatal or adult mice are infected exclusively via the peripheral, subcutaneous route with comparably high virus doses, a protective immune response is always induced. If draining lymph nodes are absent (e.g., in Aly/Aly mice7), mice will become virus carriers after peripheral infection comparably to mice with intact peripheral lymphoid organs infected intravenously. Thus, infection via periphery and staggered induction of T cells in draining lymph nodes and then spleen impairs exhaustion by rapid expansion and activation of specific T or B cells, thereby improving the relative numbers game. In contrast, early systemic spread of antigen-expressing cells throughout the lymphohemopoietic system enhances T-cell deletion. Of course the relative numbers of potentially inducible T cells versus the overall concentration and amount of antigen or antigen-expressing cells are relevant for the question of induction—versus exhaustion or ignorance. The very early experiments of Traub,11 Billingham et al.13 Lubaroff and Silvers14 have shown that small doses of antigen fail to induce tolerance even at the neonatal or pre-neonatal stage, whereas large doses are much more successful. This has more recently been reanalyzed, and the results confirm that the smaller the antigen doses at neonatal or post-neonatal times the more they fail to induce tolerance, whereas high doses are usually successful.33-35 Similarly, Moskophidis et al.16, 36 have shown that peripheral infections or low-dose intravenous infections with LCMV in general cause induction of a T-cell response, whereas high doses of the same virus intravenously caused virus persistence and deletion of T cells. Several other experiments have evaluated mouse models in which T cells are at very high concentrations; the results have shown that these mice cannot be rendered tolerant in conditions under which normal mice (not exhibiting a transgenic T-cell receptor) are readily exhausted. One example of the numbers game is the following: whereas RIP GPLCMV mice were not rendered diabetic after infection with vaccinia GPLCMV, RIP Gp × TCR Tg mice were readily rendered diabetic. Thus, increase of T-cell precursor frequencies by a factor of about 105 is efficient in causing immunopathology under conditions where the usual low precursor frequency of 10−5 cannot induce disease. If a second signal (B7) is expressed in RIP GPLCMV mice,37 then diabetes can be induced with a vaccinia recombinant virus infection, but it has not been established how this enhancement actually occurs, since RIP GP/TCR Tg plus B7 transgenic mice become diabetic spontaneously. This latter experiment suggests that sufficient transgenic T cells become activated spontaneously to be recruited to the islets and cause lethal or diabetogenic immunopathology. Again, the exact pathway of this induction remains to be analyzed. The conventional interpretation, based on the two-signal hypothesis regarding enhanced immunogenicity of extra-lymphohemopoietic B7 expression, emphasizes the possibility that peripheral (i.e., nonlymphoid organ-associated B7) expression enhances peripheral T-cell responses in islets. Immunological memory describes the “improved” behavior of the immune system in a situation in which the host has already encountered the antigen previously. The details of the mechanisms are poorly understood but hotly debated.26, 38-40 Some would like to see immunological memory simply reflecting increased precursor frequencies. In fact, all evidence points to the fact that increased precursor frequencies are antigen independent and readily demonstrable in all mice primed some time previously. We would like to see memory identified with a protective advantage that should play a role in overall survival in an evolutionary context. In fact, there is good evidence that increased precursor frequencies of memory B cells or memory T cells may by themselves not be protective.41 Instead, for protection by increased neutralizing antibody titers or by effector T cells, antigen is necessary to maintain immunity. Examples are the correlation between time after vaccination and declining neutralizing antibody titers or the fact that vaccines against TB, HIV and tumors are not effective, even though increased precursor frequencies may be demonstrable (reviewed in ref.42). From these points of view, immunity—in particular immunological memory against tumors—should be critically reviewed. Attempts at vaccinating patients against tumor antigens have recently become successful, but only in a few instances has this approach improved the clinical situation.43-46 Therefore, one must ask which parameters correlate best with protective immunological memory and are of benefit to a tumor-bearing patient: increased precursor frequencies of T and B cells or activated effector T cells and antibody-secreting B cells? In most experimental models, vaccination with tumor antigens (irrespective of whether they are tumor associated or tumor specific developmental antigens) can be shown to protect against a tumor challenge. However, this is not what is of clinical interest. Experimental tumor models only rarely use postexposure vaccination protocols and only rare examples have shown protective efficiency. Our recent experimental model26 revealed the following data relevant to immunological memory and vaccination against tumors. When mice were given a solid small tumor piece of about 2 × 106 cells as a transplant subcutaneously, the tumors usually grew; CTLs were not made tolerant, and they promptly responded against LCMV infection. About 14 days after transplantation, at a time when tumors were palpable and represented roughly 107 cells, such tumor-bearing mice were vaccinated with either dendritic cells expressing the relevant T-cell epitope or single-cell suspensions of the tumor itself, either subcutaneously or intravenously. The tumor was not rejected, and tumor growth was delayed by 1, 2 or 3 days. In contrast, if the same mice were treated every 2 to 3 days with antigen-pulsed or antigen-epitope-expressing dendritic cells, or by single cell suspensions of the tumor, for a period of about 3 to 4 four weeks, tumors were mostly rejected and did not grow again after cessation of the therapy. The tumor-bearing mice—vaccinated only once—were clearly primed, because their spleen cells and lymph node cells (taken out of the tumor-bearing mice) had primed T cells and could be restimulated in a secondary in vitro culture. Therefore, it was not a failure to prime the T-cell response that prevented tumor rejection by mice vaccinated once, but rather a failure to keep T cells activated and the response going for a long enough time to eliminate the tumor completely.26 Research in immunology is rather susceptible to beliefs and dogmas; examples like idiotypic networks or suppressor T cells reflect a painful history of misleading concepts. The two-signal (or danger) theory seems to lead immunology into a similar trap. If clearly defined predictions of a hypothesis can be falsified, the general validity of that hypothesis should be questioned. Such falsification has the greater weight the more convincing evidence can be demonstrated to be of biological relevance in the living host. As summarized here, fibroblasts without second-signal qualities can readily induce a CTL response26, 47 if such fibroblasts reach organized lymphoid tissue; they are immunologically neutral in the extralymphatic periphery. If antigens—even in great masses—stay strictly outside secondary lymphoid tissues, they are ignored by T cells without T cells being deleted, anergized, made tolerant or silenced. Cross-presentation, cross-processing or cross-priming of peripheral antigens to be efficiently presented via class I APCs are theoretically not impossible but seem biologically inefficient in viral infections and against several at not at a that would improve immune this mechanism would be not as by several This does not that under very in vitro conditions or under high doses of antigens for or inactivated such may be demonstrated is that within the of the two-signal hypothesis such key parameters have only in protection to tumor models or minor histocompatibility examples have not (or have only defined the antigen at the and of the potentially by effector CTLs in the of the and the which would The why and can be only to a and under conditions is not the data indicate that immune responses (including against are usually not as had been proposed and again by the two-signal Instead, T- and B-cell responses are regulated by dose and time of these parameters seem to be in understanding why successful, peripheral tumors are not prime for immune the of relative numbers of immunological effector cells and of tumor cells them to immune all immune the views presented that may be