Immunological Control of Neoplasia - Chapter 3

Author: Christine Ichim

Chapter 3 Immune Suppression in Cancer

Previous theories about cancer interactions with the immune system attributed a primary role for the immune system in controlling and stopping the growth of neoplasia. The concept of immune surveillance, proposed by Thomas and Burnet, advocates that immune cells are patrolling the host for mutations. Once mutations have been detected, the abnormal cells are destroyed. The theory of immune surveillance, and supporting arguments are described in the book Immunological Surveillance by Burnet (526). This theory has come under fire in the late 1970s and to some extent even until today, due to the discovery of tumour suppressor genes that controls expansion of mutated cells much more effectively than the immune response. p53 is a well described tumour suppressor gene that becomes activated when DNA damage occurs. The p53 product stops the cell cycle until DNA repair can occur, or if the damage is too extensive, the p53 product will initiate apoptosis (527). 90% of mice deficient in p53 develop tumours within 9 months (528), this is contrasted with the T cell deficient mouse that at 9 months gets 6.8% tumour (529). Therefore, at face value, the molecular tumour suppressor mechanisms seem to be much more effective at suppressing incidence of neoplasia than immunological defenses. Additionally, tumours growing in mice tend primarily to be of hematopoietic origin, this is explained by some scientists to mean the genetic abnormality that does not allow the mouse to make T cells also predisposes the mouse to chromosomal instability in its hematopoietic cells. Hence the reason mice have increased tumour incidences is not because they lack immunological surveillance mechanisms but because of an inherent predisposition to neoplasia.

Although, under normal conditions the immune response might not be the first line of defense against neoplasia, nor the most effective, the influence of the immunological response cannot be overlooked. For example, the previous chapter referred to studies where the levels of certain immune functions, such as T cell proliferation or NK cell activation, were correlated with improved prognosis. Such correlations indicate a function of the immune response in controlling the growth of an already existing cancer. Indeed, it would be more plausible for the role of the anticancer immune response to be in the control of micrometastasis as opposed to solid tumour masses, since tumour masses will likely cause immune inactivation by virtue of the immune suppressive microenvironment surrounding the tumour. Micrometastasis however would be more controllable by immune response for two reasons. First of all, the anatomical locations where the metastazing cells have migrated are not subjected to the bulk of the tumour secreted immunosuppressive factors. Secondly, by the time a cancer cell has metastasized, it possesses several additional mutations that the original cancer cells do not possess. These additional mutations possibly could increase the immunogenicity of the cell. Conversely, the mutations may allow the cancer cell to possess immune evasive mechanisms that the original cancer mass did not possess.

The importance of immune response to tumours is often underscored due to the lack of understanding of the immune evasion mechanisms used by the tumour, some of these occasions are: 1. Placing too high expectations on clinical interventions derived from our perceived ideas of how the immune response works. 2. Looking for the effects of antitumour immunity in inappropriate places. 3. Observing interactions between immune system and tumours without knowing what components of the immune system to observe. The first point is demonstrated by the poor performance of vaccine trials and the different immunotherapeutic interventions referred to in the first chapter. When the scientific community saw that chemotherapeutic methods were more successful at inducing remission, the focus of research was changed and the idea that immunotherapy was a failure became ingrained in the minds of many scientists. Today we know that many previous regimes did not work due to tumour mediated suppression of the protective response. The technology at the time of the original trials did not allow for identification, much less for intervention, in the process of neoplasia suppression of host defense functions. The second point is illustrated by the studies that claim immunotherapy failed due to its inability to facilitate antitumour responses in murine s of spontaneous tumours. Such studies dangerously claimed that spontaneous tumours lack tumour specific antigens, hence implying tumour immunotherapy is a hopeless cause. In fact, it is known today that spontaneous tumours do possess such antigens but the reason immune responses are not generated is due to the ability of these tumours to suppress the initiation of immunity. The third point refers to studies in cancers where suppression of certain host immune functions were not observed even in end stage cancer patients. The hypothesis is, if cancer patients possess normal T cell mitogenic responses but still are suffering from advancing cancer, than the effect of the T cells must be minimal and also tumour cells are not immune suppressive. Studies such as these persuaded many researchers into believing that immune response and cancer are not correlated. Today it is known that cancer cells do suppress T cell responses, but the suppression is primarily antigen specific. This makes sense from a cancer-evolutionary perspective: the cancer cell’s main objective is to survive. Therefore, the cancer will not “want” a state of systemic immune suppression that may kill the host (and the cancer with it). Instead, the cancer will “want” to suppress the immune functions that of danger only to the cancer, such as T cells with Tcr specific for the malignancy. The observations that in some patients all T cells, irrespective of specificity, are suppressed are likely to be a nonessential immunosuppressive activity of some cancer cells. The purpose of discussing the above points is to illustrate the importance of not only knowing that tumour suppression/evasion of immune function exists but, also that the understanding/misunderstanding of these mechanisms will contribute to the viability of tumour immunology as a science.

We will examine tumour-induced immune suppression by dividing the topic into two categories: antigen nonspecific immune suppression and antigen specific immune suppression. The categories are not mutually exclusive and are likely to occur simultaneously in the majorities of neoplasms. Little data is presently available at connecting specific cancer causing mutation, to the above mechanisms of immune suppression. In other words, I have not seen studies that correlate overexpression of, for example, the cancer causing ras mutation with secretion of immune suppressive factors such as TGF-B. The molecular understanding of the phenomena described below will occupy the laboratories of investigators for many years.

Antigen Nonspecific Immune Suppression

This topic was already touched upon in the discussions of the different immune cells, in cancer, however, here emphasis will be placed on the actual compounds mediating the suppression.

TGF-B

TGF-B belongs to a family of proteins involved in diverse processes such as induction of apoptosis, embryonic development, control of inflammation, and growth inhibition (530-532). As to its effects on neoplasia, TGF-B is generally an antiproliferative agent and acts as a cellular control. TGF-B is activated from a latent form by thrombospondin, which is a gene activated by p53 in response to DNA damage (533-535). Thus partly by virtue of the antiproliferative effect of TGF-B, p53 can hold DNA damaged cells in the G0 stage of the cell cycle until repair is complete. Considering these antineoplastic activities, it is surprising that TGF-B is involved in the cancer-mediated suppression of immune functions. One can explain this paradox by noting the existence of different types of TGF-B with different biological activities that is sometimes recognized by the same antibody. Thus it may be a different molecule that possesses antiproliferative activity to the neoplasm than the molecule secreted by the neoplasm to facilitate its growth. Such an explanation is likely when one considers that neoplastic cells generally possess the mutator phenotype and even single amino acid substitution in some proteins can completely alter their biological activity without effecting the portions recognized by antibodies. Another explanation for the paradoxical effects of TGF-B in cancer inhibition and immune suppression is that the molecule mediating the effects is the same but that in the early stages of neoplastic development immune response is irrelevant therefore it does not matter that p53 is activating an immune suppressant. However when the cancer mutates and is no longer responsive to the inhibitory effects of TGF-B, the molecular parts of the cancer that want to stop neoplastic progression start to secrete TGF-B in larger amounts to compensate for the fact that the cell is not inhibiting its proliferation. The larger amounts of TGF-B secreted suppress the immune function even though the cancer cell is secreting it to stop its proliferation. This hypothesis could be tested by observing whether a correlation exists between cancer progression and increase in TGF-B secretion.

The specific mechanisms of TGF-B mediated suppression of anticancer immunity occurs through a variety of mechanisms, one of them being augmentation of the Th2 response by downregulating secretion of Th1 cytokines such as IL-12 (536), IFN-( (537), or IL-2 (538). As mentioned previously, a balance exists between the Th1 and Th2 response. At conferences dealing with regulation of immune responses by cytokines, one notices the repeated references to the yin-yang belief, in that not only does Th1 immunity increase by administration of Th1 cytokines, but also, Th1 immunity is increased by decreasing Th2 cytokines. The regulation of Th1 by Th2 and vice versa is demonstrated in mice lacking essential cytokines to one of these responses, where the other response will predominate. For example, mice deficient in IL-10 tend predominately to initiate Th1 responses and develop autoimmune disease associated with over-activation of Th1 responses such as colitis (539). Likewise, mice deficient in IL-12 will tend to initiate Th2 responses (540). In addition to skewing the Th1/Th2 balance toward Th2, TGF-B is involved in altering ability of recipients to accept grafts (which, strictly speaking is potentially the effect of a Th1/Th2 modulation). An example of TGF-B’s role in halting the initiation of responses that are destructive to the target is the work of Streilein’s group with anterior chamber immune deviance, who have noticed that injection of antigen into the anterior chamber of a mouse’s eye, will skew the peripheral immune response toward that antigen so that a Th2-like response will ensue (541). The peripheral response will not trigger destruction of the reinjected antigen, although, if the primary immunization was not in the anterior chamber, than immune destruction of the antigen will occur. This concept was demonstrated elegantly when p815 cells were injected into the anterior chamber of allogeneic mice. The recipients were subsequently grafted with skin from donors possessing the same haplotype as the p815 cells, or donors possessing a third party haplotype. The grafts from the third party haplotype were rapidly rejected, while the grafts from the p815 haplotype were allowed to grow for an extended period before rejection. To demonstrate further the importance of the anterior chamber as a site of immune modulation, the experiment was repeated, yet this time the p815 cells were injected subcutaneously, graft rejection then occurred faster in the group presensitized with the p815 cells (542). The fact that aqueous humour contains large levels of TGF-B and that TGF-B generally suppresses activation of cytotoxic responses, prompted D’Orazio and Niederkorn to investigate whether TGF-B will alter to ability of ocular macrophages to promote Th1 or Th2 responses. Activating ocular macrophages in vitro without TGF-B resulted in secretion of IL-12, however when levels of TGF-B similar to those found in the anterior chamber were added to the macrophages, these macrophages secreted IL-10 and stopped secreting IL-12, thus arguing for the effect of TGF-B at switching Th1 responses to Th2 responses. To demonstrate the importance of TGF-B on macrophage induction of immune response in vivo, the investigators injected antigen pulsed TGF-B treated and antigen-pulsed TGF-B nontreated macrophages, in mice and then challenged the mouse with the same antigen. Only the injection of TGF-B treated macrophages could stop the inflammatory response to the antigen challenge, while injection of the nontreated macrophages resulted in increased inflammatory response. In mice lacking IL-10, pretreatment with TGF-B treatment macrophages did not abrogate the inflammatory response, thus arguing for the importance of IL-10, in TGF-B alterations in immune response (543).

Besides altering initiation of immune response, TGF-B can also suppress certain characteristics of already ongoing responses. For example, part of the effector phase of immune response involves ADCC through macrophages. This requires the ability of macrophages to secrete cytokines and to possess phagocytic abilities, TGF-B suppresses both (544-546). If macrophages are not phagocytosing the cellular debris, then this debris will accumulate, causing a chronic inflammatory condition. Although, TGF-B prevents inflammation in the local microenvironment, if the accumulated cellular debris enters other anatomical locations, mediators of inflammation will be secreted such as IL-1, which will in turn stimulate the hypothalamus-pituitary-adrenal axis to secrete glucacorticoids that will suppress the immune system even more, thus allowing tumour growth. Congruent with this, is the observation of activated acute phase responses in tumours of several histologies (547-549).

TGF-B elevation in cancer has also been associated with poor prognosis (550) and has been correlated to cancer progression (551), although from these studies, the effect of immune response has not been elucidated. In the future, studies correlating levels of different immune cell activation with serum cytokines, and patient prognosis should help in elucidating the interplay between TGF-B, immune activation, and the in vivo relevance to the cancer patient.

IL-10

IL-10 has been described to be the pivotal cytokine for Th2 responses because of potent anti-inflammatory and anti-cell mediated immunity actions. Indeed, the IL-10 knockout mouse possesses many properties of an animal with an uncontrolled Th1 predisposition such as autoimmune colitis, autoimmune arthritis, and lethality from exaggerated Th1 responses that in normal animals would be controlled by Th2 feedback mechanisms. IL-10 can be produced by T cells, B cells, macrophages, dendritic cells, keratinocytes and neutrophils, usually during resolution of immune response or when a Th2 response is needed for eradication of pathogens (552-555). IL-10, or cells that are innately primed to produce IL-10 upon stimulation,are found in anatomical locations where inflammation would be deleterious to the host, such as the placenta (556), testis (557), aqueous humour (543), and central nervous system (558). As mentioned previously, several pathogens such as measles and the Epstein-Barr virus secrete IL-10 analogues to evade the protective response. In view of these immune suppressive effects, one would postulate that either secreting IL-10 endogenously or influencing other cells to secrete it would be advantageous for tumours because it causes evasion of the immune response. In fact this does occur. IL-10 is secreted by gliomas (559), chronic myeloid leukemia cells (560), nasopharyngeal carcinoma (561), melanoma cells (155), squamous cell carcinoma (338), ovarian carcinoma (561), renal cell carcinoma (562), breast cancer (563), lymphoma (564), gastric carcinoma (565), soft tissue sarcoma (566), T cell leukemia (567) and bronchogenic carcinoma (577). The importance of IL-10 in enhancing growth of neoplasia, possibly through immune inhibition, is demonstrated in studies that show correlation between serum IL-10 and poor prognosis. This has been shown in lung cancer (578), and non-Hodgkin’s lymphoma (579), but not in large cell lymphoma (580). Future studies are needed to examine IL-10 levels in the tumour microenvironment and correlate them with prognosis, since in order for IL-10 to enter serum very large amounts have to be produced, this does not have to happen in order for the tumour to suppress immune functions.

IL-10 generally is believed to suppress antitumour immune attack because it directly activates Th2 cell differentiation from the Th0. In addition, IL-10 suppresses the ability for tumours to be recognized by Tc cells by virtue of IL-10's ability to downregulate MHC expression. Matsuda et al demonstrated that incubating melanoma target cells with IL-10 resulted in 100% inhibition of killing by autologous MHC 1 restricted Tc cells, which was associated with decrease in MHC 1 expression (581). IL-10 can also effect antigen presentation by downregulation of intracellular proteins needed for antigen processing such as TAP-1 and TAP-2 (582). At the level of the T cell response, tumour induced anergic T cells maintain a state of anergy based upon autocrine IL-10 secretion (583). This is consistent with findings that IL-10 is needed to expand a population of Th cells, not dissimilar to Ts cells, that antigen specifically promote tolerance (584-586). The role of these cells in cancer awaits elucidation.

PGE-2

PGE-2 is a product of inflammatory reactions that has been associated with switching to a Th2 immune response. Since cancer cells usually are usuallyt a greater amount then cells in normal tissue, most tumours are associated with inflammatory-like reactions and therefore production of PGE-2. Conversely, cancer cells also possess the ability to induce secretion of PGE-2 from immune cells entering the tumour such as macrophages. In fact, many researchers have claimed that suppressory macrophages are constant secretors of PGE-2. This product of inflammation possesses the ability to suppress Th1 cytokine production by antigen presenting cells such as dendritic cells (587) or macrophages (588). Ineffective Th1 production from these cells will push the immune response toward Th2 or even induce anergy in the T cells during antigen presentation. In vitro studies have reiterated this point since PGE-2 treatment of Th cells during activation induces them to secrete IL-4, IL-10, and IL-13, all of which are Th2 cytokines, while at the same time PGE-2 inhibits secretion of the Th1 cytokines, IL-12 and IFN-( (589). An additional effect of PGE-2 on antigen presentation is that, it can downregulate expression of MHC 1 and MHC 2, on the antigen presenting cells, this enforces the point that this mediator is a mechanism for tumour immune evasion (590, 591). P.K. Lala’s group has attempted to use PGE-2 inhibitors to improve results of tumour immunotherapy with LAK cells, these results are promising (89).

Antigen-Specific Immune Suppression

The reason tumours would want to evade the immune response is because an effective immune response would lead to their eradication. Therefore, it is possible that immune response has already destroyed many cancer cells in the tumour but the ones that survive and eventually kill the host are tumours that somehow can destroy the immune response initiated against them. Examples exist both in vitro and in vivo of tumour cells altering their phenotype due to the selective pressures of immune response, for example, patients that undergo immune therapy with LAK cells, have a much higher probability of losing expression of surface MHC 1 after the therapy. This is believed to be due to immune killing of MHC 1 expressing cells, the cells that do not express MHC 1 are the ones that proliferate and are detectable (592). While immune selection occurs because Tc cells kill cells that present antigen on MHC 1, it would be interesting to see if the cells selected have an increased sensitivity to NK killing, since NK cells preferentially destroy cells that lack MHC. It would also be interesting to use animal s such as the SCID mouse to see if activating NK cells against the tumour would select for tumours that possess MHC 1, after this selection one could adoptively transfer Tc cells into the mouse and look for destruction of tumour. Such a two step approach is similar to the bottleneck hypothesis proposed by Jon LaMarre (University of Guelph) in that the primary treatment selects the cancer cells for one phenotype and then a secondary treatment destroys the remaining cells that have switched phenotypes.

The immune system selects for cancer cells that are resistant to the immune response against them. These immune resistant cancer cells possess fascinating mechanisms to antigen specifically avoid immune response such as the induction of immune cell death, and the alteration of the immunocytes to secrete substances that will turn off other immune cells attacking the tumour. There have been made many allusions to the Th1/Th2 paradigm as it relates to cancer. Indeed, activation of Th2 cells is an antigen specific mechanism of immunodepression since Th2 cells have exquisite sensitivity to the antigen by means of the Tcr, which is antigen specific. Activation of Th2 cells that are specific for the tumour will only be activated by the tumour antigens. Therefore, these lymphocytes will only be inducing a Th2 response in the vicinity of the tumour resulting in antigen specific suppression. An even more potent mechanism of antigen specific suppression is the ability of tumour cells to kill the T cells that are infiltrating the tumour. The T cells infiltrating are primarily specific for tumour antigens and usually get activated in the tumour microenvironment. Although, many of these T cells differentiate into Th2 cells, T cell death is detectable in the tumour as well. A mechanism by which tumours induce T cell death is via expression of the fas ligand. Fas, the receptor for the fas-ligand, is usually a membrane bound protein. Activated T cells express fas, and tumour cells of various histologies have been shown to possess fas ligand. Therefore when tumour cells encounter activated T cells, the T cells die. Since T cells that enter the tumour possess the Tcr that recognizes tumour antigen, the very cells that could kill the tumour are the ones that the tumour kills (593). An interesting aside is that areas of immune suppression/privilege such as the eye, the testis and the placenta, similarly possess fas ligand that prevents the entering of activated T cells.

Evidence for the importance of antigen-specific T cell deletion also comes from observations that cancer patients possess alterations in the Tcr chains specific for certain antigens, and that these deletions are accentuated as the cancer progresses. Support for the involvement of T cell deletion in neoplasia also comes from mouse studies in which tumour development occurs simultaneously with the Tcr VB2 subset deletion (594). A very convincing experiment showing the ability of tumour cells to induce antigen specific tolerance was performed on the murine B16 melanoma . The investigators transfected the tumour with the antigen hen egg white lyzozyme (HEL), and allowed the tumour to grow in syngeneic mice. Time dependently, the T cells in tumour bearing mice could no longer respond to HEL but the response to other antigens remained intact. Furthermore, by monitoring Tcr specificity, the investigators noted a decrease in the number of HEL-specific T cells (595). This experiment in the opinion of the author is the most conclusive demonstration of the tumour cell’s ability to alter immune response.

In the late 1960s and through the 1970s a flurry of papers were published describing tumour secreted blocking factors which antigen specifically inhibits anti-tumour responses. The existence of such factors has been forgotten in the literature since the assays used for their detection were said by some to be faulty. Regardless, researchers have cloned cells that produce these blocking factors and molecular evidence suggests the activity to be mediated by soluble MHC-Antigen complexes (596). Some groups have even claimed induction of anticancer response by purifying patient plasma of blocking factors using plasmapheresis technology (Rigdon Lentz, Tennessee Oncology, Personal Communication). While further study needs to be conducted on the properties of such soluble factors, one cannot help but notice their uncanny similarity to the soluble suppressive factors described in ocular immunology (597) and the factors presumed to mediate infectious tolerance (598). Collaborations between these fields should allow for much more expedient progress.

In conclusion, tumour cells tend to evade host immune function by both antigen specific and antigen nonspecific means. Antigen specific methods of evasion destroy/impair only the part of the immune system that can destroy the tumour, whereas antigen nonspecific suppressive mechanisms suppress immune function to all antigens systemically or in the tumour’s microenvironment. It is antigen nonspecific suppressory mechanisms that allow for bacterial growth inside some solid tumours (599). This paper has touched upon some of the basic mechanisms of tumour mediated suppression. However, many others exist but could not be described due to space limitations, these include:
1. Tumour downregulation of host costimulatory molecules.
2. Tumour secretion of other immune suppressive cytokines such as IL-4, IL-13 and VEGF.
3. Tumour secretion of progesterone induced blocking factor.
4. Shedding of soluble tumour antigens so to induce tolerance.
5. Tumour covering it is self with negative sugars such as mucins to avoid cellular interactions.
6. Secretion of immune suppressive gangliosides.
7. Tumour modulation of host endocrine function.
8. Tumour suppression of thymic function.
9. Release of free oxygen intermediaries from tumour (suppresses NK function).
10. Tumour alteration of T cell signaling mechanisms.

Chapter 1 - Introduction to The Immunotherapy of Cancer
Chapter 2 - Components of Immune Response
Chapter 3 - Immune Suppression in Cancer
Chapter 4 - Immune Interactions with Chronic Myeloid Leukemia

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