How+Cancer+Eliminates+Normal+Immune+Responses

//Jim Skoumal and Kelly Hall//

**Table of Contents:**

 * 1) Project Objective:[[image:http://4.bp.blogspot.com/-eXz8is49K4g/Tah1iwIOpyI/AAAAAAAAAzg/IaWZe86w6KI/s1600/image4.png width="299" height="232" align="right" link="http://www.google.com/imgres?hl=en&sa=X&biw=1280&bih=654&tbm=isch&prmd=imvns&tbnid=37Lv5GGzCgha7M:&imgrefurl=http://valerietonnerhealthcoach.blogspot.com/2011/04/immune-system-part-one-what-is-immune.html&docid=pag79ZjnwqvFEM&imgurl=http://4.bp.blogspot.com/-eXz8is49K4g/Tah1iwIOpyI/AAAAAAAAAzg/IaWZe86w6KI/s1600/image4.png&w=599&h=472&ei=nPqeT6XNCqaJiAKzw_WnBA&zoom=1&iact=rc&dur=216&sig=110997969811797149771&page=1&tbnh=130&tbnw=158&start=0&ndsp=21&ved=1t:429,r:20,s:0,i:126&tx=101&ty=44"]]
 * 2) Overview of the Immune System
 * 3) What cells are involved
 * 4) How it functions normally
 * 5) How Cancer Cells Interact with the Immune System (Breadth)
 * 6) Evasion
 * 7) Destruction
 * 8) Tumor counterattack (Depth)
 * 9) Coexistence-will talk briefly about this, but visit Cancer and the Immune System for more detail
 * Implications and Summary
 * Bibliography

**1. Project Objective**
The purpose of this wiki is to examine the myriad ways in which cancer cells and the immune system interact. We have provided some general information about all the currently known models of cancer cell-immune cell interactions, but will focus mainly on the ways in which cancer cells actively suppress and destroy the immune system. Specifically, we will provide an in-depth analysis of the phenomenon known as "tumor counterattack" and attempt to determine if it can be considered an immune escape mechanism.

**2. Overview of the Immune System**
According to //Janeway's// //Immunobiology,// in order to effectively protect an individual against disease, the immune system must fulfill four tasks: immunological recognition, immune effector functions, immune regulation, and immunological memory (1). The first task refers to having an immune system that recognizes the presence of an antigen (something foreign in the body). The purpose of the second task is to contain the foreign bodies and, if possible, to eliminate all of them before they can do damage. In the case of viruses and bacteria, to eliminate them before they can replicate. This is done by a variety of different methods. Which effector methods are used depends on which branch of the immune system is taking part in the immune response. The third task, immune regulation, refers to the ability of the immune system to shut down when there is no longer a need for it. Lastly, the immune system must have the capability to "remember" what antigens it has fought against in the past. With this memory, the immune system can destroy antigens it has already seen more quickly and more effectively the next time they enter the body.



As mentioned above, the immune system is comprised of different branches, that of innate immunity and adaptive immunity. Their main goal is roughly the same: to seek and destroy potentially harmful antigens that get into the body. The cells of the innate branch include macrophages and neutrophils and can be thought of as soldiers in the front lines. The cells of the innate system are usually the first cells to recognize and destroy pathogens. Recognition occurs when cell surface proteins on macrophages and neutrophils bind the cell surface proteins on other cells. In the case of immune cell-antigen binding, the signal cascade that is induced by binding alerts the macrophages and neutrophils the antigen is non-self //and// dangerous. This leads to the termination of the antigen.

However, if neutrophils and macrophages cannot effectively contain the pathogen, the cells of the adaptive system are called into action. Adaptive immune cells include T-cells and B-cells. Activating the adaptive response is usually done when dendritic cells bind an antigen, ferry it to the lymph tissue where adaptive immune cells reside, and hand the antigen to a T-cell that recognizes that particular epitope. Each T-cell binds to a specific antigen epitope, so T-cells literally live to destroy only a certain antigen. Once the T-cell binds the antigen, it begins proliferating so there are a lot of the same T-cells available to fight off that particular antigen. There are many subsets of T-cells, and each subset functions slightly differently. CD8+ T-cells, also known as Cytotoxic T-cells (Tc), destroy pathogens using granzymes. CD4+ T-cells, also known as Helper T-cells (Th1 and Th2), help __#|activate__ B-cells as well as function as memory T-cells. Memory T-cells will recognize an antigen that was once present in the body and respond more quickly than a non-memory T-cell. Memory T-cells do not have to wait for the cells of the innate immune system to become overwhelmed or for an antigen presenting cell to present it with the pathogen.

Eventually, B-cells will be presented with and bind to the same antigen. They will also begin to proliferate and, in the process, produce B-cells that are even better at binding the specific antigen epitope than the original B-cell. Some B-cells remain memory cells, while others differentiate into plasma cells. Plasma B-cells are able to secrete antibodies that bind to antigens, thus marking the antigens for destruction. Antibodies also function to block receptors on the antigen that allow it to bind to the body.

Both innate and adaptive immune cells destroy antigens by using cytokines such as chemokines and cytotoxins. A cytokine "is any small protein made by a cell that affects the behavior of other cells. Cytokines act via specific cytokine receptors on the cells they affect." A subset of cytokines, chemokines are "small chemoattractant proteins that stimulate the migration and activation of cells, especially phagocytic cells and lymphocytes. They have a central role in inflammatory responses." (1) When a macrophage or neutrophil encounter an antigen, it will begin releasing chemokines to attract more innate immune cells and dendritic cells. Therefore, chemokines are similar to antibodies. The binding of antibodies to antigen receptors makes the antigen a target for cytotoxic T-cells in the area. Once the cytotoxic T-cells encounters the antigen (marked or not), it uses cytotoxins to destroy the antigen. Cytotoxins are proteins made by cytotoxic T cells and Natural Killer cells (NK) that participate in the destruction of target cells." Examples include perforins, granzymes, and granulysins." Generally, perforins punch holes in the plasma membrane of cells, which then allows the granzymes and granulysins to enter the cell and begin a signal transduction cascade that results in programmed cell death.

The most important thing to remember about the immune system is that events do not necessarily occur linearly. All of the cells and destruction mechanisms described above can occur at the same time during the same immune response. Furthermore, the chemokines and cytokines being secreted by the body can have different effects on the same cells at different times and in different locations. Many of the cytokines secreted antagonize one another, so the resulting mechanism of action depends entirely on the concentration of particular cytokines. Each breach of the body's protective layers must be looked at as individual incidents. No two immune responses are alike.

**3. How Cancer Cells Interact with the Immune System**
As described above, one of the main jobs of the immune system is to surveil the body for cells that are not supposed to be there. According to Igney, "the key cells of the immune system for tumor surveillance are T-cells" ([|2]). Over the years, many tumor cell antigen epitopes have been identified that can be recognized by T-cells. The antigen epitopes found exclusively among tumor cells are termed tumor-specific antigens. These antigens typically arise from mutations and translocations of normal genes. Examples include B-catenin, cdk4, and ras. There are also tumor-associated antigens that are expressed not only by tumor cells, but by normal cells too. In class, we discussed the importance of the tumor cell environment and how it contributes to the growth and development of the tumor. These tumor-associated antigens play a significant role in tumorigenesis by defending the cancer cells from the immune system or by secreting mitogens. Lastly, the overexpression of normal cellular proteins can also alert the immune system that a tumor is growing ([|2]). Cancer cells must find ways to stop the immune system from doing its job, which is to detect tumor-specific antigens, tumor-associated antigens, and the overproduction of normal cell proteins (among other things). The majority of the mechanisms cancer cells use to evade or destroy the immune system invovle structural and functional changes of these tumor cell antigens.

__//Ignorance//__ One way in which tumor cells escape immune effector cells is by having a phenotype that fails to activate an immune response. Essentially, the tumor cells do not appear to be "dangerous" to the immune cells so the immune system ignores them. Igney and colleagues suggest one mechanism of immunological ignorance may be due to a lack of danger signals present on or around the cancer cells (2). If the cancer cells do not have cell surface proteins that mark it as dangerous, the immune system has no reason to act. Similarly, the cancer cells may lack the needed number of adhesion molecules on its surface. So even if the immune system recognized the tumor cells as dangerous, immune effector cells would not be able to bind to the tumor. As mentioned above, immune cells must bind cell surface proteins, including adhesion molecules, in order to induce the signal transduction cascade that leads to programmed cell death. Without the correct type or number of adhesion proteins, the tumor cells can escape the deadly effects of immune cells.
 * A. Evasion**

Another way in which tumor cells can hide from immune cells is to be physically hidden. Igney and colleagues explain that the stromal cells surrounding the tumor cells may actually act as a barrier that prevents immune cells from reaching all of the cancer cells (2). If the immune cells cannot reach the tumor cells, then the tumor cells will be able to continue growing.

Lastly, there are some places in the body where the immune system does not travel. These immune priveledged sites would be a great place for tumor cells to hide out and proliferate. Even if the tumor cells have adhesion proteins and surface proteins that mark them as dangerous, immune cells would be destroyed upon entering the immune privileged site. Such sites include the brain, the eyes, and the testes.

__//Impaired antigen presentation//__ Igney and colleagues also discuss the benefits of impaired antigen presentation by tumor cells. They state "in general, defects in antigen presentation are more pronounced in metastatic lesions than in the primary tumor" (2). This suggests that the down-regulation or change of specific cell surface proteins is not the only mechanism tumor cells utilize to avoid destruction by the immune system. However, many tumor cells lack molecules critical for the binding of immune cells and antigen-presenting cells. One example is the reduced expression or complete lack of the MHC class 1 molecules (2). These molecules are required for immune cell-antigen binding to occur. Reducing the number of these molecules or changing them to prevent binding will greatly reduce the affinity immune effector cells have for the tumor cells. This can be acheived through "gene deletions or rearrangements, point mutations, and defects in transcriptional regulation [that] lead to selective loss of an MHC haplotype, locus, or allele" (2). These changes can occur to other critical molecules as well. And while complete loss of needed molecules sounds great, this can actually target the tumor cells for death by natural killer cells (NK). This again shows that cancer cells utilize a variety of different mechanisms to avoid detection by the immune system.

__//Tolerance Induction//__ Besides hiding from the immune system, tumor cells have also developed ways to make T-cells inert. The immune system has a way to protect against T-cells that do not work properly, and cancer cells have learned to use this to their advantage. When a T-cell binds to another cell, the cell's MHC molecule must bind the T-cell receptor (TCR), //and// co-stimulatory molecules on both the T-cell and the antigen must also bind. If the T-cell fails to bind the co-stimulatory molecules, it becomes anergic and non-functional. Many tumor cells lack these co-stimulatory molecules, and so induce T-cell anergy whenever T-cells bind the tumor cells (2).

Rabinovich also describes another way in which T-cells, both CD4+ T-cells and CD8+ T-cells, become anergic. Although the exact mechanism behind tumor-specific antigen-mediated T-cell anergy has yet to be discovered, it is thought that antigen presenting cells (APCs) like dendritic cells play an important role (7). According to Rabinovich, it is known that "the environmental context in which the antigen is encountered by dendritic cells greatly influences the fate of the T-cell. While an antigen encountered by dendritic cells in an inflammatory context triggers [T-cell] maturation to a phenotype capable of generating a strong immune response, antigen captured by dendritic cells in a non-inflammatory environment would fail to elicit productive T-cell responses, leading instead to the development of T-cell tolerance" (7). Unfortunately, most T-cells are presented with antigens by APCs in a non-inflammatory environment. Since tumor cells are often capable of secreting immunosuppresive factors (described below), tumor cells are able to use the environment-dependent immune response to their advantage.

An even more clever approach by tumor cells is to force the T-cell to respond differently and less effectively than it normally would. Immune deviation is the process by which "the immune response is driven toward a Th2 humoral response away from the Th1 response required for efficient tumor rejection by cytotoxic T cells" (2). As mentioned in the overview of the immune system section, there are different types of T-cells. According to Igney and colleagues, Th1 cells are better at inducing programmed cell death than Th2 cells (2). Th2 cells are often involved in the down-regulation of an immune response and the activation of B-cells (1). Therefore, if the tumor cells forced the T-cells to produce a Th2 reponse, there is a greater chance that the tumor cells will survive because the Th2 response tends to shut down immune effector cells. Also some types of CD4+ T-cells have been shown to suppress the cytotoxic T-cell response (2). The immune deviation response is not well understood, but it is thought that the cytokines TGF-beta and IL-10 play an important role in the event. More research is needed to fully elucidate the mechanism of immune deviation.

__//Apoptosis Resistance//__ Resisting cell death is one of the hallmarks of cancer, and apoptosis is just one form of cell death. Clearly, cancer cells have found ways to circumvent this form of programmed cell death. Effector cells of the immune system use two main methods to induce cell death. Through the death receptor pathway, T-cells express the death ligand CD95L. When the CD95 receptor present on another cell binds the CD95 ligand, the apoptotic signal transduction cascade is set in motion - Refer to the image below(2). According to Igney and colleagues, NK cells also use the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) specifically for tumor cells (2). TRAIL functions similarly to CD95.

The other way to kill cells involves the use of granule exocytosis. "Granzymes are neutral serine proteases that can activate caspases in the target cell" (2). This is how CD8+ cytotoxic T-cells and natural killer cells destroy pathogens. Using this method, T-cells secrete perforin and granzymes toward the target cell, which trigger the cell death signal transduction cascade. Although the two mechanisms appear different, the same programmed cell death signal is activated in both. At some point in the signal transduction pathways, the caspases activate the death substrates that give rise to the cellular changes associated with apoptosis.

The red proteins in the diagram at right are proteins tumor cells use to inhibit apopotosis at various points. FLIPs interfere with the initation of apoptosis by binding directly to the receptor that induces cell death. Some Bcl-2 proteins are anti-apoptotic, and prevent the mitochondria from releasing enzymes that assist in apoptosis. Lastly, IAPs (inhibitor of apoptosis proteins) inhibit apoptosis by binding to the caspases, preventing them from cleaving proteins needed in apoptosis. All of these proteins are found to be over-expressed in many mouse and human tumor models, which may explain why it is so challenging to get rid of certain cancers (2).

There are many other ways in which tumor cells can inhibit apoptosis. One mechanism is to express decoy death ligands and death receptors. Research shows that some tumor cells express soluble forms of CD95-like ligands. These pseudo death ligands can bind to death receptors and competitively inhibit the real death ligands expressed by T-cells. Similarly, tumor cells can also express molecules that are similar to CD95 receptors (2). Yet when the CD95 death ligand binds to the pseudo receptors, the apoptosis signal tranduction pathway is not activated. The use of similar, inert forms of death ligands and death receptors can block the real forms of death receptors and death ligands, respectively.

And while using pseudo proteins in a very clever mechanism, there are other ways to competitively inhibit the initiation of apoptosis. Mutations at the transcriptional, translational, and post-translational level can also result in the inhibition of apoptosis initiation. Small morphological or biochemical changes in proteins may prevent normal death ligands from binding to mutated death receptors. There are many ways in which tumor cells can evade detection and destruction by the immune system, and scientists will undoubtedly uncover more in the future.

While evading the immune system is an important step in tumorigenesis, there are times when evasion is simply not possible or sufficient to sustain tumorigenesis. Although there are fewer known mechanisms of immune destruction by tumor cells, we will explore two of them here.
 * B. Destruction**

__"//Counterattack"//__ Because T-cells are the primary immune cells involved in cancer surveillance, it makes sense that tumor cells have evolved a mechanism to induce T-cell apoptosis. According to Igney and colleagues, "repetetive stimulation of T-cells with [an] antigen induces apoptosis, a process referred to as activation-induced cell death (AICD)" (2). In other words, if a T-cell binds to the epitope of a tumor too many times, the T-cell will commit suicide. This process is seen most often with repeated binding of CD95/CD95L, which is also known as Fas/FasL and APO-1/APO-1L (8). For the purpose of continuity, we will refer to the death receptor and death ligand as CD95 and CD95L, respectively.

In order to better understand tumor counterattack, more information about the expression of CD95/CD95L and their role in the immune system is needed. The CD95 receptor is expressed in many cell types throughout the body. Any cell expressing CD95 is subject to programmed cell death. However, only a few cells are typically allowed to initiate apoptosis via the CD95/CD95L cell death pathway. These cells are characterized by the expression of CD95L and are predominantly activated T-cells (8). Therefore, the expression of CD95L is tightly regulated at the transcriptional level by various transcription factors. Igney and Krammer provide three possible ways in which CD95L can be transcribed: 1) An intracellular increase in calcium, which causes the de-phosphorylation of NFAT proteins. The de-phosphorylated NFAT proteins can then bind the CD95L promoter region. 2) Activation of mitogen-activated protein kinases (MAPK) can induce CD95L transcription by activating AP-1 transcription factors through the JNK pathway. AP-1 activity eventually leads to the upregulation of CD95L. 3) Lastly, early growth response (EGR) 2 and 3, NFkB, and the transcription factor SP-1 can contribute to the transcription of CD95L (8). The diagram at left illustrates the different pathways and transcription factors that contribute to CD95L expression. Needless to say, cancer cells have found a way to initiate one or more of the signal transduction cascades in order to express the apoptosis-inducing ligand.

There are also three forms of CD95L. As we learned in class, each form has a specific function. Membrane-bound CD95L is found on the surface of cells and "is the primary mediator of apoptosis" (8). Some of the membranous CD95 ligands are stored in intracellular microvesicles. These can be secreted into the intercellular space in response to physiological stimuli. Their exact function is not well understood. Lastly, a soluble form of CD95L can be generated when matrix metalloproteases cleave the membrane-bound form (8). The soluble CD95L has been shown to induce an immune response from cytotoxic T-cells. However, it should be noted that contradictory immune responses have been demonstrated for the soluble CD95L. Both pro- and anti-apoptotic mechanisms have been found for the human soluble CD95L, while the murine CD95L has been shown only to be anti-apoptotic by competitively inhibiting CD95/CD95L binding (8). Now that the structure and expression of CD95 and CD95L have been described, the regulation and purpose of the proteins can be explained.

Although it may seem counter-productive for over-stimulated T-cells to die, this process exists to protect self cells from a potential auto-immune attack. "Apoptosis, triggered via the CD95 system, plays a fundamental role in the regulation of T-cell homeostasis in the periphery" (8). During development in the thymus, T-cell binding affinities are constantly being monitored (1). Before leaving the thymus, T-cells go through processes called positive selection and negative selection (1). While there is simply no use for T-cells that cannot recognize and bind antigens, it is imperative that T-cells that bind too tightly to self cells are destroyed before they can become activated and proliferate. The T-cells that have a higher affinity for self cells than they should are deleted via CD95/CD95L AICD (8). The T-cells that have a high affinity for antigens and a low affinity for self cells are allowed to survive and are released into the lymphatic system, or periphery (1).

While deletirous T-cells are typically eliminated before they reach the periphery, T-cell regulation is not yet complete. As mentioned previously, if a T-cell binds an antigen, it undergoes clonal expansion. But once the antigens have been destroyed and removed, there is no longer a need for such a high concentration of that antigen-specific T-cell. This is where CD95/CD95L AICD comes back into play. According to Igney and Krammer, T-cells are resistant to CD95/CD95L AICD during the expansion phase (8). This is logical because activated T-cells cannot replicate if the number of their progeny are being carefully controlled through destruction. However, once the T-cells that have recently undergone clonal expansion have been activated, they become sensitive to CD95/CD95L AICD (8). The activated T-cells now express CD95L, likely through one of the transcriptional regulation mechanisms described above. This allows them to destroy unwanted cells in the body. It also allows them to be destroyed if they turn out to bind too tightly with self cells. Although the progenitor T-cell was carefully selected for in the thymus, its progeny did not undergo positive or negative selection in the periphery. Some of the activated progeny may be too self-reactive and are thus destroyed by T-cells or other immune cells via CD95/CD95L (8). "Humans or mice with deleterious mutations of CD95 (//lpr// mutation) or CD95L (//gld// mutation) display a phenotype of accumulation of abberant T-cells, leading to lymphadenopathy, splenomegaly, and an autoimmune disease that closely resembles system lupus erythematosus" (8). Without wildtype CD95 receptors and/or CD95 ligands, the lymph nodes and spleen become swollen from excessive numbers of T-cells. This includes the auto-reactive T-cells which were not destroyed, resulting in an autoimmune disease.

This disease model demonstrates the power and importance of CD95/CD95L expression and regulation, especially regarding tumor CD95L expression and regulation. Tumor cells not only passively resist the immune system through the use of CD95/CD95L interactions, they are also capable of the elimination of lymphocytes in a process known as "tumor counterattack". When CD95-sensitive lymphocytes come into contact with CD95L present on tumor cells, CD95/CD95L-mediated apoptosis begins and ultimately destroys the lymphocytes. In cases where tumor cells express CD95L, the newly activated T-cells are destroyed regardless of the T-cells' affinity for self cells. Tumor cells can therefore "over-use" CD95L to kill all activated, tumor-specific T-cells that bind to the tumor mass (8). However, "although many studies of tumor counterattack have been published, the results are contradictory and therefore do not clarify whether tumor counterattack is a relevant immune escape mechanism in vivo" (2).

For example, Igney and colleagues write, “Immunosuppression in vivo was directly demonstrated in allogeneic mice injected with CD95L-transfected colon carcinoma cells. Alloantibodies were virtually completely abolished and allospecific cytotoxic T lymphocytes and helper T-cells were reduced” ([|2]). This mechanism can be used to explain the immune-privileged sites in the human body such as the eyes and testis. The lack of the immune system in the eyes allows for the high success of corneal transplantations. The lack of functional CD95L-expression, according to Igney, “resulted in inflammation and invasion of ocular tissue by cells without signs of apoptosis. CD95-mediated apoptosis of lymphoid cells was necessary for tolerance induction following antigen injection into the anterior chamber of the eye” ([|2]). Igney and colleagues obviously observed the successful counterattack attempts of the tumor cells.

Hanhe and researchers saw demonstrated similar results. In their sensitized mouse model, Hanhe and colleagues compared tumor formation between wildtype mice, CD95 receptor knockout mice (//lpr)// and CD95 ligand knockout mice (//gld//) up to six days after daily carcinogen injections (9). Their initial results (Panel C of the diagram at right) indicate that tumor formation occurs more rapidly in wildtype mice and mice lacking with T-cells lacking CD95L than it does in mice with T-cells that lack the CD95 receptor. In other words, tumor cells expressing CD95L were not able to kill T-cells lacking the CD95 receptor. This presumably allowed T-cells to kill tumor cells, thus slowing tumor growth in //lpr// mice. In contrast, T-cells lacking the CD95 ligand were not able to destroy tumor cells as effectively. Tumors were seen earlier and in more //gld// mice than in //lpr// mice. This was demonstrated again in a second experiment (Panel D).

However, many studies have been published that contradict the tumor counterattack hypothesis. A variety of studies have found that tumor cells over-expressing CD95L result in neutrophil invasion and an inflammatory immune response (8). This was demonstrated when CD95L-expressing tumor cells were transplanted into the pancreas of allogenic mice resulted in graft rejection (8). However //in vitro// studies of the same type were found to result in T-cell apoptosis and tumor growth. There is clearly a disconnect between laboratory experiments and real life cases. Two other studies also suggest the soluble form of CD95L is chemotactic for neutrophils, which may help explain the results of the previous studies. It is thought that CD95L acts on surrounding macrophages, making them secrete IL-1. IL-1 is used by macrophages to call neutrophils to an infected site (1). The image at left shows the hypothesized mechanism of CD95L+ tumor rejection via macrophages. Once IL-1 is produced, neutrophils are called to the tumor site, recognize the tumor as foreign, and then destroy the tumor cells. However, others have found the exact opposite results. Instead of the rejection of tumors expressing soluble CD95L, soluble CD95L conferred a growth advantage over tumors that expressed the membrane-bound form of CD05L(8). In summary, there is an equal amount of support for the belief that tumor counterattack is real and the belief that it is not real.

Although "no study has demonstrated conclusively that a tumor (or graft), by CD95L expression, deleted anti-tumor-specific lymphocytes, escaped the immune response, and thus had a growth advantage in vivo", Igney and Krammer provide reasons as to why (2). According to them, most animal experiments use CD95L-transfected tumor cells that express unnaturally high levels of CD95L (8). It has therefore been suggested that the overexpression of CD95L on experimental tumors actually results in rejection of the tumor by neutrophils (8). Physiological CD95L levels may not induce the neutrophilic responses seen in experimental studies, although this is not yet known because CD95L expression levels in human tumors have not been quantified (8).

Another explanation for the contradictory results may rely on the __#|cytotoxic__ activity of the experimental CD95L-transfected tumor cells. In one experiment, researchers used assays to show that slowly growing CD95L tumor cells had actually lost the ability to induce apoptosis in infiltrating T-cells (8). As we discussed in class, cell lines used in laboratory experiments are often unnatural in that certain features have been selected for so the cells can be used over and over again. It is reasonable to assume that some of the properties of the tumor cells used in these experiments are different from the tumor cells found naturally in cancer patients. For example, if the number of variations in tumor cells increases with time, then the "age" of the experimental tumor cell lines may be an important, overlooked factor. If the experimental tumor cells have divided more times and acquired different characteristics from the original tumor cells, the experimental tumor cells may not respond like naturally occurring tumor cells.

Furthermore, Igney and Krammer explain the importance of timing when it comes to expressing CD95L. T-cell sensitivity depends on the activation status of T-cells. Tumors that constitutively express CD95L, which include many of the CD95L-transfected tumor cells in experiments, may be more likely to invoke a neutrophil response and be rejected than naturally occurring tumor cells (8). That being said, "induction of CD95L expression at later time-points after transplant in established tumors also led to tumor rejection" (8). Clearly, more experiments need to be done in order to fully elucidate the reason that some results favor tumor counterattack as an immune escape mechanism and others contest it.

Despite the results in Igney's test, the validity of this mechanism is still in question. Other tests indicate the expression of CD95L on tumors acts as a pro-inflammatory, attracting neutrophils to tumors. The exact reasoning for these extremely contradictory results has not been determined, but other studies show that the microenvironment, timing of interaction with t-cells and level of t-cell co-stimulation effect the inflammatory response and may hold the key to better understanding the process. Several other molecules such as the death ligand TRAIL, RCAS1 and Chemokine show potential for T cell deletion. But like CD95L, the experimental results are conflicting ([|3]).

__//Expression of Immunosuppresive Factors and Molecules//__ Another destruction strategy is the expression of immunosuppressive factors by either the tumor cells, epithelial cells, or stromal cells. One of the most distinct factors is the cytokine TGF-β, which effects the proliferation, activation, and differentiation of innate and adaptive immune cells. TGF-β ultimately inhibits the anti-tumor immune response. In addition, tumor cells produce vascular endothelial growth factor (VEGF) that both aids in angiogenesis and inhibits differentiation of progenitors into dendritic cells. This is beneficial to the tumor cells because, without APCs like dendritic cells, the adaptive immune response cannot be efficiently activated. “Further immunosuppressive factors expressed by malignant cells are prostaglandins, interleukin (IL)-10, macrophage-colony stimulating factor (MCSF), and soluble tumor gangliosides” ([|2]). While all of these cytokines can function as immunosuppressive factors, they can also aid immune cells. For example, IL-10 can suppress the antigen presentation capacity of APCs, thus suppressing the immune system. However, it also stimulates T-cells and mast cells, as well as B-cell proliferation (6). Again, it is important to remember the effects of cytokines are dependent upon type, concentration, and environment.

The purpose of an inflammatory immune response is to contain the antigenic cells in a small area to keep them from spreading to the rest of the body. This containment also aids in more efficient, effective destruction of the antigens because they are localized. As mentioned previously, both innate and adaptive effector immune cells use chemokines and cytotoxins to induce programmed cell death of any cell it deems "foreign". Some of these cytokines, such as TGF-β and IL-1 attribute to inflammation. And while the inflammation is meant to harm the antigenic cells, there is evidence that it can actually help tumor cells survive and proliferate.
 * C. Coexistance and Tumor Growth**

The current data suggest the tumor cells that are able to survive an immune attack (likely using one, if not multiple mechanisms described throughout this wiki) are able to use some of the cytokines secreted by the immune cells to their advantage. For example, Murdoch and researchers suggest that the tumor environment can cause some immune cells to secrete pro-angiogenic factors. The surviving tumor cells can then use thees pro-angiogenic factors to create the vasculature they need to grow and survive (4). Specifically, mast cells, eosinophils, and tumor-associated macrophages may be induced to secrete these angiogenic factors in response to hypoxia, acidosis, and high lactate (4). Murdoch and colleagues also found that neutrophils called to the site of a tumor can secrete matrix metalloproteinase 9 (MMP9). MMP9 degrades the extracellular matrix (ECM) and releases vascular endothelial growth factor (VEGF), which also promotes angiogenesis. Any tumor cells that are able to survive the inflammatory response would potentially be able to use the angiogenic factors secreted by immune cells during that response.

Furthermore, in the event that the foreign cells and/or the inflammatory response damages immune effector cells, secreted proliferation signals intended for immune cells may be used by tumor cells as well. If more effector cells are needed during an immune response, existing ones will secrete proliferative signals to induce the differentiation of effector "stem cells" (not true stem cells) into more effector cells (1). Because tumor cells are simply self cells that have grown out of control, they will also posess the receptors for proliferative growth factors. As we have learned in many biology courses, when antagonistic cytokines are present in the same area, the one present in the higher concentration is likely to "win". In this case, if tumor cells are receiving more proliferative signals than they are death signals, they are more likely to continue growing than to be killed (5). This may be another mechanism by which tumor cells use the immune system to their advantage.

The role of cytokines secreted by immune cells have been shown to be multifacted. In some instances, an inflammatory immune response leads to the destruction of foreign cells, while in other cases tumor cells use the same cytokines for tumorigenesis (most specifically, angiogenesis). For more information about the inhancement of tumor cell growth via the immune system, please visit Cancer and the Immune System.

4. Implications and Summary
In summary, the CD95/CD95L pathway regulates apoptosis through activation-induced cell death (AICD). The mechanism is initiated through cells presenting CD95L. This pathway allows for T-cells to eliminate unwanted cells, while at the same time providing a fail-safe to prevent T-cells from attacking self cells. The expression of CD95L is usually limited to a few cells such as T-cells, but cancerous tumors have evolved the ability to initiate one of the three major transcription factors controlling CD95L expression. This allows tumor cells to present CD95L to the attacking T-cells, causing the T-cells to apoptose (A simple figure showing both typical T-cell elimination and then tumor counterattack is to the left.) While this phenomenon has been witnessed, there have not been enough conclusive studies to confirm whether tumor cells are capable of deleting anti-tumor-specific lymphocytes through the CD95/CD95L pathway. However, it is likely that a large number of other factors influence the ability of tumor cells to use "counterattack". Because it is impossible to mimic the activities occurring in a live cell in a lab, the //in vitro// results will likely always differ slightly from the //in vivo// results. Additional studies are required for the full understanding of this process, the importance of additional research is described below.

Due to the the lack of understanding of tumors expressing CD95L, it has come to light that perhaps improper therapies have been used on tumors. Certain chemotherapies act to increase the expression of CD95L by activating the immune system and, subsequently mitogenic cytokines. Not only would this be ineffective against tumors expressing CD95L, but the treatment would also inhibit T-cells (through the process described above) allowing for tumor counterattack. A proper treatment could be a pharmaceutical that renders T-cells immune to CD95L binding, allowing the immune system to handle the tumor on its own without additional drug therapy. Of course, this treatment has the potential to cause autoimmune-like side effects in that it will also prevent the body from killing self-recognizing T-cells. This will need to be addressed for the safe treatment of cancer via blocking apoptosis of tumor-specific T-cells. All in all, it is our opinion that tumor counterattack is a valid mechanism of T-cell destruction.

Not only is it prudent to understand this mechanism better to better treat tumors, but there are possible medical applications for the CD95/CD95L system. Research is currently being undertaken to apply this process towards increasing the success of organ transplants and skin grafts. The phenomenon is observed in the CD95/CD95L-mediated immune privileged areas allows for a high success rate of cornea transplants. This same idea can be applied to other organs in the body. In the graph to the right we see the results of a study done to try and develop a 'buffer' to implement between host and donor cells to allow for successful transplantation. The study developed the buffer by using the CD95/L mechanism to induce apoptosis of T-cells and prevent graft-versus-host disease (GVHD). We see here that with the buffer, the subjects survived an additional 12 days. With further development this may allow for much higher success rates of organ transplants and skin grafts, eliminating much of the stress and worry of waiting on the organ donor list. Further research might yield further applications in the field of medicine, including useful tumor chemotherapies. In addition to boosting the successfulness of tissue grafts, there is a possible application of knowledge to autoimmune diseases. Some autoimmune diseases, the most notable being autoimmune lymphoproliferative syndrome (ALPS), work through the CD95/CD95L pathway to cause harm to the human body. These types of diseases are hard to treat because they deal with the complexities of the human immune system, but hopefully with further understanding of CD95, advancements can be made to treat diseases like ALPS.

>> Nov. 2007. Web. []. >>
 * 5. Bibliography**
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 * 6) Hanhe, M., Rimoldi, D., & Schroter, M. (1996). Melanoma cell expression of fas(apo-1/CD95) ligand: implications for tumor immune escape. //Science, 274,// 1363-1366. Retrieved from https://dl-web.dropbox.com/get/Bio%20179%202012/Student%20Papers/For%20Kelly%20Hall/4.pdf?w=1f1ffd4d