第15讲 Cancer and the Immune System

HEADS UP!

The immune system has limited ability to protect us against cells when they first become cancerous. First, there is a built-in conflict between providing surveillance against cancer cells, and guarding against autoimmunity. Second, cancer cells mutate rapidly, making them a "moving target." And third, tumors can create a "self-protective" environment in which immune surveillance is compromised. 

INTRODUCTION

In this lecture, we are going to discuss how the immune system deals with cancer. Because you may not have had a cancer course, I will begin by discussing some general properties of cancer cells. After all, it's important to know the enemy.

CANCER IS A CONTROL SYSTEM PROBLEM

Cancer arises when multiple control systems within a single cell are corrupted. These are of two basic types: systems that promote cell growth (proliferation), and safeguard systems that protect against "irresponsible" cell growth. Controlled properly, cell proliferation is a good

thing. After all, an adult human is made up of trillions of cells, so a lot of proliferation must take place between the time we are a single fertilized egg and the time we are full-grown. However, once a human reaches adulthood, most cell proliferation ceases. For example, when the cells in your kidney have proliferated to make that organ exactly the right size, kidney cells stop proliferating. On the other hand, skin cells and cells that line our body cavities (e.g., our intestines) must proliferate almost continuously to replenish cells that are lost as these surfaces are eroded by normal wear and tear. All this cell proliferation, from cradle to grave, must be carefully controlled to insure that the right amount of proliferation occurs at the right places in the body – and at the right time.

Usually, the growth-promoting systems within our cells work just fi ne. However, occasionally one of these systems may malfunction, and a cell may begin to proliferate inappropriately. When this happens, that cell has taken the first step toward becoming a cancer cell. Because these growth-promoting systems are made up of proteins, malfunctions occur when gene expression is altered, usually as a result of a mutation. A gene which, when mutated, can cause a cell to proliferate inappropriately is called a proto-oncogene. And the mutated version of such a gene is called an oncogene. The important point here is that uncontrolled cell growth can result when a normal cellular gene is mutated.

To protect against malfunctions in the control systems that promote cell proliferation, our cells are equipped with internal safeguard systems. These safeguards are of two general types: systems that help prevent mutations and systems that deal with mutations once they occur. Cells have a number of different repair systems that can fi x damaged DNA, helping safeguard against mutations. These DNA repair systems are especially important, because mutations occur continuously in the DNA of all our cells. In fact, it is estimated that, on average, each of our cells suffers about 25,000 mutational events every day. Fortunately, repair systems work nonstop, and if the DNA damage is relatively small, it can be repaired immediately as part of the "maintenance" repair program. 

Sometimes, however, the maintenance repair systems may miss a mutation, especially when there are many mutations and the repair systems are overwhelmed.

When this happens, a second safeguard system comes into play – one that monitors unrepaired mutations. If the mutations are not extensive, this safeguard system stops the cell from proliferating to give the repair systems more time to do their thing. However, if the genetic damage is severe, the safeguard system will trigger the cell to commit suicide, eliminating the possibility that it will become a cancer cell. One of the important components of such a safeguard system is a protein called p53. Proteins like p53, which help safeguard against uncontrolled cell growth, are called tumor suppressors, and the genes that encode them are called anti-oncogenes or tumor suppressor genes. Mutations in the gene for p53 have been detected in the majority of human tumors, and scientists have created mice with mutant p53 genes. In contrast to normal mice, which rarely get cancer, mice that lack functional p53 proteins usually die of cancer before they are seven months old. So, if you are ever asked to give up one gene, don't pick p53!

The take-home lesson is that every normal cell has both proto-oncogenes and tumor suppressor genes.

Where things get dangerous is when proto-oncogenes are mutated, so that the cell proliferates inappropriately, and tumor suppressor genes are mutated, so that the cell can't defend itself against proto-oncogenes "gone wrong." Indeed, cancer results when multiple control systems, both growth-promoting and safeguard, are corrupted within a single cell. It is estimated that between four and seven such mutations are required to produce most common cancers. This is the reason why cancer is a disease which generally strikes later in life: It usually takes a long time to accumulate the multiple mutations required to inappropriately activate growth-promoting systems and to disable safeguard systems.

Mutations that affect growth-promoting systems and safeguard systems can occur in any order. However, one type of mutation that is especially insidious is a genetic alteration which disrupts a safeguard system involved in repairing mutated DNA. When this happens, the mutation rate in a cell can soar, making it much more likely that the cell will accrue the multiple mutations required to turn it into a cancer cell. This type of "mutation-accelerating" defect is found in most (perhaps all) cancer cells. Indeed, one of the hallmarks of a cancer cell is a genetically unstable condition in which cellular genes are constantly mutating.

CLASSIFICATION OF CANCER CELLS

Cancer cells can be grouped into two general categories: non-blood-cell cancers (usually referred to as solid tumors) and blood cell cancers. Solid tumors are further classified according to the cell type from which they arise. Carcinomas, the most common tumors in humans, are cancers of epithelial cells, and include lung, breast, colon, and cervical cancer. These cancers generally kill by metastasizing to a vital organ, where they grow and crowd the organ until it can no longer function properly.

Humans also get cancers of the connective and structural tissues, although these sarcomas are relatively rare compared to carcinomas. Perhaps the best-known example of a sarcoma is bone cancer (osteosarcoma).

Blood cell cancers make up the other class of human cancers, and the most frequent of these are leukemias and lymphomas. Blood cell cancers arise when descendants of blood stem cells, which normally should mature into lymphocytes or myeloid cells (e.g., neutrophils), stop maturing and just continue proliferating. In a real sense, these blood cells refuse to "grow up" – and that's the problem. In leukemia, the immature cells fill up the bone marrow and prevent other blood cells from maturing. As a result, the patient usually dies from anemia (due to a scarcity of red blood cells) or from infections (due to a deficit of immune system cells). In lymphoma, large "clusters" of immature cells form in lymph nodes and other secondary lymphoid organs – clusters that in some ways resemble solid tumors. Lymphoma patients usually succumb to infections or organ malfunction.

There is another way to classify human cancers: spontaneous and virus-associated. Most human tumors are "spontaneous." They arise when a single cell happens to accumulate a collection of mutations that causes it to acquire the properties of a cancer cell. These mutations can result from errors made when cellular DNA is copied to be passed down to daughter cells, or from the effects of mutagenic compounds (carcinogens). Such mutagens can be byproducts of normal cellular metabolism, or can be present in the air we breathe and the food we eat. Mutations can also be caused by radiation (including UV light) or by errors made in assembling the segments of DNA that make up the B and T cell receptors. As we go through life, these mutations occur "spontaneously." However, there are certain factors that can accelerate the rate of mutation and increase the chances that a cell will become cancerous: cigarette smoking, a fatty diet, an increased radiation exposure from living at high altitude, working in a plutonium processing plant, and so on.

Some viruses produce proteins that can interfere with the proper functioning of growth-promoting and safeguard systems. Infection with these special tumor viruses decreases the total number of cellular genes which must be mutated to turn a normal cell into a cancer cell. Consequently, a tumor virus infection can be an accelerating factor for cancer. For example, essentially all human cervical cancers involve an infection by the human papillomavirus. This sexually transmitted virus infects cells that line the uterine cervix and expresses viral proteins in these cells that can disable two safeguard systems, including the safeguards provided by p53. Like-wise, hepatitis B virus can establish a chronic infection of liver cells, can inactivate p53, and can act as an accelerating factor for liver cancer.

The hallmark of virus-associated cancer is that only a small fraction of infected individuals actually get cancer, yet for those who do, virus or viral genes usually can be recovered from their tumors. For example, less than 1% of women infected with genital human papillomavirus will ever get cancer of the cervix, yet human papillomavirus genes have been found in over 90% of all cervical carcinomas examined. The reason for this, of course, is that the virus can't cause cancer by itself – it can only accelerate the process that involves the accumulation of cancer-causing mutations. About one fifth of all human cancers have a viral infection as an accelerating factor.

IMMUNE SURVEILLANCE AGAINST CANCER

From this introduction, it should be clear that powerful defenses exist within the cell (e.g., tumor suppressor proteins) to deal harshly with most wannabe cancer cells. But does the immune system of a healthy human provide significant protection against cancerous cells which might go on to form a tumor? To try to answer this question, let's examine the roles which various immune system cells might play in cancer surveillance – keeping in mind that their ability to provide meaningful surveillance may depend critically on the type of cancer.

CTLs and spontaneous tumors

The majority of human cancers are spontaneous tumors that are not of blood cell origin. It has been proposed that killer T cells might provide surveillance against these solid tumors, preventing their formation. Let's try to evaluate this possibility.

The activation problem

Imagine that a heavy smoker finally accumulates enough mutations in the cells of his lungs to turn one of them into a cancer cell. Remember, it only takes one bad cell to make a cancer. And let's suppose that because of these mutations, this cell expresses antigens that could be recognized as foreign by CTLs. Now let me ask you a question: Where are this man's naive T cells while the tumor is starting to grow in his lung? That's right. They are circulating through the blood, lymph, and secondary lymphoid organs. Do they leave this circulation pattern to enter the tissues of the lung? No, not until after they have been activated.

So right away, in terms of immune surveillance, we have a "traffic problem." To make self tolerance work, naive T cells are not allowed out into the tissues where they might encounter self antigens that were not present in the thymus during tolerance induction. As a result, it's unlikely that virgin T cells ever would "see" tumor antigens expressed in the lung – because they just don't go there. What we have here is a serious conflict between the need to preserve tolerance of self (and avoid autoimmune disease) and the need to provide surveillance against tumors that arise, as most tumors do, out in the tissues. And tolerance usually wins.

Now, sometimes virgin T cells do disobey the traffic laws and wander out into the tissues. So you might imagine that this kind of adventure could give some T cells a chance to look at the tumor that's growing in this guy's lung, and be activated. But wait! What is required for T cell activation? First of all, killer T cells must recognize antigens which are produced within a cell and presented by class I MHC molecules on the surface of that cell.

This means that the cancer cell itself must do the antigen presentation. So far, so good. However, CTLs also require co-stimulation from the cell that presents the antigen. Is this lung tumor cell going to provide that co- stimulation?

I don't think so! This isn't an antigen presenting cell, after all. It's a plain old lung cell, and lung cells usually don't express co-stimulatory molecules like B7. Consequently, if a virgin CTL goes rogue and breaks the traffic laws, enters the lung, and recognizes a tumor antigen displayed by class I MHC molecules on a cancer cell, that CTL most likely will be anergized or killed – because the cancer cell will not provide the co-stimulation the CTL needs for survival.

Again we see a conflict between tolerance of self and tumor surveillance. The two-key system of specific recognition plus co-stimulation was set up so that T cells which recognize self antigens out in the tissues, but which do not receive proper co-stimulation, will be anergized or killed to prevent autoimmunity. Unfortunately, this same two-key system makes it very difficult for CTLs to be activated by tumor cells that arise in the tissues. So the bottom line is that a CTL would have to perform "unnatural acts" to be activated by a tumor that is beginning to grow out in the tissues: It would have to break the traffic laws, and somehow avoid being anergized or killed. This could happen, of course, but it would be very inefficient compared to the activation of CTLs in response to, for example, a viral infection.

You might ask, "Why, during evolution, was such a premium placed on avoiding autoimmune disease that the immune system's ability to defend against cancer was compromised?" What we need to remember is that our immune system evolved to protect humans until we are past our "breeding age." Autoimmune disease can be devastating to a young person, but cancer usually is a disease that affects people later in life. Consequently, evolutionary pressure to protect humans of child-bearing age resulted in an immune system that sacrifices a robust defense against cancer in favor of protection against autoimmune disease.

The mutation problem

A possible solution to the activation problem is that cancer cells from the primary tumor might metastasize to a lymph node, where T cells could be activated. However, by the time this happens, the original tumor probably will have become quite large. Even a tumor that weighs only about half an ounce will contain more than 10 billion cancer cells – more cells than there are people on our planet! This poses a major problem for immune surveillance, because cancer cells usually mutate like crazy, and with so many cells mutating, it is likely that some of these mutations will prevent recognition or presentation of tumor antigens. For example, the gene encoding the tumor antigen itself might mutate so that the tumor antigen no longer can be recognized by activated CTLs, or no longer will fit properly into the groove of an MHC molecule for presentation. Also, genes that encode the TAP transporters can mutate in a tumor cell, with the result that tumor antigens will not be efficiently transported for loading onto class I MHC molecules. Tumor cells also can mutate so that they stop producing the particular MHC molecules that CTLs are restricted to recognize. This happens quite frequently: About 15% of the tumors that have been examined have lost expression of at least one of their MHC molecules. Indeed, a tumor cell's high mutation rate is its greatest advantage over the immune system, and usually keeps these cells one step ahead of surveillance by CTLs.

Cancer cells fight back

There is another difficulty which tumor-specific CTLs must face in providing suveillance against solid tumors: Cancer cells fight back. Once a solid tumor has been established, the cancer cells can modify the environment in the neighborhood of the tumor to make it more difficult for tumor-specific CTLs to operate. In Lecture 8, I mentioned an inhibitory receptor, PD-1, which is found on the surface of activated T cells. The natural function of this checkpoint protein is to restrain CTLs so that the immune response does not become over-exuberant.

However, many types of cancer cells express ligands for these immunosuppressive proteins, and ligation of PD-1 on T cells can impair their function. The result is that a growing tumor is able to "shield" itself from killing by tumor-specific CTLs.

Many tumors also express high levels of indoleamine 2,3-dioxygenase. This enzyme catalyzes the metabolism of the essential amino acid tryptophan, resulting in the rapid consumption of tryptophan from the tumor environment.

And when killer T cells are starved for tryptophan, they stop proliferating and become anergic. In addition, tumor cells can influence helper T cells in their neighborhood to become regulatory T cells. Exactly how this is accomplished is not well understood, but the resulting iTregs secrete TGFβ and IL-10, creating an immunosuppressive environment in which CTLs function poorly.

My conclusion is that killer T cells provide limited protection against solid tumors when these cells first become cancerous because it is very difficult to activate CTLs early in the course of the disease. Later, when the tumor becomes larger, killer T cells may be activated.

However, at this late stage, CTLs are relatively ineffective at eradicating the tumor. The high mutation rate of cancer cells helps them escape immunosurveillance, and the tumor can create an immunosuppressive environment that reduces the effectiveness of tumor-specific killer T cells. Consequently, even when it occurs, CTL surveillance against solid tumors usually is a case of "too little, too late."

CTLs and cancerous blood cells

Okay, so CTLs probably don't provide serious surveillance against non-blood-cell, spontaneous tumors, especially when they first arise. That's a real bummer, because these make up the majority of human tumors. But what about blood cell cancers such as leukemia and lymphoma? Maybe CTLs are useful against them. After all, immunosuppressed humans do have higher frequencies of leukemia and lymphoma than do humans with healthy immune systems. This suggests that there might be something fundamentally different about the way the immune system views tumors in tissues and organs versus the way it sees blood cells that have become cancerous. Let's take a look at what these differences might be.

One of the problems that CTLs have in providing surveillance against tumors that arise in tissues is that these tumors simply are not on the normal traffic pattern of virgin T cells – and it's hard to imagine how a CTL could be activated by a cancer it doesn't see. In contrast, most blood cell cancers are found in the blood, lymph, and secondary lymphoid organs, and this is ideal for viewing by CTLs, which pass through these areas all the time. Thus, in the case of blood cell cancers, the traffic patterns of cancer cells and virgin T cells actually intersect. Moreover, in contrast to tumors in tissues, which are usually unable to supply the co-stimulation required for activation of virgin T cells, some cancerous blood cells actually express high levels of B7, and therefore can provide the necessary co-stimulation.

Also, on average, cancerous blood cells have fewer mutations than do most solid tumors. For this reason, the immune system might have a relatively easy time dealing with blood cell cancers because the likelihood of "escape mutations" might be less than with highly mutated solid tumors.

These properties of blood cell cancers suggest that CTLs may provide surveillance against some of them.

Unfortunately, this surveillance must be incomplete, because people with otherwise healthy immune systems still get leukemias and lymphomas.

CTLs and virus-associated cancers

Certain viral infections can predispose a person to particular types of cancer. Because killer T cells are good at defending against viral infections, it is easy to imagine that CTLs might provide surveillance against virus-associated tumors. Unfortunately, this surveillance is probably quite limited. Here's why.

Most viruses cause "acute" infections in which all the virus-infected cells are rather quickly destroyed by the immune system. Consequently, viruses which only cause acute infections do not play a role in cancer – because a dead cell isn't going to make a tumor.

This explains why most viral infections are not associated with human cancer.

There are viruses, however, which can evade the immune system and cause long-term (sometimes lifelong) infections (e.g., hepatitis B virus and the human papillomavirus). Indeed, all viruses which have been shown to play a role in causing cancer are able to establish chronic infections during which they "hide" from the immune system. CTLs cannot destroy virus-infected cells while they are hiding, and because these hidden cells are the very ones which eventually become cancerous, it can be argued that CTLs do not provide effective surveillance against virus-associated cancer.

Of course, you might propose that without killer T cells, more cells would be infected during a virus attack, thereby increasing the number of cells in which the virus might be able to establish a long-term, hidden infection.

And this probably is true. In fact, this may help explain why humans with deficient immune systems have higher than normal rates of virus-associated tumors. However, the bottom line is that CTLs cannot provide significant surveillance against virus-infected cells that have become cancerous, because these cancers only result from long-term viral infections – infections which CTLs cannot detect or cannot deal with effectively.

IMMUNE SURVEILLANCE BY MACROPHAGES AND NK CELLS

Macrophages and natural killer cells may provide surveillance against some cancers. Hyperactivated macrophages secrete TNF and express it on their surface.

Either form of TNF can kill certain types of tumor cells in the test tube. This brings up an important point: What happens in the test tube is not always the same as what happens in an animal. For example, there are mouse sarcoma cells that are very resistant to killing by TNF in the test tube. In contrast, when live mice that have these same sarcomas are treated with TNF, their tumors are rapidly destroyed. Studies of this phenomenon showed that the reason TNF is able to kill the tumor when it is in the animal is that this cytokine actually attacks the blood vessels that feed the tumor, cutting off the blood supply, and causing the tumor cells to starve to death. This type of death is called necrosis, and it was this observation that led scientists to name this cytokine "tumor necrosis factor."

In humans, there are examples of cancer therapies in which activated macrophages are likely to play a major role in tumor rejection. One such therapy involves injecting the tumor with bacille Calmette–Guérin (BCG), a cousin of the bacterium that causes tuberculosis. BCG

hyperactivates macrophages, and when it is injected directly into a tumor (e.g., a melanoma), the tumor fills up with highly activated macrophages that can destroy the cancer. In fact, one way of treating bladder cancer is to inject it with BCG – a treatment which is quite effective in eliminating superficial tumors, probably through the action of hyperactivated macrophages.

But how do macrophages tell the difference between normal cells and cancer cells? The answer to this question is not known for certain, but evidence suggests that macrophages recognize tumor cells that have unusual cell surface molecules. One of the duties of macrophages in the spleen is to test red blood cells to see if they have been damaged or are old. Macrophages use their sense of "feel" to determine which red cells are past their prime.

And when they find an old one, they eat it. What macrophages feel for is a fat molecule called phosphatidylserine. This particular fat is usually found on the inside of young red blood cells, but flips to the outside when the cells get old. Like old red blood cells, tumor cells also tend to have unusual surface molecules, and in fact, some express phosphatidylserine on their surface. It is believed that the abnormal expression of surface molecules on tumor cells may allow activated macrophages to differentiate between cancer cells and normal cells.

Natural killer cells target cells that express low levels of class I MHC molecules and that display unusual surface molecules (e.g., proteins which indicate that the target cells are "stressed"). In the test tube, natural killer cells can destroy some tumor cells, and there is also evidence that NK cells can kill cancer cells in the body. Certainly, there would be a number of advantages to having macrophages and NK cells provide surveillance against wannabe cancer cells. First, unlike CTLs, which take a week or more to get cranked up, macrophages and NK cells are quick-acting. This is an important consideration, because the longer abnormal cells have to proliferate, the greater is the likelihood they will mutate to take on the characteristics of metastatic cancer cells. In addition, once a tumor becomes large, it is much more difficult for the immune system to deal with. So you would like the weapons that protect against cancer cells to be ready to go just as soon as the cells start to get a little weird.

You would also want anti-tumor weapons to be focused on diverse targets, because a single target (e.g., the MHC–peptide combination seen by a killer T cell) can be mutated, rendering the target unrecognizable.

Both NK cells and macrophages recognize diverse target structures, so the chance of them being fooled by a single mutation is small. In addition, macrophages are located out in the tissues where most tumors arise, so they could intercept cancer cells at an early stage. And with immune surveillance, as with real estate, location is everything.

There are problems, however, with macrophages and NK cells providing surveillance against cancer. Macrophages need to be hyperactivated before they can kill cancer cells. That's what the BCG treatments do: They hyperactivate macrophages by causing inflammation. So if a wannabe cancer cell arises at a site of inflammation where macrophages are already hyperactivated, that's great. But if there's no inflammatory reaction going on, macrophages will probably remain in a resting state and simply ignore the cancer cells. Unlike macrophages, which are found in large numbers in our tissues, most NK cells are found in the blood. Like neutrophils, NK cells are "on call." And the cells which do the calling are activated macrophages and dendritic cells that are responding to an invasion. So unless there is an inflammatory reaction going on in the tissues, most NK cells will just continue to circulate in the blood.

As a tumor grows, it eventually becomes so large that the neighboring blood vessels cannot provide the nutrients and oxygen required for continued growth, and some of the cancer cells begin to die. Cancer cells also die when they accumulate mutations that are lethal. Consequently, at a later stage in the growth of a tumor, dying cancer cells may provide the signals required to activate macrophages – which can then recruit natural killer cells from the blood. So at this point, macrophages and NK cells may play a role in destroying at least some of the tumor cells. In addition, because NK cells do not need to be activated to kill, natural killer cells that are circulating in the blood may be able to destroy either blood cell cancers or cancer cells that are metastasizing through the blood from a primary tumor. 

REVIEW

Although it is certain that human cells have built-in safeguards to help protect them from becoming cancerous, it is not nearly so clear what role the immune system plays in protecting us against this terrible disease. The immune system probably is able to defend against some virus-associated and blood cell cancers. Also, natural killer cells and macrophages can recognize and kill some tumor cells – those which have unusual molecules on their surface. And NK cells may reduce the frequency of metastases or help slow the metastatic process once a primary tumor has formed. Consequently, macrophages and NK cells may be useful against certain types of cancer.

Unfortunately, it is unlikely that killer T cells provide significant surveillance against most solid tumors in humans. There are several reasons for this. First there is the activation problem. Many safeguards are in place to protect humans against autoimmunity, and these safeguards make it very difficult for cancer-specific CTLs to be activated – especially during the early stages of tumor development. Virgin T cells are activated in the secondary lymphoid organs. Consequently, the normal traffic pattern of naive T cells keeps them from coming in contact with cancer cells in the tissues. In addition, most cancer cells cannot supply the co-stimulation required to activate killer T cells, so even a "chance encounter" between a naive T cell and a tumor cell out in the tissues isn't likely to result in activation.

Another obstacle to cancer surveillance by killer T cells is that, because of their high mutation rate, cancer cells represent a "moving target." Even if a CTL can be activated so that it can attack some cells in a tumor, it is very likely that there will be other cancer cells within that tumor which have mutated so that they are invisible to that killer T cell. In addition, rapidly mutating tumor cells can create an immunosuppressive environment which can interfere with the immune response, and make CTLs ineffective against solid tumors.

It is also unlikely that the immune system provides significant surveillance against virus-associated cancers. These cancers arise in cells in which the virus has established a "stealth" infection – making the infected cells invisible to the immune system.