Chapter 15 Crowd Control: Tumor Immunology and Immunotherapy It is by no means inconceivable that small accumulations oftumour cells may develop and, because of their possession of new antigenic potentialities, provoke an effective immunological reaction with regression of the tumour and no clinical hint of its existence. Macfarlane Burnet, immunologist, 1957 Throughout this text, we have studied various defenses that the body erects against the appearance of cancerous growths. Many of these defenses are inherent in cells, more specifically in their hard-wired regulatory circuitry. The most obvious of these are the controls imposed on cells by the apoptotic machinery, which is poised to trigger the death of cells that are misbehaving or suffering certain types of damage or physiologic stress. The pRb circuit and the DNA repair apparatus are similarly configured to frustrate the designs of incipient cancer cells. The organization of tissues also places constraints on how incipient cancer cells can proliferate. For example, normal epithelial cells that lose their tethering to the basement membrane activate the form of apoptosis that is called anoikis. This mechanism limits the ability of epithelial cells to stray from their normal locations within tissues and grow in ectopic (i.e., abnormal) sites. At the same time, the special status afforded to stem cells and their genomes (Section 12.3) also reduces the probability of mutant cancer cells' gaining a foothold within a tissue. 655 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy Beyond these cell- and tissue-specific mechanisms, mammals may have another line of defense-the immune system. The immune system is highly effective in detecting and eliminating foreign infectious agents, including viruses, bacteria, and fungi, from our tissues. One of the major questions in cancer research over the last half century has been whether the immune system can also recognize cancer cells as foreigners and proceed to kill them. Actually, evidence is rapidly accumulating that the immune system does indeed contribute to the body's multilayered defenses against tumors. The difficulties associated with establishing this type of anti-cancer defense are apparent from the outset: the immune system is organized to recognize and eliminate foreign agents from the body while leaving the body's own tissues unmolested. Cancer cells, however, are native to the body and are, in many respects, indistinguishable from the body's normal cells. How can cancer cells be recognized by the immune system as being different and, therefore, appropriate targets of immune-mediated killing? We will wrestle with this problem and its ramifications repeatedly throughout this chapter. The field of tumor immunology, more than any other area of cancer research, remains in great flux, with basic concepts still a matter of great debate. Consequently, in this chapter, you will find many observations and conclusions to be much more tentatively stated than elsewhere in this book and subject, no doubt, to future revision. Still, this is an area of cancer biology that is well worth our time and study, since it holds great promise for new insights into cancer pathogenesis and new ways of treating hwnan tumors. Research conducted on mammals over the past three decades has.revealed an immune system of great complexity and subtlety. Before we enter into discussions of its anti-tumor functions, we need to take an excursion into the workings of the general immune system. An understanding of its mechanisms of action, at least in outline, is a prerequisite for engaging the three major questions that will occupy us in this chapter. First, what specific molecular and cellular mechanisms enable the immune system to recognize and attack incipient cancer cells? Second, do these immune mechanisms represent effective defenses that prevent the appearance oftwnors? Third, how can the immune system be mobilized by oncologists to attack tumors once they have formed? (An introduction to immunology will occupy our attention in Sections 15.1 through 15.6; an overview will be provided in Figure 15.14.) 15.1 The immune system functions in complex ways to destroy foreign invaders and abnormal cells in the body's tissues The mammalian immune system launches several types of attack against foreign infectious agents and the body's own cells that happen to be infected ""ith such agents. It identifies its targets by recognizing specific molecular entitiesantigens-that are made by these agents. Having done so, the immune system undertakes to neutralize or destroy the infectious particles (bacterial and fungal cells, virus particles) , as well as infected cells displaying these antigens. To the extent that the immune system also functions to ward off cancer, one assumes that it exploits many of the same mechanisms that it uses to eliminate foreign infectious agents. The most familiar of the immunological defense strategies involves the humoral immune response-the arm of the immune system that generates soluble antibody molecules capable of specifically recognizing and binding antigens (Figure 15.1). Thus, a virus particle or bacterium displaying antigens on its surface may rapidly become coated with antibody molecules, which may result in the neutralization of these pathogens (Figure 15.2). Similarly, an infected cell 656 (C) '.1-- light Function of the humoral immune response lig ht (L) chain (B) constant region di su lfide bonds (A) Figure 15.1 Structure of antibody molecules and their binding to antigens The most abundant antibody molecule in the plasma is the immunoglobulin y (lgG) molecule. (A) X-ray crystallography of an IgG molecule reveals the symmetry that allows the two antigen-binding domains (top left, top right) to bind two antigen molecules Simultaneously. (B) IgG molecules are divided into two functional regi ons. One region is designed to recognize and bind antigen molecules. Because the IgG molecules in plasma can recognize an essentially unlimited number of antigens, IgG molecules have a comparable diversity of structures in their antigen-binding portions, which are call ed their variable domains (red), to recognize this diversity The remainder of the IgG molecule is termed its constant region (blue) and is invariant am ong all IgG molecules of a given subclass, e.g, all IgG 1 molecules. An IgG molecule as a whole is a heterotetramer composed of two light (L) chains and two heavy (H) chains. Two separate antigen-recognizing and binding pockets are displayed (top left, top right), each composed of an H- and an L-chain N-terminal domain (concave shapes). (C) The detailed struct ure of an antigen-antibody complex is seen here in this space-filling molecular model in which the antigen-binding domains of the heavy chain (purple) and light chain (yel/ow) are seen to contact the antigenic molecule, in this case the chicken egg-white lysozyme molecule (light blue). Only parts of the variable regions of the heavy and light chains are shown here. (A glutamine residue, red, is indicated that is important for the hydrogen bonding of the antigen to the antibody molecule.) (From CA Janeway Jr. et ai, Immunobiology, 6th ed . New York Garland Science, 2005 ) may display on its surface the antigens made by the agents that have infected it and its surface may become coated with antibodies that recognize and bind these antigens. Once a mammalian cell or an infectious agent is coated by antibody molecules, it may be recognized, engulfed, and destroyed by phagocytic t- A ~ l'--J;). ~ ; . . - < ~ lJ).. ant igen (chicken egg-White lysozyme) heavy (H) chain (L) chain Figure 15.2 Neutralization by antibody molecules (A) Virus particles (red) can become coated by antibody molecules (blue) developed by the immune system of an infected host. This coating neutralizes (inactivates) the infecti vity of the particles by blocking their adsorption to host cells . (B) Similarly, a bacterium displaying certain surface antigens (red) can also be prevented from adhering to host cells (A) antibody prevents viral adsorption (B) antibody prevents bacterial adherence by bound antibody molecules. 657 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) bacterium Fc receptors ~ -o macrophage - -{ ~ lysosome (B) Fc receptors I(\ I / NK cell activated~ ~ ~ ~ ~ ~ - -NK cell o 0 0 targeted mammalian cell (e) Figure 15.3 Coating of pathogens by antibody molecules and their elimination by effector cells of the immune system The coating of viruses, bacteria, and mammalian cells by antibody molecules is often the prelude to their being phagocytosed (engulfed) or destroyed by cytotoxic cell s of the immune system. (A) The coating of a bacterium (red) by antibody molecules (yellow) may provoke a macrophage to use specialized receptors on its surface, termed Fc receptors (green), to recognize and bind the constant regions of the antibody molecules (which are not involved in antigen recognition; see Figure 15.1). This often result s in the phagocytosis of the antibodycoated bacterium and its eventual destruction in Iysoso mes within the cytoplasm of the macrophage. (B) A mammalian cell (gray) becomes coated by antibody molecules (blue) that recognize and bind antigens (red) on its surface. A type of lymphocyte termed a natural killer (NK) cell then uses its Fc cell surface receptors (green) to bind the constant regions of the coating antibody molecules. This binding results in acti vation of the N K cell , which proceeds to destroy the targeted cell, using cytotoxic granules (purple dots), whose contents it introduces into the targeted cell, to do so. (C) Sheep red blood cells were treated with an antibody that recognizes an antigen displayed on their surface. As seen in this scanning electron micrograph, a large number of them have become adsorbed to a macrophage via the Fc receptors on the surface of the latter. (A and B, from CA Janeway Jr. et ai, Immunobiology, 6th ed. New York Garland Science, 2005; C, from J. Sw anson, University of Michigan.) cells, such as macrophages, or killed by cytotoxic cells, such as natural killer (NK) cells (Figure 15.3). Importantly, these immune cells do not, on their own, have the ability to recognize specific foreign antigens. Instead, the antibody molecules that have bound to antigens on the surfaces of viruses, bacteria, or mammalian cells alert these immune cells to the presence of targets that should be destroyed. The other arm of the immune system involves the cellular immune response. This response is mounted when specialized cytotoxic cells are developed by the immune system that can, on their own, recognize and directly attack other cells displaying certain antigens on their surface. In this case, soluble antibodies are not required as intermediaries to recognize antigens displayed by targeted cells, since cytotoxic cells of the T-lymphocyte lineage (CTLs) have developed their own antigen-recognizing machinery in the form of T-cell receptors (TCRs), which they use to identify cells bearing particular antigens; such cells are then targeted for destruction by the cytotoxic T lymphocytes (Figure 15.4). 658 Antigen presentation and the immune response We can also depict the immune system in another dimension: many of the responses of the immune system to an infectious agent (e.g., a specific strain of virus) and its antigens depend on a previous encounter with this agent. The immune system has been "educated" through the initial encounter to recognize certain antigens displayed by this agent and to mount a vigorous counterattack against it in the event that it encounters this agent a second time; this represents the adaptive immune response. At the same time, other cellular components of the immune system are naturally endowed with the ability to recognize certain infectious agents or abnormal cells and thus do not require prior exposure and education; this inborn ability is termed the innate immune response. For example, the natural killer (NK) cells cited above have the ability to recognize specific cell-surface molecules displayed by aberrant cells, even without having encountered such cells previously. 15.2 The adaptive immune response leads to antibody production Adaptive immune responses begin when infectious particles or abnormal cells are engulfed by specialized phagocytic cells of the immune system, notably macrophages and dendritic cells (DCs) (Figure 15.5). Having ingested these objects or fragments thereof, the phagocytic cells are then charged with the task Figure 15.4 Cytotoxic T lymphocytesof presenting the ingested contents to other cellular components of the immune The cellular arm of the immune response system. More specifically, these cells must inform the immune system of the set results in the formation of cytotoxic cells, of antigens that were associated with the particles that they have ingested. This such as cytotoxic T cells (T c's, CTLs) that presentation of ingested antigens by phagocytic cells takes place in the lymph are able to recognize and kill other cells nodes, to which these cells migrate following uptake of antigen. (As discussed in displaying certain antigens on their detail below, the antigens are presented in the lymph nodes to various types of surface. (A) CTLs develop antibody-like T cells.) molecules on their surface termed T-cell receptors (TCRs). A di verse array of TCRs In order to educate the immune system, these antigen-presenting cells (APCs) are developed during the development first digest the particles that they have phagocytosed (i.e., ingested outright) or of the immune system, paralleling the endocytosed (i.e., bound via cell surface receptors and then internalized). This development of a diverse repertoire of soluble antibodies. Each CTL displays a digestion, which is carried out in specialized cytoplasmic vesicles, snips internalparticular antigen-recognizing TCR.ized proteins into small oligopeptides that are 18-22 amino acid residues long. (B) Seen here is a CTL (upper right, The oligopeptides are then loaded onto the major histocompatibility complex ist panel) that has already used its TCR(MHC) class II molecules as the latter are making their way to the surface of APCs to recognize and bind to a target cell (diagonally below it to the left). The cytotoxic granules w ithin this CTL (red spot) begin over a period of minutes to (A) T-cell receptors /! migrate toward the point of contact between the killer and its victim. By 40 minutes, the contents of these granules (such as granzymes; see Section 9.14) have been introduced into t he target cell, which has already advar.ced into apoptosis and begun to disintegrate. (From C.A. Janeway Jr. et ai, Immunobiology, 6th ed . cytotoxic T cells (CTLs) New York: Garland Science, 2005) target cell CTL 'j,' ... f '/',,' . ', ' ,.,'. .' /I '..:' .. , ...., e"" "'--\'," . .. "/ ,' . . # l ; I .. \. "/-, J \ ",. 1.., .... '...' " .'}. r'; . , .... .I.' . .. f - l. "', - .-......:-.. ... ' '" .. . .' . . "."\"'''''''' .:, ,-'0 ........,' \.... -. , . \ 11....t:....;to:'!I'. time =0 after 1 minute after 4 minutes after 40 minutes 659 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) antigens ~ .' . .," -antigen uptake by Langerhans cells leave Langerhans cells enter Langerhans cells in the skin and enter the the lymph node to become the skin lymphatic system dendritic cells expressing B7 (B) B7-positive dendritic cells stimulate naive T cells Figure 15.5 Antigen presentation by dendritic cells The immune syste m becomes aware of infectious agents and their antigens largely through the acti ons of ant igen-presenting cells (APCs), notably dendritic cells. (A) Here we see a drawing of specialized phagocytic cells (i.e., Langerhans cells, yellow) residing in the skin, which take up antigens (red dots) by phagocytosi s and then migrate to the lymph nodes (light blue), w here they mature into dendriti c cells. In the lymph nodes, these cells confront T cells (dark blue circles), to which they present antigens; this results in the functional activation of the T cells and the subsequent mounting of a specific immune response against cells and viruses that display these antigens. (B) The dendritic cells take their name from their mUltiple arms extending out from the cell body. (From CA. Janeway Jr. et aI., Immunobiology, 6th ed. New York Garland Science, 2005. ) (Figure 15.6) . More specifically, the oligopeptide fragments become attached to the specialized antigen-presenting domains (Figure 15.7A) of MHC class II molecules. (In humans, the MHC molecules are often termed HLA, or human leukocyte antigen, molecules, but we will use the more generic term, MHC, throughout this chapter to refer to both human and murine molecules of this type.) The class II MHC molecules function much like a street hawker's hands used for displaying wares to passers-by. In this case, the wares are oligo peptide antigens captured by the APCs and the intended customers are other cells of the immune system, specifically a class of lymphocytes termed hel per T cells (TH cells) , often called CD4+ cells to reflect a specific cell surface antigen that they display ~ reticulum MHC class 1\ Figure 15.6 Antigen processing by antigen-presenting cells in the endoplasmic reticulum, which then move to the cell surface, After phagocytes, notably dendritic cells and macrophages, have allowing the MHC class II molecules to display the oligopeptide internalized potential antigenic particles (red oblongs), these are fragments on their surface and present the oligopeptide fragments fragmented into oligopeptides (red dots) by proteolysis. The to T cells in the lymph nodes. (From CA Janeway Jr. et al., resulting oligopeptides are then loaded onto MHC class II molecules Immunobiology, 6th ed. New York: Garland Science, 2005.) 660 Antigen presentation and the immune response oligopeptide antigen oligopeptide antigen Figure 15.7 Antigen presentation by MHC molecules (A) The structure of the antigen-presenting groove of an MHC class II molecule is shown here as determined by X-ray crystallography. The oligopeptide antigen (stick figure, in color) that is bound via hydrogen bonds to the "palm " of the MHC molecule (ribbon diagram) is shown w ith its N-terminus to the left and C-terminus to the right. The oligopepti de antigen together wit h the nearby amino acid residues of the MHC molecu le form the molecular structure that is recognized by other immune cells, which may, for example, use T-cell receptors to do so. (B) A very similar arrangement characterizes the structure of the antigen-presenting domain of MHC class I molecules. (From C.A. Janeway Jr. et al., Immunobiology, 6th ed. New York: Garland Science, 2005.) (Figure 15.8). Because macro phages and dendritic cells are specialized to use their MHC class JI molecules to present antigens scavenged from their environment, immunologists sometimes call them "professional" APCs, to distinguish them from other types of cells that are not specialized for this type of antigen presentation. Note that it is the combined molecular structures formed by the class II ectodomains (t he "hands") and their bound oligopeptide antigens (the "wares") that are presented to helper T (TR) cells (see Figure 15.7). Antigen presentation to certain helper T cells provokes the latter to activate, in turn, the B cells that can manufacture immunoglobulin (antibody) molecules that specifically recognize and bind the particular antigen (see Figure 15.8). The subsequent maturation of these B cells yields a population of cells (called plasma cells) that actively secrete this particular antibody species into the circulation, that is, antibody molecules that are specialized to recognize and bind the particular antigen that originally triggered this series of responses. (Dendritic cells, once again functioning as APCs, can also activate cytotoxic T cells-not indicated in Figure 15.8.) This system works well when confronting infectious agents such as virus particles, bacteria, and fungi in the extracellular spaces. Thus, these infectious agents can be internalized by the professional antigen-presenting cells, and the peptides deriving from the ingested agents can be presented again to the outside world. The antibody molecules that are eventually formed by B cells and their descendants as a result of this antigen presentation can recognize and bind the infectious particles and thereby neutralize them (see Figure 15.2). By the same token, we can imagine that cancer cells displaying certain distinctive antigenic proteins on their surfaces might also provoke an antibody response by the immune system and become coated by antibody molecules bound to these cell surface antigenic molecules. 661 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) dendritic cell productive interaction between APC and T H cell TH cell antibody molecules activatio'n differentiation TCR secreted (] n V !J B-cell plasma cell activation (B) TH cells Figure 15.8 Immunocyte encounters within lymph nodes Dendritic cells interact directly wit h helper T cells and present antigen to them in the lymph nodes. (A) Dendritic cells engulf, process, and present antigenic oligopeptide fragments (red dots; see Figure 156) on their surface to T cells in the lymph nodes, using their MHC class II molecules (gray) to do so. Here, a dendritic cell meets a number of T cells (above), known hereafter as helper T (T ~ cell s. Each of them displays its own distinCt T-ce" receptor (TCR; green) on its surface. However, in the f irst three encounters, none of the T H cells' T-ce" receptors recognizes and binds the antigen being presented by the MHC II molecule of the dendritic cell. Nonetheless, on occasion, the dendritic cell succeeds in finding a T H cell whose T-cell receptor does indeed recogni ze the oligopeptide antigen being presented by the dendritic cell's MHC class II molecules (belovv) This causes the T H to become activated; the T H cell leaves the dendritic cell and proceeds to search for B cells that also display on their surface the same antigen in t he cont ext of M HC II. When and if the T H finds such a B cell (light yellow, 2nd diagram from righ t), it activates the Bee", which proliferates and, having differenti ated into a plasma cell (light brown), begins to release antibody molecules that are capable of recognizing thi s oligopeptide ant igen. (B) Multiphoton microscopy reveals the capsule of a mouse lymph node (blue) and a number of recently arrived, dye-labeled dendritic cells (red dots) as well as dye- labeled T cells (green dots) to which antigen wi ll be presented by the dendritic cells. The two cell types are largel y segregated from one another within the lymph node, and their mechanisms of trafficking and interact ion within the lymph node remain poorly understood. The T H cells have arrived in the lymph node from the venous circulation and have extravasated via diapedesis (Sidebar 14.3) in order to take up residence in the node. (B, from TR. Mempel, S.E. Henrickson and U.H. Von Andrian, Nature 427: 154-1 59, 2004) 662 --Cytotoxic cells and the immune response (A) antibody-antigen \ cell surface complexes channel inserted in plasma membrane (B) The antibodies coating a cell or infectious agent may elicit an alternative type of immune attack: a set of proteins in the plasma, termed complement, will recognize the constant regions of antibody molecules tethered to the surface of a cell (including bacterial, fungal, and mammalian cells). bind to these antibody molecules, and proceed to punch holes in the adjacent plasma membrane, thereby killing the cell (Figure 15.9). This series of steps leading to adaptive humoral responses tells us something important about the molecular structure of the antigens that are immunogenic, that is, that elicit immune responses: they are not intact proteins, but instead are oligo peptide fragments derived from the cleavage of much larger proteins (see Figures 15.6 and 15.7). (The major exceptions to this generality are certain complex carbohydrate chains and linked side chains that may, under some circumstances, also be immunogenic.) 15.3 Another adaptive immune response leads to the formation of cytotoxic cells The type of immunologic response described above fails to deal effectively with infectious agents that have entered into cells and are therefore shielded by the plasma membrane from scrutiny. Similarly, in the case of cancer cells, the humoral response system will fail to recognize aberrant cellular proteins that are hiding deep within these cells. In principle, such shielding should create a serious problem for the immune system, which needs to monitor what is going on inside cells in addition to its task of monitoring the contents of the extracellular spaces and the surfaces of cells. The problem is solved by an antigen-presenting mechanism that echoes the one used by the professional antigen-presenting cells (APCs) described above. Actually, this other antigen-presenting mechanism is the more widespread of the two, since it is used by the great majority of cell types throughout the body. It works like this (Figure 15.10): rather than being used for their normally designated functions, a portion of the proteins synthesized within cells (by some Figure 15.9 Complement-mediated killing (A) Antigen-antibody complexes (red spheres) formed by the binding of antibody molecules to cell surface antigens (left) can attract complement proteins present in the plasma (ye//ow, green, purple) and induce them to form complexes that lead, through a series of steps, to the formation by other complement proteins of channel s in the plasma membrane of the cell (right) at a site adjacent to w here the antigen-antibody complexes initially formed . (B) The resulting channels, seen here in this electron micrograph, destroy the integrity of the barrier functions of the plasma membrane and lead rapidly to cell death. (From CA Janeway Jr. et al., Immunobiology, 6th ed. New York Garland Science, 2005) 663 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) to cell surface, .cytosol ..... membrane (B)f Og re 15.10 Display of intracellular antigens by MHC class I molecules vesicle _____ MHC class I endoplasmic reticu lum oroteasome .. plasma -q ..I peptide fragments :.. s: all cell types throughout the body, including cancer cells, routinely divert G OO L on of their recentl y synt hesized proteins to the antigen-presenting acmnery. (A) Some of the recently synthesized proteins in the cytosol are .l1ed t o proteasomes (purple, yellow), in which they are broken down into gopeptides (red dots); resulting oligopeptides are then introduced into the e1doplasmic reticulum, where they may encounter MHC class I molecules yellow) that bind them relati vely tightl y (see Figure 15.7B). Thi s will cause the mul ti-protein complexes to move via membranous vesicles to the cell surface, w here these protein complexes serve to display to the immune system fragments of the proteins that are being synthesized within the cell. The overall process of displaying these antigens is similar to that undertaken by MHC class II molecules (Figure 15.6); however, MHC class II antigen presentation is the speciality of "professional antigen-presenting cells", such as macrophages, dendritic cells, and B cells, while MHC class I presentation is undertaken routinel y by almost all cell t ypes in the body. (B) A broad spectrum of oligopeptide fragments deri ving from v MHC class I a large number of cellular proteins (here represented as four distinct protein species) are displayed simultaneously by cells using their MHC class I proteins (A, from CA Janeway Jr. et ai, Immunobiology, 6th ed. New York Garland Science, 2005.) accounts, as much as one-third in certain cells) is routinely diverted to specialized proteasomes. There these proteins are cleaved into oligopeptides. These cleavage products, of 8 to 11 amino acid residues in length, are then attached to MHC molecules en route to the cell surface and displayed on the outside of cells by the other major class of antigen-presenting molecules-the MHC class I molecules (see Figure 15.7B). Included among the intracellular peptides displayed by the MHC class I molecules are those synthesized normally by cells as well as those made by foreign infectious agents within the cell, such as viruses and bacteria. This external presentation of internal antigens occurs routinely and continuously, whether or not foreign proteins happen to be present within a cell. The display by a cell of certain oligopeptide antigens on its surface (via its MHC class I molecules) may attract the attention of cytotoxic T cells (Tc's, also called cytotoxicT lymphocytes, CTLs, or CD8+ cells), which proceed to kill this cell (see Figure 15.4). The origins of this killing can be traced back to the actions of helper T cells. Recall that some helper T cells are able to activate the humoral immune response by interacting with and stimulating antibody-producing B cells (see o - cellular proteins . . -. , .. . . .' . ..". .. ..o .ee . ,_ ..". . ...- , ........ . .. 664 Cytotoxic cells and the immune response Figure 15.11 Activation of cytotoxic T cells by helper T cells In addition to inducing B cells to make antibody molecules (Figure 158), helper T celis (TH) of a second subtype (blue) can activate the precursors of cytotoxic T celis (light red, bottom) to become active cytotoxic T cells (termed Tc 's or CTLs, red) that can use their T-cell receptors (TCRs) to recognize and bind antigens presented on the surfaces of many cell types throughout the body by MHC class I molecules. This recognition results in attack on the antigen-displaying cell (gray, top), as shown by the micrographs of Figure 15.4B. The Tc 's often use cytotoxic granules (black dots) containing perforin and granzymes to kill targeted cells (Figure 15.12) Figure 15.8). Now, we encounter a second, independent function of helper T cells: some of them can contribute to the activation of cytotoxic T cells, which are specialized to recognize and kill target cells displaying the particular oligopeptide antigen that initially provoked an immune response (Figure 15.11) . This attack on antigen-displaying cells by cytotoxic lymphocytes represents the cellular arm of the adaptive immune response. The capacity of helperT cells to facilitate development of both humoral and cellular immune responses reflects the ability of distinct subpopulations ofTH cells to produce and release the soluble immune factors known as cytokines: TH'S that promote hwnoral immunity (by stimulating B cells) produce interleukin-4 OL-4), while TH'S that promote cell-mediated immunity (by stimulating cytotoxic T cells) secrete interferon-y (IFN-y) . Cytotoxic T cells can kill their cellular victims through two separate mechanisms. They can expose their intended victims to certain toxic proteins (Figure 15.12A and B) . One of these, periorin, punches holes in the plasma membrane of a targeted cell . These holes then enable granzymes released by the cytotoxic cells to enter into the cytoplasm of the victim. As described earlier (Section 9.14), once in the cytoplasm of the targeted cell, granzymes cleave and thereby activate pro-apoptotic caspases. The second killing mechanism, also discussed in Section 9.14, involves the Fas death receptor, which is displayed on many cell types throughout the body. Cytotoxic T cells can present the ligand of the Fas receptor, termed FasL, to their intended victims. FasL then activates the Fas death receptors on the surfaces of the targeted cells, thereby activating their extrinsic apoptotic pathway (Figure 15.12C) . Killing by cytotoxic T cells can play an important role in limiting the infectious spread of viruses. For example, a recently infected cell in which a virus is actively replicating will use its MHC class I molecules to display oligo peptide antigens derived from cleaved viral proteins. This antigen display will warn the immune system that abnormal proteins are being produced deep within the cell. If the immune system is functioning well, its cytotoxic T cells will recognize the viral oligopeptide antigens displayed by the cell's MHC class I molecules and kill this cell long before the virus has had a chance to multiply and release progeny virus particles. This means that the immune system actually uses two arms of the adaptive immune response to limit viral infections: the cellular response is used to kill virus-infected cells, while the humoral response is used to neutralize virus particles that have been released into extracellular spaces, including the circulation, by coating these particles with antibody molecules (see Figure 15.2A). As we will see, the anti-viral responses are important means by which the immune system blocks the appearance of virus-induced human tumors. antigen-displayi ng target cell MHC TH cell L activation [ active T c maturation T c precursor 665 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) (8) (C) targeted ce lis "as FADD death. -X domains .. - -J pro- .fl 8 ",::::'i Q lb active cClspase: 3 . o . .target cell pro- 0. . . .. caspase 3 .. .0 . f apoptosis (0 ) (E) 60 min-15.4 The innate immune response does not require prior sensitization Ninety-nine percent of the animal species on the planet do not possess adaptive immune responses to protect them from attack by pathogens. These organisms rely on innate immunological responses for such protection. Importantly, this ancient, widespread innate immunity system has been conserved during the 666 Cytotoxic cells and the immune response Figure 15.12 Mechanisms of cell killing by cytotoxic lymphocytes (A) This electron micrograph of a cytotoxic T lymphocyte (T(, CTL) reveals a series of lytic granules in its cytoplasm (pink arrows, left panel). When contact is made with a targeted cell (which was initially recognized by the T-cell receptors borne by the T C), these granules release perforin, which forms cylindrical channels in the plasma membrane of the target (center cel/, right panel); pro-apoptotic proteins such as granzymes (see Section 9.14), which are also carried in these granules, are then introduced through these channels into the cytoplasm of the targeted cell, where they initiate the apoptotic cascade by cleaving procaspases. (B) In the absence of a cellular target, the lytic granules (green, yellow), w hich contain perforin and granzymes, are scattered throughout the cytoplasm of cytotoxic T lymphocytes (Tcs; upper panel) In the lower panel, a synapse has been formed with a targeted cell (left), and the lytic granules have congregated at the synapse in preparation for killing the targeted cell. (C) An alternative mechanism of killing cells that have been targeted for destruction depends on the display of FasL (orange) by the T c (top, pink). FasL, which is a trimer, then engages the Fas receptor (brown) displayed by the targeted cell (bottom cell, gray) and triggers receptor trimerization and resulting activation of the ext rinsic apoptot ic cascade in the targeted cell via the sequential activation of caspases 8 and 3 (see al so Figure 9.31). (D) NK cells are programmed to recognize and kill other cells, including cancer cells, that do not display normal levels of MHC class I molecules on their surface. This scanning electron micrograph (SEM) reveals that NK cells (colorized green), one of which has spread a portion of its cytoplasm across the surface of a human ductal breast carcinoma cell in the initial stage of such an attack. (E) This SEM reveal s the initial attack of an NK cell (left panel, smaller cell) on a leukemia cell. Sixty minutes later, the NK cell has caused extensive damage to the plasma membrane of the leukemia cell, which has fragmented and rolled up its plasma membrane in response t o this attack (right panel). (A, C, and E, from C A Janeway Jr. et ai., Immunobiology, 6th ed. New York: Garland Science, 2005; B, from R.H. Clark, J.C Stinchcombe, A. Day et ai., Nat. Immunol. 4:1111-1120, 2003; D, courtesy of S.C Watkins and R. Herberman .) evolution of mammals and continues to pLaya critical role in various immunological responses. The cellular components of the innate immune response are able to recognize and attack foreign particles and aberrant cells without having been "educated" through prior exposure to these agents. Thus, these immunocytes (cells of the immune system) "instinctively" recognize aberrant cells, such as cancer cells, in the body's tissues and target these cells for attack and destruction. Instead of recognizing specific antigens, the cells mediating innate immunity recognize characteristic molecular patterns that are present on the surfaces of infectious agents (or transformed cells) but are not displayed by normal cells. A major component of the innate response is the natural killer (NK) cell. It is likely that many initial encounters of the immune system with cancer cells are made by NK cells. As we will discuss in greater detail later, the NK cells recognize configurations of cell surface proteins displayed by a wide variety of cancer cell types. Hence, NK cells are "pre-programmed" to recognize cancer cells and to eliminate them from the body's tissues. In addition to NK cells, yet other cellular components of the innate immune system, including macrophages and neutrophils, are likely to contribute to mounting innate immune responses against cancer cells. After an NK cell has initiated the innate immune response by recognizing and attacking a target cell (Figure 15.120 and E), it sends out cytokine signals, notably interferon-y (IFN-y), in order to recruit yet other immune cells, including macrophages, to the site of attack. The actions of this second wave of 667 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy Figure 15.13 Destruction of normal tissues by autoimmune attack Extensive tissue damage can be wrought b an immune system that has been pro oked to attack the body's normal tissues. In principle, the same immune mechanisms that are at work here can also 0 , erate to attack and destroy malignant ti ssues. (A) A normal pancrea ic islet (i .e , islet of Langerhans) in a mouse (left panel) is composed largely 0 ' InSuli n-secreting pcells (light brown) ith a small number of a cells at its periphery (dark brown). The pancreas of a mouse sufferi ng from diabetes resulting f rom autoimmune attack on pcells is seen 0 have lost almost all of them (right panel). (B) A normal glomerulus in the kidney (center of left panel), which contains a complex network of tubules, is responsib le for the filtering of plasma and the production of urine. In the disease of systemic lupus erythematosus (SLE), an autoimmune attack on the basement membrane located beneath the epithelial cells of the glomerulus results in the accumulation of antibody protein and the invasion of a variety of inflammatory cells; together, these eventually destroy the architecture of the glomeruli (right) and thus kidney function. (A, from CA Janeway Jr. et ai, Immunobiology, 6th ed . New York Garland Science, 2005; B, courtesy of A.B. Fogo. ) immunocytes will often enable the immune system to mount more specific and ultimately more effective responses, in particular, adaptive humoral and cellular responses. For example, large numbers of cytotoxic T cells can be mobilized by the adaptive immune response to efficiently kill cancer cells. 15.5 The need to distinguish self from non-self results in immune tolerance The immune system is finely tuned and highly specific. Most critically, it must be able to distinguish foreign proteins (e.g., those made by invading infectious agents) from those proteins that are normally made by the body's own cells. As a consequence, if the oligopeptides displayed by one of the normal cells in the body are similar or identical to those routinely encountered by the immune system, this cell will remain unmolested by the various arms of the immune system-one of the manifestations of immune tolerance. In fact, immune tolerance represents the major puzzle of current immunological research: How does the immune system learn to discriminate foreign proteins and peptides from the body's normal repertoire of proteins? Immunologists often refer to this behavior as the ability of the immune system to discriminate between "nonself" and "self." A variety of mechanisms operating during the development of the immune system ensure that any T cells and B cells that happen to recognize self-antigens are eliminated; alternatively, if such cells escape elimination, their actions will be strongly suppressed. Failure to delete such self-reactive or auto-reactive lymphocytes from the large pool of lymphocytes in the body results in the survival of immune cells that may target the body's own normal tissues. Should such auto-reactive cells actually do so, this breakdown of tolerance may lead to autoimmune diseases, such as rheumatoid arthritis, ulcerative colitis, and lupus erythematosus, in which the immune system dispatches antibodies and cytotoxic cells to attack normal cells and tissues (Figure 15.13). (A) pancreatic islet (8) kidney glomerulus ~ ~ ~ normal autoimmune destruction 668 Loss of tolerance and autoimmunity Immune tolerance raises a simple and obvious point that will dominate the discussions that follow: How does the immune system, which is designed to be tolerant of the body's own cells, recognize and attack cancer cells, which are, to a great extent, very similar at the biochemical level to cells that are normally present in the body? And if it does undertake attacks against cancer cells, including those transformed by tumor viruses, how might these cells evade and thwart the attacks launched by various arms of the immune system (see Sidebar 36 OJ? 15.6 Regulatory T cells are able to suppress major components of the adaptive immune response Research beginning in the 1990s has described an entirely new class ofT cells that have come to be called regulatory T cells (Treg cells or simply Tregs) . Indirect evidence suggesting their existence came from the observation that in normal individuals, a significant proportion of cytotoxic T cells (CTLs) recognize normal tissue antigens presented by these individuals' MHC class I molecules-a situation that should lead directly to extensive immune attack on normal tissues and resulting autoimmune disease. However, such attacks do not occur, apparently because of suppression of these cells' actions by some unknown agents. The discovery ofTreg cells seems to have largely solved this problem, since these cells are able to block the actions of the cytotoxic T cells that are scattered throughout our tissues. Indeed, in genetically altered mice lacking Treg cells, lethal autoinunune disease develops; a comparably aggressive, ultimately fatal autoimmune disease has also been documented in humans who are unable to make Tregs. Like T helper (TH) cells, the Tregs display the C04 antigen on their surface. However, the Tregs are distinguished by their additional display of the C025 surface antigen and their expression of a transcription factor, termed FOXP3, that programs their development. Because Tregs express antigen-specific T-cell receptors (TCRs; see Figure 15.4), they can specifically block the actions of those cytotoxic T lymphocytes whose TCRs recognize the same antigens. In addition, when located in the lymph nodes, the Tregs can prevent the activation ofTH cells by dendritic cells. It appears that the T regS must be in close proximity with the T Hand T c cells that they suppress, and that the release of T G F - ~ and interleukin-10 (IL-lO) by the Tregs is often used to inhibit or kill these other types ofT lymphocytes. Research on TIegS is still in its infancy. However, it is possible that their behavior holds the key to understanding the pathogenesis of a number of autoimmune diseases. At the same time, the actions of Tregs may explain how many types of tumor cells can thrive in the presence of large numbers of CTLs that should, by all rights, be able to eliminate them-a topic pursued later in this chapter. An overview of the various components of the immune system that we have covered until now is provided in Figure 15.14. 15.7 The immunosurveillance theory is born and then suffers major setbacks As suggested by the quotation at the beginning of this chapter, the notion that the immune system is able to defend us against cancer is an old one. Burnet's 1957 speculation about the immune system's role in monitoring tissues for the presence of tumors, together with other speculations made by Lewis Thomas, represented the first instance that the notion of the immunosurveillance of cancer was clearly articulated. 669 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy At the time, infecting microorganisms, specifically, bacteria, viruses, and fungi, were known to be strongly immunogenic, in that they usually provoke an immune response that leads to their total eradication by various arms of the immune system. By analogy, it was plausible that the immune system continuously monitors its tissues for the presence of cancer cells. Having identified them-so this thinking went-the immune system would treat these cancer cells as foreign invaders and eliminate them long before they had a chance to proliferate and form life-threatening tumors. Early attempts in the 1950s to test this model were not definitive. When tumors were removed from some mice and implanted in others, the tumors were rapidly destroyed in a way that gave clear indication of the actions of vigorous host immune responses. Soon it became clear, however, that this rejection had nothing to do with the neoplastic nature of the tumor cells. Instead, their elimination was a consequence of what came to be called allograft rejection. Thus, cells and tissues from one strain of mice are invariably recognized as being foreign when implanted in mice of a second strain. This is a consequence of the fact that the humoral cellular immunity specific ___ __ ________ _____ __ _ __ nonspecific(adaptive) " ..- (innate) , proteinsantigen cells,- controllers -, effectors present!ng cellscell surface antibody , binds antigen regulatory T NKcells, I complement(Treg) cytotoxic dendritic celis, : .. .. __-----,,.--A____-----i macrophages . I proliferation -st imul ate stimulate killing of targetplasma cells ,cells 1 t t innate Fc recognition receptors secrete antibodies - - - -- -- - - - - - - -- --- - ----: -- - -- - - - - - - -- .... - -- - -- - - - -- - - - - - - -- -- - -1- --- - -- -'j! ._----------- .. -----_._--------------- ---- ... --_._. __ ..... -.': immunity neutralize pathogens killing of target cells __ Figure 15.14 Overview of the humoral and cellular arms of th e immune system The humoral immune system (left) is"driven IJ\ ere actions of B cells that develop millions of distinct antigenSUE::: ' ( antibody molecules through the rearrangement of antibodyE1(OC( g genes and the diversification of the sequences encoding : f 2 c'l- ge'1-combining sites in the V regions of antibody molecules. - r6 'lumo-al response depends on activation by helper T (TH) cells, .', ([) oeoends, in turn, on their prior activation in the lymph nodes art gen-presenting cell s, largely dendritic cells. The latter process bl prole r s lOW oligopeptides t hat are recognized by the T-cell receptors 01 Tli cells, whi ch proceed to activate B cells that have developed, by chance, anti bodies that recognize the oligopeptide antigens. T-cell receptors (TCRs) are used as well by cytotoxic T cells (Tcl cells (also te rmed CTLs), which rely on these receptors to recognize and kill target cell s displaying cognate antigens. The activation of the Tc cells also depends on prior stimulation by TH cells. A third class of T cells that also expresses antigen-specific T-cell receptors are the regulatory T cells, often called Tre9 s. These play important roles In suppressing the actions of both Tc and TH cells and thereby 670 prevent inappropriate activation of immune responses that might otherwise lead to a breakdown of tolerance and resulting autoimmune disease. These various manifestations of adaptive immunity are augmented by arms of the innate immune'system (right), specifically cell types that can aid in the elimination of pathogens and cancer cells without any prior" education" through previous exposure to these entities. Thus, natural killer (NK) cells are primed to kill many types of cancer cells because of the abnormal configuration of cellsurface molecules displayed by these cells; macrophages are also capable of recognizing and killing many cellular pathogens without any prior exposure to these agents. Although macrophages and NK cells cannot themselves recognize most cell-surface antigens, the coating of potential target cells by antibody molecules (produced by the adaptive immune response) will attract macrophages and NK cells, which will use their Fe receptors to bind to the constant (C) regions of antibody molecules and proceed to kill the antibodycoated cells. Similarly, the complex group of plasma proteins known as complement may also recognize antibody molecules bound to the surface of a cell and then kill this cell by inserting channels in its plasma membrane. Rejection of histoincompatible tumors cells of various strains of mice display distinct, genetically templated major histocompatibility (MHC) molecules on their surfaces. (In this instance, however, it is not the bound oligopeptide antigens that evoke an immune response but the MHC molecules themselves, which vary slightly in structure from one strain of mouse to another.) For example, engrafted cancer cells from BALBI c mice were recognized as being of foreign origin (and were therefore histoincompatible) when introduced into C57/BL6 mice, and vice versa (Figure 15.15). These graft rejections from dissimilar, allogeneic (i.e., genetically distinct) mouse strains were not observed when tumor cells of BALB/c origin were grafted into BALB/c hosts, that is, into syngeneic hosts that, by definition, shared an identical genetic background and identical histocompatibility antigens with the engrafted cells. [In fact, the term histocompatibility derives from the observation that tissue ("histo-") from mice of one inbred strain can be grafted and established in the bodies of other members of the same genetic strain and are in this sense "compatible."] The observed rejections of allogeneic tumors represented a detour for the young field of tumor immunology, since they shed no light on how the immune system of a mouse or human host would recognize cancer cells that arise in its own tissues. Still, this early work did make one profoundly important point: in addition to eliminating microbes and various types of viruses, the immune system is capable of destroying mammalian cells that it recognizes as foreign or, quite pOSSibly, as othervvise abnormal. As an additional corollary, these observations of immune function led to the conclusion that cancer cells could never be transmitted from one individual to another (but see Sidebar 37 . , and Figure 15.16). An alternative strategy was then embraced for studying the immunosurveillance problem. If the immune system were indeed responsible for suppressing the appearance of tumors, animals with compromised immune systems should suffer increased rates of cancer. Such cancers, which originated within their own bodies-so-called autochthonous tumors-were, of course, of the same histocompatibility type as the remaining tissues in these animals. In these situations, the issue of histocompatibility (and -incompatibility) was rendered irrelevant. 1( . carcinogen.. BALB!c ! tumors C57/BL6 ! tumors tumo, celiAQ- tumm cell n inject cells inject cells Figure 15.15 Syngeneic mice and MHC variability The use of inbred strains of mice has revealed that major determinants of the immunogenicity of the cells of these mice (and of mammals in general) are the MHC class I molecules. These molecules are highly polymorphic w ithin a species, indicating that one individual (or one inbred strain of mice) almost al ways has a different set of MHC class I molecules from another (red, blue cell surface molecules) Therefore, if a tumor arises within a BALB/C mouse, it is often transplantable into a syngeneic host, i.e., another BALB/c mouse, but not into an rl rl allogeneic host, such as a C57/BL6 tumors in no tumors in no tumors in tumors in mouse. The converse is true for tumors syngeneic allogeneic allogeneic syngeneic host host host host ariSing in C57/BL6 mice. 671 --Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy Figure 15.16 Regression of CTVS and re-expression of MHC antigens One exception to the rule of the nontransmissibility of cancer from one organism to another comes from canine transmissible venereal sarcoma; its cells are transferred from one dog to another via sexual intercourse. The transferred cells initially form a vigorously grovving tumor, which is, however, rejected after several months (see also Sidebar 37 . ) (A) While the canine transmissible venereal sarcoma (CTVS) cells initially express very 10 levels of MHC class I proteins, aft er 12 eeks of tu mor growth these proteins begin to be fe-expressed, as seen here in t he tumors borne b hree dogs. (8) Thi s re-expression results, at least in part, from s'gnals released by tumorinfiltra: 'og lymphocytes (TILs) Fresh ( ulwre med'um has li ttle effect on the express o ~ 0 HC class I (green) or class I red) prot eins by CTVS cells (left) aaors re eased by TILs isolated from o'oc;:'esslog tumors (1 st 12 weeks) also a e Ie effect (middle). However, "2SorS released by TILs from regressing : VTlors (12-21 weeks) potently induce He protein expression (right) by cultured CTVS cells. Such re-expression appears to be responsible for tumor regression in vivo. (From yw. Hsiao, K. W. Liao, S.w. Hung, and R.M. Chu, } Immuno!. 172:1508-1514,2004) (A) (8) "D 25 40 Q) Vl -- ... Vl Q) 20 ~ ~ 0.. -- 30 x ~ ~ Q) 15 V ' I ~ u ~ Ol 20I '+- . ~ ~ 10 o ~ '+0 ;oR ~ 10o 0.. x 5 xQ) "D c: Q) 0.1 I + medium from TILs from regressing tumors co-cultured with CTVS + medium from TILs from progressing tumors co-cultured with CTVS dog 1 dog 2 dog 3 0 3 6 9 12 15 18 21 weeks after implantation + fresh medium In the late 1960s, immunocompromised mice of the Nude strain first became available to cancer researchers. These mice lack a functional thymus-the tissue in which the T lymphocytes of the immune system initially develop. Their lack of hair, another distinct phenotype of this strain, gave them their name (see Figure 3.13). The research that followed in the early and mid -1970s revealed that these mice are no more susceptible to spontaneously arising or chemically induced autochthonous tumors than are their normal, wild-type littermates. So, the immunosurveillance theory suffered a major setback, having failed a major critical test of its validity. It lost credibility and retreated from the main arena of cancer research for two decades. But this rejection was premature. Only years later did it become apparent that mice of the Nude strain, while lacking many of their normal T lymphocytes, retain other components of their immune system in an intact form. For example, some types ofT cells may be able to develop outside the thymus, the normal site of maturation of these cells. In addition, a very important type of immune cell-the natural killer (NK) cell-is able to develop totally outside the thymus, and thus NK cells are present in large numbers in Nude mice. In the 1980s, researchers began to accumulate evidence that NK cells are actually very important in recognizing and killing a variety of abnormal cells, including cancer cells. So in the end, the lessons taught by the low cancer rates of Nude mice were of limited value, since these mice did indeed continue to harbor functionally important components of the inunune system. Still, Nude mice, as well as other types of immunocompromised mice, have proven to be of great value in cancer research (Sidebar 38 0 ). Evidence also began to accumulate that certain chemically induced tumors in mice were antigenic and could be recognized and eliminated by the immune system. For example, in one set of experiments, cells from a 3-methylcholanthrene (3MC)-induced tumor were irradiated prior to injection into mouse hosts in order to prevent the proliferation of these cells in the hosts (Figure 15.17). (The chemically induced tumor had been induced in the same strain of mice as these hosts.) Subsequently, the mice received a second injection of live tumor cells originating from the same tumor or from a second 3MC-induced tumor; the cells originating from the same tumor did not grow, while the cells from the second tumor did grow and form a new tumor. This indicated that the two tumors were antigenically different and that the initial exposure to dead cancer cells had immunized the mice against live cells originating in the same tumor. Hence, tumor cells could have distinctive antigens, and under certain conditions, these antigens could provoke the immune system to attack and kill such cells. 672 Cancer susceptibility and immune function 15.8 Use of genetically altered mice leads to a resurrection of the immunosurveillance theory In the mid-1990s, several lines of research gave new life to the long-discredited immunosurveillance theory. These experiments derived from the then recently gained ability to create genetically altered strains of mice at will. This technology (see Sidebar 7.10) was exploited to create mice whose genomes lacked one or more of the genes known to play critical roles in the functioning of the immune system. One group of experiments used mice that were rendered incapable of expressing the receptor for interferon-y (IFN-y) through targeted inactivation of the responsible gene in their germ line. Like growth factors, IFN-yis a diffusible protein factor that conveys signals from one cell to another and induces responses in cells by binding and activating its cognate cell surface receptor. Importantly, IFN-y has not been found to be released by cells other than those of the immune system. Consequently, any changes observed following deletion of the IFN-y receptor gene from the mouse genome could be attributed to defects associated with immune cells and their interactions with the remaining cells in the body. Strikingly, mice tha t lack the IFN -yreceptor in all of their cells were found to be 10 to 20 times more susceptible to tumor induction by the chemical carcinogen 3 -methylcholan th rene. In another set of experiments, tumor cells were forced to express a dominantnegative IFN-y receptor, rendering them unresponsive to the IFN-y released by various types of immunocytes. These cells were then injected into wild-type mice and found to be more tumorigenic than tumor cells carrying the corresponding wild -type receptor. This particular experiment suggested that the IFNY receptor displayed by cancer cells enables them to respond to IFN -y released by immunocytes, and that this response usually prevents or retards the growth of tumors formed bv these cells. These striking effects of IFN-y could be associated, at least in part, with the actions of the natural killer cells. The NK cells were discovered and named because of their innate ability to recognize tumor cells as abnormal and to eliminate them. Once NK cells identify cancer cells as targets for elimination, they release IFN -y in the vicinity of the targeted cells. The released IFN -y, in turn, elicits several distinct responses. As mentioned earlier, IFN -y enables the NK cells to call in other types of immune cells to assist in killing targeted cancer cells, thereby amplifying the immune system's response. Among the responding immune cells are macrophages, which aid not only by killing the cancer cells directly but also indirectly, by functioning as professional antigen-presenting cells (APCs) that process and display antigenic molecules derived from the corpses of their victims (see Figure 15.6). At the same time, IFN-y stimulates targeted cancer cells to display on their surfaces increased levels of class I MHC molecules that may carry oligopeptide antigens capable of provoking further, highly specific adaptive immune responses. This helps to explain why transformed cells lacking the IFN-yreceptor are more tumorigenic than counterpart cells that do display this receptor. All of these responses seemed to be defective in genetically altered mice lacking the IFN-y receptor; such mice were also found to have an increased susceptibility to certain types of spontaneously arising tumors. When taken together, these experiments provided compelling validation of the idea that immune surveillance plays a critical role in tumorigenesis, at least in chemically induced tumors of mice. Further support of the immunosurveillance theory came from mice that had been deprived of the gene encoding perforin, the protein used by lymphocytes irradiated tumor cells immunize mouse with irradiated tumor cells n .. inject viable cells inject viable cells of the same tumor from a second, independently induced tumorI ! ~ O host response host response rejects tumor permits proliferation cells and of cells and prevents tumor growth tumor formation Figure 15.17 Immunization of mice by exposure to killed cancer cells Mice were initially injected with irradiated, killed cancer cells (red) deriving from one chemically induced tumor. When these mice were subsequently injected with li ve cells from the same tumor, the cells failed to grow (lower left). However, when these mice were injected with live cells from a second tumor (blue), the cells proliferated and formed a tumor mass (lower right). The reciprocal experiment (not shown) yields the opposite results, i.e., injection with killed blue cancer cells rendered mice immune to the blue tumor but not to the red tumor. 673 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy (A) (8) ta-chain locus T cell ~ - c h a i n locus '1;, Figure 15.18 RAG proteins and TCR gene rearrangement The RAG-1 and RAG-2 proteins are responsi bl e for the rearrangement of DNA segments that leads to the formation of both antibody molecules and T-cell receptors (TCRs). (A) This diagram illustrates the organization of the unrearranged genes encoding the a and ~ chains of the TCR. The RAG-1 and RAG-2 proteins rearrange the germ-line versions of these two genes through the deletion of intragenic segments and the attendant fusion of previ ously di stantly linked DNA segments within the a and within the ~ gene. Rearrangement within the a gene is achieved when the RAG proteins juxtapose an L segment encoding a leader sequence (L) at the N-terminus of the a chain with one of the 70 to 80 Va gene segments (red) and one of the 61 Ja segments (yellow); since the choice of individual Va and Ja segments to be fused is essentially random, this results in a large number of combinations of joined Va- Ja segments and a comparably large number of distinct antigen-binding pockets. A similar set of rearrangements occurring independently on a different chromosome results in the formation of the ~ chain-encoding segments of the TCR. Since the antigenrecognition domains of the TCR are created cooperatively by the amino acid sequences encoded by the a and ~ chains, these RAG-1!2-mediated rearrangements of the TCR-encoding genes are able to create a vast number of distinct antigenbinding domains. (8) The TCR (above) on the surface of a cytotoxic T cell (Te, CTL) enables the T cell to recognize a specific oligopeptide antigen (yellow) displayed by an MHC class I molecule (below; see also Figure 15.78) on the surface of a potential target cell; such recognition and binding by the TCR is achieved by its Va and Vp domains (colored loops) whose generation is described in panel A. (Confusingly, the Va domain of the TCR is created by juxtaposition of Va and J a DNA segments of the a-chain locus, while the Vp domain is created by fusion of the Vp, Dp, and Jp DNA segments of the ~ chain locus, all illustrated in panel A.) Once such recognition has occurred, it may result in the killing of the target cell (see Figure 1512). Since TCRs are also used by T Hand Tregs for other immune functions (not shown; see Figure 15.14), loss of TCRs caused by inactivation of a RAG gene leads to crippling of many components of the multi-faceted cellular immune response as well as the inactivation of the humoral response, which dependends on RAG 1!2-mediated rearrangement of antibody genes. (From c.A. Janeway Jr. et ai, Immunobiology, 6th ed. New York: Garland Science, 2005.) and NK ceUs to mediate killing of targeted cells. Recall that perforin is used by cytotoxic cells to create channels in the plasma membrane of their victims, allowing the entrance of apoptosis-inducing granzymes (see Figure 15.12A). Mutant mice lacking the ability to make perforin showed an elevated incidence of spontaneous tumors and were also more susceptible to developing tumors following exposure to 3-methylcholanthrene. Similarly, increased cancer susceptibility was registered in genetically altered mice that lacked the RAG-lor RAG-2 proteins; these two proteins are responsible for rearranging the genes encoding soluble antibody molecules as well as those encoding the antigen-recognizing T-cell receptors (TCRs) displayed on the surfaces ofT cells (Figure 15.18). Such RAG-lor -2-negative mice lack T lymphocytes, B lymphocytes, y8 T cells (not discussed further in this chapter), and a subclass of NK cells called NKT cells. As a consequence, these mice have severely compromised adaptive immune responses. ~ target cell 674 Cancer susceptibility and immune function For example, in one experiment, 3-MC treatment caused 30 of 52 RAG-2-i- mice to develop sarcomas, while only 11 of 57 wild-type mice of the same genetic background and treated in parallel formed these tumors. The mutant mice were also found to be far more susceptible to spontaneously arising cancers. Thus, 50% of older (18-month-old) RAG-2-negative mice developed spontaneous gastrointestinal malignancies-a twnor that is otherwise rare in wild-type mice ofthis age. Arguably the most persuasive evidence supporting the role of immunosurveillance in cancer prevention comes from detailed studies of the 3-MC-induced sarcomas growing in either R A G ~ i - or wild-type mice. When tumor cells prepared from these two groups of sarcomas were grafted into new R A G ~ i - hosts, both groups of sarcomas seeded tumors in these new hosts with high efficiency (Figure 15.19). A very different outcome was observed, however, when tumor cells were transplanted into syngeneic vvild-type (and thus immunocompetent) hosts. Cells from 17 tumors that had previously been induced in wild-type mice all succeeded in generating tumors in their new hosts. In contrast, cells from 8 of 20 tumors that had previously been induced by 3-MC in RAG2-i- mice failed to form tumors, being rejected by the immune systems of these wild-type hosts (see Figure 15.19). These observations open our eyes to an entirely new dimension of tumor immunology. They suggest that when 3-MC-transformed cells arise in an immunocompetent host, those that happen to be strongly immunogenic (and thus capable of provoking some type of immune response) are effectively eliminated by the host, resulting in the survival and outgrowth of only those cancer cells that happen to be weakly imm,unogenic. The latter then multiply and form tumors in their original hosts and, later on, succeed in doing so when transplanted into other immunocompetent hosts. Hence, these tumors represent a subset of those that originally arose in the primary hosts. The missing, strongly immunogenic twnors are apparently eliminated early in tumor progression by host immune systems and therefore never see the light of day (see Figure 15.19). In contrast, when 3-MC-transformed cells arise in an immunocompromised host (see Figure 15.19A), two classes of tumors are initially formed, as beforethose that are strongly immunogenic and those that are weakly immunogenic; both types of tumor cells survive in an immunodeficient host. Later, when these tumors are transplanted into immunocompetent hosts, those that are strongly immunogenic fail to form tumors, while those that are weakly immunogenic succeed in doing so. We conclude that in wild-type mice, a functional immune system plays an important and effective role in eliminating a significant fraction of the tumors that are initially induced by 3-MC. These observations indicate that the immune system of these mice plays an active role in determining the identities of tumors that arise and the antigens that they express. This active intervention in the phenotype of tumors has been termed immunoediting, to indicate the weeding out of some tumors and the tolerance of others. Immunoediting can be thought of as a type of Darwinian selection, in which the selective force is created by the directed attacks of the immune system on incipient tumors. 15.9 The human immune system plays a critical role in warding off various types of human cancer Because the biology of mice and humans differs in so many respects, we need to interpret the results described above with caution when attempting to understand the role of the human immune system in defending us against cancer. In 675 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy addition, the chemical carcinogens used in the experiments described above may well create tumors in mice that are far more antigenic or immunogenic than spontaneously arising human tumors (to be discussed in Section 15.12). Figure 15.19 Effects of immune function on the development of anti-tumor immune responses Both wild-type (wt) and RAG2-1immunocompromised mice were exposed to the potent carcinogen 3-methylcholanthrene (3-MC) (A) When the tumors induced in the RAG2-/- mice were transplanted back into RAGL'- hosts, they all formed tumors (above). However, when the tumors induced in the RAG;z--I- mice were transplanted back into wild-type hosts, 8 of 20 tumors failed to form (below) Each line presents the kinetics of growth of a single implanted tumor. (B) This experiment and other experiments using tumors induced in wt mice (not shown) are summarized here. Following exposure to 3-MC, the wt mice developed fewer tumors (blue) than did the RAG2-/- mutants (blue and red) The tumors from the two groups of mice were excised and cells from each were converted to a cell line that could be propagated in vitro. Cells from each of these cell lines were then transplanted back into either wt mice or RAG2-/- mutant mice. Cells from all of the tumors that appeared initially in the wt mice (bl ue) were able to form new tumors in both w t and RAGL'- hosts (left). However, cells from all of the tumors that arose and grew initiall y in the RAGL'mice were able to form new tumors in RAGL'- mice (red, blue), but only some of these (blue) were able to form new tumors in the wt mice (right). These experiments suggested that 3-MC initially induced t wo types of tumor cells in all of the mice strongly immunogenic (red) and weakly immunogenic (blue). Both red and blue cells formed tumors in the RAG2-/- mice, but only blue cells formed tumors in the wt mice, since any initially formed red tumor cells were eliminated by the functional immune systems of these mice. This meant that the tumors that did arise in wt mice were already selected for being weakly immunogenic and thus capable of forming new tumors in other wt mice. (A, from V. Shankaran, H. Ikeda, AT Bruce et al., Nature 410 1107- 1111, 2001) (B) (A) 25 20 15 E.s 10 ~ o ~ , . E t :, .. ". 3 20" : :;, " .. / c.. mice engrafted with 24D3 lymphocytes"" ::J::J::J a:l tumor cell Iysates figure 15.23 Specificity of the antigen display by a chemically induced fibrosarcoma Mice of the BALBIc strain were immunized with Iysates of cells of the 3-methylcholanthrene-induced Meth A fibrosarcoma line. A line of antigen-presenting lymphocytes, termed 24D3, was developed from these mice. (A) As gauged by their incorporation of 3H-thymidine, proliferation of these lymphocytes in culture was stimulated by addition of Meth A cell Iysates (2nd bar). However, Iysates prepared from 14 other tumor cell lines, including other methylcholanthrene-induced sarcomas, UV-induced squamous cell skin carcinomas, lymphomas, a melanoma, and a lung carcinoma, failed to stimulate proliferation of these lymphocytes (remaining channels). In the absence of antigen (1st channel) no proliferation was seen. (B) When a clonal population of these 24D3 lymphocytes was introduced into BALBIc hosts, the formation of tumors by subsequent ly injected Meth A fibrosarcoma cells was fully blocked (left panel). However, the formation of tumors by an unrelated fibrosarcoma line, termed CMS5, was unaffected by the presence of these lymphocytes (right panel). (From T. Matsutake and P.K. Srivastava, Proc Natl. Acad. Sci. USA 983992-3997, 2001) 686 Tumor-associated transplantation antigens their descendants expressing low levels of TSTA will survive long enough to be studied by an experimenter, greatly complicating the biochemical isolation and identification of the TSTA protein. In recent years, several of the genes encoding 3-MC-induced TSTAs have nevertheless been isolated by gene cloning procedures. In one cloning strategy, the oligopeptides that were bound to MHC class I molecules on the surfaces of 3MC-transformed cells and served as targets for immune recognition were eluted from the MHC molecules, purified, and subjected to amino acid sequencing. The resulting amino acid sequences were then used to predict the nucleotide sequences of the encoding genes, which made possible the cloning of these genes. Sequence analyses of the TSTA-encoding genes cloned from these tumors showed that the genes were all point-mutated alleles of normal cellular genes encoding various cellular proteins, none involved in any obvious way in the transformation of these cells (Sidebar 41 . ). . These observations suggest that during the course of chemical carcinogenesis, the 3-MC carcinogen, a known point mutagen (Section 12.6), mutates both a proto-oncogene (often the K-ras gene) in target cells and additional genes that, as mutant alleles, specify TSTAs; the latter genes are struck at random-innocent bystanders tl1at play no causal role in tumorigenesis but happen to have been damaged by the large doses of mutagenic carcinogen used to provoke tumor formation. Importantly, the behavior of these chemically induced TSTAs is quite different from that of the ISlAs resulting from tumor virus infection. For example, SV40 virus can be used to induce a sarcoma in a mouse. Subsequent removal of this SV40-induced sarcoma will result in immunization of the mouse against subsequently inoculated tumor cells that derive from this particular SV40-induced sarcoma as well as a!l y other tumors that have been induced by SV40 virus. In this instance, there is indeed a cross-immunity established, in that all the SV40induced tumor cells seem to share a common TSTA or set of TSTAs. It happens that the dominant ISlA responsible for this cross-immunity is a familiar protein: it is the virus-encoded large T oncoprotein, which is expressed at significant levels in all SV40 virus-transformed cells. This contrasts with the behavior of a group of 3-MC-induced cancers, where each tumor expresses its own unique TSTA or set ofTSTAs. Observations like these raise the question whether similar mechanisms operate during human tumorigenesis. Thus, do the highly mutable genomes of cancer cells (Chapter 12) generate mutant, antigenic proteins as inadvertent by-products of the mutagenesis that drives tumor progression (see Sidebar 15. I)? Or are the 3-MC-induced TSTAs artifacts of the high doses of mutagenic carcinogens used in many mouse tumorigenesis experiments that do not accurately reflect the mutagenic processes that create human tumors? 15.13 Tumor-associated transplantation antigens may also evoke anti-tumor immunity As noted above, tumor-associated transplantation antigens (TATAs) represent normal cellular proteins that, for one reason or another, have failed to induce tolerance. When these normal proteins are expressed by tumors they evoke a measurable immune response, often involving both the humoral and cellular arms of the immune system. For a variety of reasons, the antigenicity of melanomas has been more intensively studied than that of all other human tumors (Sidebar 42 . ). Much of their antigenicity stems from their display of certain TATAs. Melanoma cells may Sidebar 15.1 Microsatellite instability often leads to more immunogenic tumors As described in Section 12.4, defects in the DNA mismatch repair machinery create the condition of microsatellite instability (MiN), which leads to mutations accumulating in hundreds, possibly thousands of cellular genes within tumor cell genomes. Among other consequences, these mutations generate shifts in the reading frames of many of these genes. The resulting mutant alleles often encode novel amino acid sequences, sometimes termed "frameshift pep tides," some of which may function as potent tumor-specific antigens. This logic predicts that the 15% of human colorectal cancers that exhibit microsateJlite instability should interact with the host immune system differently from the majority of colorectal cancers that show no MIN and instead exhibit chromosomal instability (CIN). In fact, the MIN tumors show a markedly higher degree of tumor-infiltrating lymphocytes (TILs) and a lower degree of metastasis. Moreover, antigen presentation by their MHC class I proteins . is compromised Jar more frequently than in colorectal tumors with chromosomal instability (60% vs. 30%), suggesting that the MIN tumors are under greater pressure to evade killing by various arms of the immune system, and that they undertake the immunoevasive maneuver of blocking antigen presentation by their cell-surface MHC class I proteins. Taken together, these observations suggest that the MIN that enables some colorectal tumors to evolve more rapidly can exact a price in the markedly higher immunogenicity of these tumors. 687 Chapter 15: Crowd Control: Tumor Immunology and Immunotherapy Figure 15.24 Normal proteins displayed as tumor-associated antigens on melanoma cells (A) The tyrosinase enzyme, which is involved in pigment production in melanocytes and melanomas, is detected here in the melanocytes of the skin (red, located Just above the basement membrane in the skin) through the use of a monoclonal antibody; it is not detectable in other normal tissues. Its expression by melanoma cells can cause them to become immunogenic and the target of ki lli ng by cytotoxic lymphocytes. (B) The spermatogonia in the testis have been stai ned here with a monoclonal antibody agai nst the MAGE-l antigen; normall y this antigen is seen onl y in the placenta. Its expression has been detected in a variety of human tumor types and has been studied in detail in melanomas because it is often immunogenic when expressed by these tumors. (A, from VT. Chen, E. Stockert, S. Tsang et al., Proc. Nat!. Acad. Sci. USA 928125-8129,1995; B, from JL Cheville and Pc. Roche, Mod. Pathol. 12974-978, 1999 ) overexpress certain proteins that are present in their normal melanocyte precursors, albeit at lower levels. Such lineage-specific proteins are sometimes cal.Ied differentiation antigens, implying that their display is a vestige of the differentiation program that previously governed the behavior of the normal cellular precursors of tumor cells. Included among the melanoma TATAs are transferrin, tyrosinase (Figure 15.24A), gpl00, Melan-A/MART-l, and gp75. The display of these differentiation antigens by melanoma cells often provokes a vigorous response by the immune system, which results in a very peculiar form of autoimmune disease-vitiligo-the depigmentation of large areas of skin seen in some melanoma patients (Figure 15.25) . This depigmentation is a specific response to the presence of a melanoma. For example, when 104 renal carcinoma patients were treated with the cytokine interleukin-2 OL-2) in order to enhance their anti-tumor immune responses, none developed vitiligo; in contrast, of 74 melanoma patients who were treated Similarly, 11 developed vitiligo. In these melanoma patients, it is clear that the immune response provoked by the melanoma TATAs leads, as a by-product, to attack and destruction of normal melanocytes, which also express these antigens. This type of vitiligo is formally analogous to the paraneoplastic syndromes discussed earlier (Sidebar 400), in which the display by tumors of cellular proteins results in the destruction of normal tissues that also happen to express these proteins. Significantly, melanoma patients showing vitiligo usually survive for longer periods than those who don't-suggesting that their immune systems are effective in controlling the melanomas, at least for a period of time. (For example, in a large population of melanoma patients described in 1987, 75% were still alive five years after initial diagnosis; among the subgroup of these patients who exhibited concomitant vitiligo, 86% survived for this period of time.) The antigenicity of human melanoma cells may also derive from their display of the other major subclass of TATAs, the oncofetal antigens-literaHy those antigens that are displayed during embryogenesis and once again by tumors. Included among these are the antigens called either cancer germ-line or cancertestis (eT) antigens, to reflect their normal e>-''}Jression in the germ cells of the testis and the fetal ovary. The genes for a number of these antigens, such as (A) tyrosinase antigen (8) MAGE-1 antigen r--.............. skin melanocytes testes spermatagonis 688 Tumor-associated transplantation antigens Figure 15.25 Autoimmune depigmentation provoked by melanomas The melanoma patient shown here, who was dark-skinned prior to the onset of melanoma, has lost almost all of his skin pigment except for several isolated areas (face, armpit) due to the autoimmune attack inci ted by the melanoma cell s. The condition of pigment loss, known as vitiligo, is often correlated with a longer survival of melanoma patients. (Courtesy of A.N. Houghton.) MAGE-1 (Figure 15.24B), MAGE-3, BAGE, GAGE-I, and GAGE-2, have been cloned (Sidebar 43 0 ). By 2003,44 genes or gene families encoding a total of 89 distinct cancer-testis antigens had been identified. As an aside, it seems that the absence of immune responses against these antigens in males is likely due to the fact that several of the cell types in the testes do not express MHC class I molecules and are thereby prevented from presenting their internal contents to the immune system. (Of course, females may never express these