Cancer and organ transplantation are two situations in which the immune response to human cells that are genetically distinct from the normal self has important clinical consequences. In order for cancers to grow, they have to evade host immunity, and effective methods of enhancing patients’ immune responses against tumors, called cancer immunotherapy, have transformed clinical oncology. In organ transplantation, the situation is the reverse: immune responses against grafted tissues from other people are a major barrier to successful transplantation, and suppressing these responses is a central focus of transplantation medicine. Because of the importance of the immune system in host responses to tumors and transplants, tumor immunology and transplantation immunology have become subspecialties in which researchers and clinicians come together to address both fundamental and clinical questions.
Immune responses against tumors and transplants share several characteristics. These are situations in which the immune system is not responding to microbes, as it usually does, but to noninfectious cells that are perceived as foreign. The antigens that mark tumors and transplants as foreign may be expressed in virtually any cell type that is the target of malignant transformation or is grafted from one individual to another. Therefore, immune responses against tumors and transplants may be directed against diverse cell types. Also, the immune system uses the same major mechanism, the activation of cytotoxic T lymphocytes (CTLs), to kill both tumor cells and the cells of tissue transplants.
In this chapter we focus on the following questions:
What are the antigens in tumors and tissue transplants that are recognized as foreign by the immune system?
How does the immune system recognize and react to tumors and transplants?
How can immune responses to tumors and grafts be manipulated to enhance tumor rejection and inhibit graft rejection?
We discuss tumor immunity first and then transplantation, and we point out the principles common to both.
Immune Responses Against Tumors
For over a century scientists have proposed that a physiologic function of the adaptive immune system is to prevent the outgrowth of transformed cells and to destroy these cells before they become harmful tumors. Control and elimination of malignant cells by the immune system is called tumor immune surveillance . Several lines of evidence support the idea that immune surveillance against tumors is important for preventing tumor growth ( Fig. 10.1 ). However, the fact that common malignant tumors develop in immunocompetent individuals indicates that tumor immunity is often incapable of preventing tumor growth or is easily overwhelmed by rapidly growing tumors. Furthermore, biologists now consider the ability to evade immune destruction as a fundamental feature (“hallmark”) of cancers. This has led to the growing realization that the immune response to tumors is often dominated by tolerance or regulation, not by effective immunity. The field of tumor immunology has focused on defining the types of tumor antigens against which the immune system reacts, understanding the nature of the immune responses to tumors and mechanisms by which tumors evade them, and developing strategies for maximally enhancing antitumor immunity.
Malignant tumors express various types of molecules that may be recognized by the immune system as foreign antigens ( Fig. 10.2 ). Protein antigens that elicit CTL responses are the most relevant for protective antitumor immunity. These tumor antigens have to be present in the cytosol of tumor cells in order to be recognized by CD8 + CTLs. The tumor antigens that elicit immune responses can be classified into several groups:
Neoantigens encoded by randomly mutated genes . Recent sequencing of tumor genomes has revealed that common human tumors harbor a large number of mutations in diverse genes, reflecting the genetic instability of malignant cells. These mutations usually play no role in tumorigenesis and are called passenger mutations. Many of these mutations result in expression of mutated proteins, called neoantigens because they are newly expressed in the tumor cells but not in the normal cells of origin of the tumor. Because T cells only recognize peptides bound to major histocompatibility complex (MHC) molecules, mutated tumor proteins can be recognized only if peptides carrying the mutated amino acid sequences can bind to the patients’ MHC alleles. Tumor neoantigens may not induce tolerance because they are not present in normal cells, and are the most common targets of tumor-specific adaptive immune responses. In fact, the number of these mutations in human cancers correlates with the strength of the antitumor immune responses patients mount and the effectiveness of immunotherapies that enhance those responses. In experimental tumors induced by chemical carcinogens or radiation, the tumor antigens are also mainly random mutants of normal cellular proteins.
Products of oncogenes or mutated tumor suppressor genes . Some tumor antigens are products of mutations, called driver mutations, in genes that are involved in the process of malignant transformation. The driver mutations that encode tumor antigens may be amino acid substitutions, deletions, or new sequences generated by gene translocations, all of which can be seen as foreign.
Aberrantly or overexpressed expressed structurally normal proteins . In several human tumors, antigens that elicit immune responses are normal (unmutated) proteins whose expression is dysregulated in the tumors, sometimes as a consequence of epigenetic changes such as demethylation of the promoters in genes encoding these proteins, and sometimes by gene amplification. These structurally normal self antigens would not be expected to elicit immune responses, but their aberrant expression may be enough to make them immunogenic. For example, self proteins that are expressed only in embryonic tissues may not induce tolerance in adults, and the same proteins expressed in tumors may be recognized as foreign by the immune system.
Viral antigens . In tumors caused by oncogenic viruses, the tumor antigens may be products of the viruses.
Immune Mechanisms of Tumor Rejection
The principal immune mechanism of tumor eradication is killing of tumor cells by CTLs specific for tumor antigens . The majority of tumor neoantigens that elicit immune responses in tumor-bearing individuals are endogenously synthesized cytosolic or nuclear proteins that are processed by proteasomes and displayed as class I MHC–associated peptides. Therefore, these antigens are recognized by class I MHC–restricted CD8 + CTLs, whose function is to kill cells producing the antigens. The role of CTLs in tumor rejection has been established in animal models: tumors can be destroyed by transferring tumor-reactive CD8 + T cells into the tumor-bearing animals. Studies of many human tumors indicate that abundant CTL infiltration predicts a more favorable clinical course compared with tumors with sparse CTLs.
CTL responses against tumors are initiated by recognition of tumor antigens on host antigen-presenting cells (APCs). The APCs ingest tumor cells or their antigens and present the antigens to naive CD8 + T cells in draining lymph nodes ( Fig. 10.3 ). Tumors may arise from virtually any nucleated cell type in any tissue, and, like all nucleated cells, they usually express class I MHC molecules, but often they do not express costimulators or class II MHC molecules. We know, however, that the activation of naive CD8 + T cells to proliferate and differentiate into active CTLs requires recognition of antigen (class I MHC–associated peptide) on dendritic cells in secondary lymphoid organs and also costimulation and/or help from class II MHC–restricted CD4 + T cells (see Chapter 5 ). How, then, can tumors of different cell types stimulate CTL responses? The likely answer is that tumor cells or their proteins are ingested by the host’s dendritic cells, transported to lymph nodes draining the site of the tumor, and the protein antigens of the tumor cells are processed and displayed by class I MHC molecules on the host dendritic cells. This process, called cross-presentation or cross-priming, was introduced in Chapter 3 (see Fig. 3.16) . Dendritic cells can also present peptides derived from ingested tumor antigens on class II MHC molecules. Thus, tumor antigens may be recognized by CD8 + T cells and by CD4 + T cells.
At the same time that dendritic cells are presenting tumor antigens, they may express costimulators that provide signals for the activation of the T cells. It is not known how tumors induce the expression of costimulators on APCs because, as discussed in Chapter 5 , the physiologic stimuli for the induction of costimulators are usually microbes, and tumors are generally sterile. A likely possibility is that tumor cells die if their growth outstrips their blood and nutrient supply, and adjacent normal tissue cells may be injured and die due to the invasive tumor. These dying cells release products (damage-associated molecular patterns; see Chapter 2 ) that stimulate innate responses. The activation of APCs to express costimulators is part of these responses.
Once naive CD8 + T cells have differentiated into effector CTLs, they are able to migrate back to any site where the tumor is growing, and kill tumor cells expressing the relevant antigens without a requirement for costimulation or T cell help.
Immune mechanisms in addition to CTLs may play a role in tumor rejection. Antitumor CD4 + T cell responses have been detected in patients, and increased numbers of CD4 + effector T cells, especially Th1 cells, in tumor infiltrates are associated with good prognosis. Antitumor antibodies are also detectable in some cancer patients, but whether these antibodies protect individuals against tumor growth has not been established. Experimental studies have shown that activated macrophages and natural killer (NK) cells are capable of killing tumor cells, and Th1 responses work largely by activating macrophages, but the protective role of these effector mechanisms in tumor-bearing patients is not clearly established.
Evasion of Immune Responses by Tumors
Immune responses often fail to check tumor growth because cancers evade immune recognition or resist immune effector mechanisms. The immune system faces daunting challenges in combating malignant tumors, because immune responses must kill all the tumor cells in order to be effective, and tumors can grow rapidly. Often, the growth of the tumor simply outstrips immune defenses. Not surprisingly, tumor cells that evade the host immune response are selected to survive and grow. Tumors use several mechanisms to avoid destruction by the immune system ( Fig. 10.4 ):
Some tumors stop expressing class I MHC molecules or molecules involved in antigen processing or MHC assembly, so they cannot display antigens to CD8 + T cells. Mutations affecting class I MHC–associated antigen presentation are likely more effective at immune evasion than loss of tumor neoantigens because any tumor may express many immunogenic antigens, all of which would have to be mutated or lost, whereas mutation in any component of antigen presentation will lead to failure to present all antigens.
Tumors engage pathways that inhibit T cell activation. For example, many tumors express PD-L1, a ligand for the T cell inhibitory receptor programmed cell death protein 1 (PD-1). Furthermore, tumors, being persistent, cause repeated stimulation of T cells specific for tumor antigens. The result is that the T cells develop an exhausted state, in which they express high levels of PD-1, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), and other inhibitory molecules, and become unresponsive to antigen.
Factors in the tumor microenvironment may impair the ability of dendritic cells to induce strong antitumor immune responses. For example, dendritic cells that capture tumor antigens often express only low levels of B7 costimulators, resulting in preferential engagement of the inhibitory receptor CTLA-4 on naive T cells in the draining lymph nodes, rather than the stimulatory receptor CD28 (see Chapter 9 ). Some tumors may induce regulatory T cells, which also suppress antitumor immune responses. Myeloid-derived suppressor cells, which are developmentally related to neutrophils and monocytes but have mainly antiinflammatory functions, are abundant in tumors, and are believed to contribute to immunosuppression.
Some tumors may secrete immunosuppressive cytokines, such as transforming growth factor β.
The main strategies for cancer immunotherapy currently in practice include introduction of antitumor antibodies and autologous T cells that recognize tumor antigens and enhancing patients’ own antitumor immune responses with antibodies that block immune checkpoints and vaccination . Until recently, most treatment protocols for disseminated cancers, which cannot be cured surgically, relied on chemotherapy and irradiation, both of which damage normal nontumor tissues and are associated with serious toxicities. Because the immune response is highly specific, it has long been hoped that tumor-specific immunity may be used to selectively eradicate tumors without injuring the patient. Only recently has the promise of cancer immunotherapy been realized in patients. The history of cancer immunotherapy illustrates how the initial, often empirical, approaches have been largely supplanted by rational strategies based on our improved understanding of immune responses ( Fig. 10.5 ).
Passive Immunotherapy With Monoclonal Antibodies
A strategy for tumor immunotherapy which has been in practice for a limited number of tumors for decades relies on the injection of monoclonal antibodies which target cancer cells for immune destruction or inhibition of growth ( Fig. 10.6A ). Monoclonal antibodies against various tumor antigens have been used in many cancers. The antibodies bind to antigens on the surface of the tumors (not the neoantigens produced inside cells) and activate host effector mechanisms, such as phagocytes, NK cells, or the complement system, that destroy the tumor cells. For example, an antibody specific for CD20, which is expressed on B cells, is used to treat B cell tumors, usually in combination with chemotherapy. Although normal B cells are also depleted, their function can be replaced by administration of pooled immunoglobulin from normal donors. Because CD20 is not expressed by hematopoietic stem cells, normal B cells are replenished after the antibody treatment is stopped. Other monoclonal antibodies that are used in cancer therapy may work by blocking growth factor signaling (e.g., anti-Her2/Neu for breast cancer and anti–EGF-receptor antibody for various tumors) or by inhibiting angiogenesis (e.g., antibody against the vascular endothelial growth factor for colon cancer and other tumors).
Adoptive T Cell Therapy
Tumor immunologists have attempted to enhance antitumor immunity by removing T cells from cancer patients, activating the cells ex vivo so there are more of them and they are more potent effector cells, and transferring the cells back into the patient. Many variations of this approach, called adoptive T cell therapy, have been tried.
Adoptive therapy with autologous tumor-specific T cells. T cells specific for tumor antigens can be detected in the circulation and among tumor-infiltrating lymphocytes of cancer patients. T cells can be isolated from the blood or tumor biopsies of a patient, expanded by culture with growth factors, and injected back into the same patient (see Fig. 10-6A ). Presumably, this expanded T cell population contains activated tumor-specific CTLs, which migrate into the tumor and destroy it. This approach, which has been combined with administration of T cell-stimulating cytokines such as interleukin-2 (IL-2) and traditional chemotherapy, has shown inconsistent results among different patients and tumors. One likely reason is that the frequency of tumor-specific T cells is too low to be effective in these lymphocyte populations.
Chimeric antigen receptor (CAR) expressing T cells. In a more recent modification of adoptive T cell ther apy, blood T cells from cancer patients are transduced with viral vectors that express a chimeric antigen receptor (CAR), which recognizes a tumor antigen and provides potent signals to activate the T cells (see Fig. 10-6B ). The CARs currently in use have a single chain antibody-like extracellular portion with both heavy- and light-chain variable domains, which together form the binding site for a tumor antigen ( Fig. 10-7 ). The specificity of the endogenous T cell receptors (TCRs) of the transduced T cells is irrelevant to the effectiveness of this approach. The use of this antibody-based antigen recognition structure avoids the limitations of MHC restriction of TCRs and permits the use of the same CAR in many different patients, regardless of the human leukocyte antigen (HLA) alleles they express. Furthermore, tumors cannot evade CAR-T cells by downregulating MHC expression. In order to work in T cells, the CARs have intracellular signaling domains of both TCR complex proteins, for example the ITAMs of the TCR complex ζ protein, and the signaling domains of costimulatory receptors such as CD28 and CD137. Therefore, upon antigen binding, these receptors provide both antigen recognition (via the extracellular immunoglobulin [Ig] domain) and activating signals (via the introduced cytoplasmic domains). CAR-expressing T cells are expanded ex vivo and transferred back into the patient, where they recognize the antigen on the tumor cells and become activated to kill the cells. CAR-T cell therapy targeting the B cell protein CD19, and more recently CD20, has shown remarkable efficacy in treating and even curing B cell-derived leukemias and lymphomas that are refractory to other therapies. CARs with other specificities for different tumors are in development and clinical trials. The most serious toxicity associated with CAR-T cell therapy is a cytokine release syndrome, mediated by massive amounts of inflammatory cytokines, including IL-6, interferon-γ, and others, that are released because all of the injected T cells recognize and are activated by the patients’ tumor cells. These cytokines cause high fever, hypotension, tissue edema, neurologic derangements, and multi-organ failure. The severity of the syndrome can be mitigated by treatment with anticytokine antibodies. CAR-T cell therapy may also be complicated by on-target, off-tumor toxicities, if the CAR-T cells are specific for an antigen present on normal cells as well as tumors. In the case of CD19- or CD20-specific CARs, the therapy results in depletion of normal B cells, requiring antibody replacement therapy to prevent immunodeficiency. Such replacement may not be feasible for other tissues that are destroyed because of the reactivity of the CAR. Although CAR-T cell therapy is effective against leukemias and tumors in the blood (to which the injected T cells have ready access), it has so far not been successful in solid tumors because of difficulties in getting T cells into the tumor sites and the challenge of selecting optimal tumor antigens to target without injuring normal tissues.
Immune Checkpoint Blockade
Blocking inhibitory receptors on T cells or their ligands stimulates antitumor immune responses . The realization that tumors evade immune attack by engaging regulatory mechanisms that suppress immune responses has led to a novel and remarkably effective new strategy for tumor immunotherapy. The principle of this strategy is to boost host immune responses against tumors by blocking normal inhibitory signals for T cells, thus removing the brakes (checkpoints) on the immune response ( Fig. 10.8 ). This has been accomplished with blocking monoclonal antibodies specific for the T cell inhibitory molecules CTLA-4 and PD-1, first approved for treating metastatic melanoma in 2011 and 2014, respectively. Since then, the use of anti-PD-1 or anti-PD-L1 antibodies has expanded to many different cancer types. The most remarkable feature of these therapies is that they have dramatically improved the chances of survival of patients with advanced, widely metastatic tumors, which previously were almost 100% lethal within months to a few years. The efficacy of antibodies specific for other T cell inhibitory molecules, such as LAG-3 and TIM-3, are being tested in clinical trials. There are several novel features of immune checkpoint blockade and limitations that still need to be overcome to enhance their usefulness.
Although the efficacy of checkpoint blockade therapies for many advanced tumors is superior to any previous form of therapy, only a subset of patients (25% to 40% at most) respond to this treatment. The reasons for this poor response are not well understood. Nonresponding tumors may induce T cell expression of checkpoint molecules other than the ones being targeted therapeutically, or they may rely on evasion mechanisms other than engaging these inhibitory receptors. Oncologists and immunologists are currently investigating which biomarkers will predict responsiveness to different checkpoint blockade approaches.
One of the most reliable indictors that a tumor will respond to checkpoint blockade therapy is if it carries a high number of mutations, which correlates with high numbers of neoantigens and host T cells that can respond to those antigens. In fact, tumors that have deficiencies in mismatch repair enzymes, which normally correct errors in DNA replication that lead to point mutations, have the highest mutation burdens of all cancers, and these cancers are the most likely to respond to checkpoint blockade therapy. Remarkably, anti-PD-1 therapy is now approved for any recurrent or metastatic tumor with mismatch repair deficiencies, regardless of the cell of origin or histologic type of tumor. This is a paradigm shift in how cancer treatments are chosen.
The combined use of different checkpoint inhibitors, or one inhibitor with other modes of therapy, will likely be necessary to achieve higher rates of therapeutic success. The first approved example of this is the combined use of anti-CTLA-4 and anti-PD-1 to treat melanomas, which was shown to be more effective than anti-CTL-4 alone. This reflects the fact that the mechanisms by which CTLA-4 and PD-1 inhibit T cell activation are different (see Fig. 10.8 ). There are numerous ongoing or planned clinical trials using combinations of checkpoint blockade together with other strategies, such as small molecule kinase inhibitors, oncolytic viral infection of tumors, and other immune stimulants.
The most common toxicities associated with checkpoint blockade are autoimmune damage to organs. This is predictable, because the physiologic function of the inhibitory receptors targeted is to maintain tolerance to self antigens (see Chapter 9 ). A wide range of organs may be affected, including colon, lungs, endocrine organs, heart, and skin, each requiring different clinical interventions, sometimes including cessation of the life-saving tumor immunotherapy.