Tumor Immunology and Immunotherapy

T Lymphocytes and Natural Killer Cells

Bone marrow–derived progenitor cells enter the thymus from which T cells eventually emerge. In the thymus, an enormous repertoire of TCRs is randomly generated by recombinations and mutations in their α and β chains. Progenitors with TCRs of high affinity for self-antigens undergo deletion (negative selection). Some of those with low affinity for self-antigens survive and are positively selected so that a significant percentage of self-reactive T cells emerge from the thymus. Only a very small percentage of the cells entering and proliferating within the thymus survive this education process. Several types of T cells emerge into the periphery. CD8+ T cells recognize antigen in the context of MHC class I molecules, express αβ TCRs, are commonly referred to as cytotoxic T cells, and produce a number of cytokines. CD4+ T cells recognize antigen in the context of MHC class II molecules. There are several subsets of CD4+ T cells ( Fig. 30.1 ). Among the better recognized are Th1 cells (helper type 1 T cells) that secrete IL-2, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) and Th2 cells that produce IL-4, IL-5, IL-6, IL-10, and IL-13. Th1 cells promote cytotoxicity and inflammation and combat intracellular pathogens, whereas Th2 cells assist in the stimulation of B cells for antibody production, allergic responses, and addressing extracellular pathogens. T helper cells will favor Th1 (cell-mediated) or Th2 (humoral) immune responses. A subset of CD4 regulatory cells (T regulatory [Treg] cells) plays a critical role in dampening autoimmunity. These Treg cells constitute 5% to 10% of CD4+ cells, express the transcription factor Foxp3, and dominantly suppress autoimmune responses; mutation of the Foxp3 gene in humans and mice leads to multiorgan autoimmune disease. Another T-cell subtype is the so-called Th17 cell, which preferentially produces IL-17, IL-21, and IL-22 and is important in the pathogenesis of autoimmune diseases.

CD4+ T cells also play an important role in the initiation and maintenance of CD8+ T-cell responses. They may do this through a variety of mechanisms. Activated CD4+ T cells can interact with dendritic cells (DCs), which are professional antigen-presenting cells, through an interaction between the CD40 receptor and its ligand CD40L. This activation or “licensing” of DCs allows these antigen-presenting cells to promote the differentiation of CD8+ T cells and to establish a durable memory T-cell response. CD4+ T cells also produce IL-2 and IFN-γ, which could potentially support CD8 function. Thus, CD4+ T cells are important in shaping a productive antitumor response.

Another T-cell subset (γδ) represents a minor population (1%–10%) of CD3+ T cells that is even more enriched in mucosal epithelium and expresses TCRs that recognize bacterial and viral antigens. Natural killer T (NKT) cells express phenotypic markers of T and NK cells and express a specific family of TCRs that recognize glycolipid antigens presented by CD1d molecules. These NKT cells are thought to help initiate T-cell responses through the production of large amounts of the cytokines IFN-γ and IL-4.

Mature T cells have a broad repertoire of αβ TCRs with diverse antigen specificity. This diversity is generated during T-cell differentiation by a process of gene rearrangement of variable (V), joining (J), and diversity (D) gene segments. TCRs are composed of α and β chains; it is estimated that recombination events could potentially yield a repertoire exceeding 10 ¹² unique TCRs. These TCRs recognize antigen in the context of MHC proteins found on the surface of cells for MHC class I, proteins within the cell cytoplasm are digested in the proteasome complex into short peptide fragments (8–12 amino acid residues), which are transported to the cell surface bound in the groove of MHC class I molecules; the specific peptide sequence presented is determined by the MHC (in humans, also called human leukocyte antigen [HLA]) allele. These class I–presented peptides are usually recognized by CD8+ T cells. MHC class II extracellular antigens are internalized by antigen-presenting cells into endosomes, where they are degraded into small peptides and mounted on MHC class II molecules for display on the cell surface and typically recognized by CD4 cells. These two pathways provide the immune system with continuous surveillance for intracellular pathogens, such as viruses, and foreign cells as well as noxious proteins and pathogens in extracellular milieu. The activation of resting T cells requires engagement of the correct MHC-peptide complex by the TCR (so-called signal 1) and additional costimulatory signals (signal 2). Professional antigen-presenting cells (DCs) provide either CD80 or CD86 (B7 family genes), which engages the CD28 receptor on the T cell, a requirement for T-cell activation. T cells then upregulate another receptor, CTLA-4, which also binds B7 but with a higher affinity than CD28. Engagement of CTLA-4 induces an inhibitory signal that downregulates T-cell activation. This is a natural immunomodulatory mechanism for dampening immune responses. Monoclonal antibodies that bind to CTLA-4 can block this interaction and inhibit the negative regulatory signaling ( Fig. 30.2 ). Studies in human subjects have demonstrated that CTLA-4 blockade can break peripheral tolerance to self-antigens and induce both antitumor and anti-“self” (autoimmune) responses.

Another important pathway is the PD-1/PD-L1 axis in which the inhibitory ligand (PD-L1), commonly expressed in many cancers, can engage T-cell PD-1, thereby abrogating lymphocyte activation ( Fig. 30.2 ). Interruption of this negative signaling benefits a significant proportion of patients with several solid tumors.

Although much emphasis in antitumor immunity has been focused on adaptive responses (T lymphocytes and antibodies), effector cells of the innate immune system, specifically NK cells, can act alone or in concert with adaptive immunity. ^, NK cells can recognize and kill target cells without prior sensitization. These cells express activating and inhibitory cell surface receptors and, when their activating receptors are engaged without concomitant ligation of their inhibitory receptors, can kill targets directly. NK cells have been traditionally viewed as providing a first line of defense against virally infected cells. NK cells can also interact with the adaptive immune system. They can modulate the function of professional antigen-presenting cells (e.g., DCs), promote the generation of Th1 responses, and potentially dampen autoimmune immunopathologic changes. Because their inhibitory receptors engage MHC molecules, NK cells specifically recognize cells that have lost MHC class I molecules, which can occur during viral infections or malignant transformation. NK cells are strongly activated by exogenous cytokines such as IL-2 and have been termed “lymphokine-activated killer (LAK) cells.” LAK cells have greatly enhanced cytotoxicity for a much broader range of target cells.

The cytotoxic T cell (CTL) response is initiated by engagement of their TCR with professional antigen-presenting cells (e.g., DCs) that have processed and presented cognate antigen in the context of MHC class I. This activation event enables resting CD8 T cells to proliferate and differentiate into effector CTL with significant alterations in migratory behavior and function. These CTLs traffic to tumor sites, and their TCR engage antigen peptide presented on the surface of tumor cells and are able to deliver death signals. CTLs are able to kill multiple tumor cells, termed “serial killing”: The mechanisms of killing include ligation of so-called “death receptors” (FasL/CDaSL, Apo2L/TRAIL) or cytolytic granule (perforin, granulysin) exocytosis. ^, They also elaborate proinflammatory cytokines, which may, through various mechanisms, promote antitumor activity through recruitment of the other cytotoxic effectors such as NK cells.

Antigen-Presenting Cells

DCs are professional antigen-presenting cells whose role is to take up, process, and present antigen to the immune system ; they are essential during the initial activation of resting T cells. There are different subtypes of DCs with specialized functions that depend on their anatomic location. DCs are found in lymphoid tissues, in the skin, and on the mucosal surfaces of many organs. DCs in the gastrointestinal tract can sample bacteria in the intestinal lumen and initiate secretory immunoglobulin A (IgA) responses. In the lung, DCs help maintain tolerance to inhaled allergens. In the peripheral blood, DC precursors can migrate to sites of inflammation and initiate immune responses. DC function is powerfully modulated by a variety of receptors including Toll-like receptors (TLRs) and surface C-type lectin receptors. DCs at different stages of differentiation vary in their ability to migrate, to take up antigen by phagocytosis, and to effectively stimulate T cells. Immature DCs patrol their environment, sampling by pinocytosis and receptor-mediated endocytosis. Extracellular antigens are taken up into endosomes, which fuse with protease-containing lysosomes, and within these compartments antigens are cleaved into peptides that can bind to MHC class II molecules and be delivered to the cell surface. Proteins in the cytoplasmic compartment of antigen-presenting cells are degraded by the proteosome and actively transported into the endoplasmic reticulum, where they are loaded onto MHC class I molecules and delivered to the cell surface. Some exogenous or environmental antigens can also find their way into the MHC class I antigen-presentation pathway; this is termed “cross-presentation” and is an important mechanism for generating CD8+ class I–restricted T-cell responses. DCs can acquire antigen in the periphery and travel to lymph nodes, where they interact with T cells and present antigen. DCs originate from pluripotent stem cells from bone marrow, enter the blood, and localize to almost all tissues and lymphoid organs. Myeloid DCs (these include DCs found in deep epithelial tissues and Langerhans cells present in the epidermis) and plasmacytoid DCs are a major source of type I IFN.

DCs have cell surface receptors termed “pattern recognition receptors” that screen the environment for pathogens. The TLR family is the best characterized; these can recognize bacterial products (e.g., lipopolysaccharide, flagellin), viral products such as double-stranded RNA, and specific cytosine-guanine (CpG)–rich DNA motifs, more common in microbial genomes. These signals, along with various proinflammatory cytokines, can deliver a danger signal to DCs that establishes the context within which they see and present antigens. TLR signaling drives immature DCs into a more mature phenotype with much higher expression of MHC, costimulatory molecules, and DC-derived cytokines (such as IL-12). Immature DCs are migratory and highly efficient in antigen capture, whereas mature DCs are less mobile but more efficient in processing and presenting antigen in an immunostimulatory context.

Distinct sets of molecules govern migration of DCs to and from the periphery and to lymph nodes. Prominent among these signals are a variety of chemokines and their receptors (e.g., CCR7, CCL19, CCL21). Signals that induce maturation of immature DCs include CD40 ligand delivered by T cells as well as signals by NK cells, a variety of proinflammatory cytokines (e.g., IL-1, TNF, IL-6), and engagement of TLR and C-type lectins. The context of antigen presentation and the maturational phenotype of DCs will determine and shape the type of T-cell response. Immature DCs have the potential to be tolerogenic, perhaps because they present antigen without an appropriate costimulatory second signal. Activated mature DCs have greater potency in activating and expanding antigen-reactive T cells. This is an oversimplified overview of the complex central role of various DC subsets that orchestrate adaptive and innate antitumor responses.

Antibody

Cell surface and circulating antigens can be recognized by immunoglobulins (antibody molecules). Immunoglobulins serve as membrane-associated receptors on the surface of B cells, which can then be secreted as soluble molecules as these cells differentiate into plasma cells. There are five distinct classes of immunoglobulin molecules: IgG, IgA, IgM, IgD, and IgE. There are several isotypes of IgG and IgA. The basic structure of antibody molecules includes two identical light and two identical heavy polypeptide chains linked by interchain disulfide bridges. Variable regions within the heavy and light chains create a so-called hypervariable region responsible for antigen binding. Antibody binding to antigen is reversible and of variable avidity. The C-terminal portion of certain antibody classes can bind to Fc receptors, which are expressed among a range of mononuclear cells. Antibody binding to antigen and engagement of these effector cells can trigger phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC).

The complement system is composed of a series of plasma proteins, many of which exist as proenzymes that require cleavage for activation. Surface-bound IgG and IgM antibodies can activate complement through the so-called classical pathway, a byproduct of which is the assembly of complement proteins that effect transmembrane pore formation in target cells. Complement byproducts can also promote chemotaxis of mononuclear cells that release cytokines. Thus, complement activation not only can kill targets but can also label them as pathogens for elimination. The alternative pathway allows complement activation without antibody.

Tumor Antigens

A molecular understanding of tumor recognition has been achieved only recently. The first molecularly defined antigen recognized by a tumor-reactive T cell was only discovered in 1991. This advance first required elucidation of the biology of antigen processing and presentation and its interaction with MHC molecules, which occurred in the late 1980s. These discoveries demonstrated that any intracytoplasmic protein was a candidate to be degraded by the proteasome complex, bound to MHC class I molecules, and displayed on the cell surface for T-cell recognition, which was crucial to our early understanding of tumor antigens. Mature T cells express the CD8 or CD4 coreceptor, which binds to invariant portions on all class I or class II MHC molecules, respectively. This additional ligation increases the affinity of the T-cell interaction with the antigen-presenting cell. Therefore, T cells expressing CD4 typically recognize antigens presented by MHC class II molecules, and CD8+ T cells usually recognize class I–presented antigens.

Cancer cells can overexpress or abnormally express a variety of normal cellular proteins. Because the human T-cell repertoire can sometimes recognize self-proteins, some of these self-proteins could potentially serve as targets for immune-based therapies. As discussed in detail later, gene products resulting from tumor-specific mutations are much better T-cell targets precisely because they are “nonself” xenoantigens never before experienced during thymic selection. One characteristic of an ideal cancer antigen is its immunogenicity, or its ability to elicit a T cell or antibody response. A gene product associated with the neoplastic process (e.g., a growth factor receptor) with a high degree of specific expression by malignant cells may prove to be an excellent target because it cannot be deleted or downregulated by the tumor cells under selective immune pressure (so-called antigen-loss variants). General classifications of known tumor-associated antigens include:

• Lineage-specific tumor antigens associated with tissue differentiation or function, such as the melanocyte-melanoma lineage antigens MART-1/Melan-A (MART-1), gp100 protein, mda-7 protein, tyrosinase and tyrosinase-related protein (TRP-1 and TRP-2), the prostate antigens (prostate-specific membrane antigen and prostate-specific antigen), and carcinoembryonic antigen

• A class of proteins expressed during ontogeny and in adult germline tissues and tumors (cancer-testis or cancer-germline antigens)

• Epitopes derived from genes specifically mutated in tumor cells (so called neoantigens)

• Epitopes derived from oncoviral processes, such as human papillomavirus oncoproteins E6 and E7 or Epstein-Barr virus–derived proteins; and

• Nonmutated proteins with tumor-selective expression contributing to the malignant phenotype, including HER2/neu and hTERT.

A cytotoxic response to any nonmutated self-protein has a risk of causing autoimmune toxicity. Directing immune responses to tumor-specific mutated antigens avoids this risk, but the patient-specific nature of such mutations hampers the generation of reagents for use in multiple patients.

Immunosuppressive Tumor Microenvironment

There is abundant evidence that cancer cells have acquired an array of defense mechanisms to thwart their destruction by the immune system. These are summarized in Box 30.1 . Most human cancers present peptide epitopes in the context of MHC molecules that can be recognized by antigen-reactive T cells, but tumor cells themselves do not present antigen in an immunostimulatory context. Human T cells require additional signaling through costimulatory molecules, such as CD80/86 (B7 family), for optimal T-cell activation and expansion. Without these other signals, T cells can become anergic. Tumor cells can also downregulate antigen expression by a variety of mechanisms, such as epigenetic silencing, loss of MHC expression, and loss of function of the intracellular machinery that processes and transports peptides to the cell surface.

The immune system also has complex and generally fine-tuned down regulatory signaling to modulate responses. Contraction of an acute immune response after 1 to 2 weeks may be appropriate for a viral infection but will be counterproductive to rejecting a large mass of malignant tissue. Autoimmune and allograft immune responses represent the types of chronic ongoing processes that would favor antitumor immune responses and underscore the need for a better understanding of the basic biology of immune regulation.

In addition to CTLA-4 signaling (see earlier), negative signaling can also be transduced through PD-1. DCs express the programmed death receptor ligand PD-L1 (or B7-H1); its expression by DCs can skew T cells toward an unresponsive phenotype. ^, DCs found within the tumor microenvironment have been shown to express high levels of PD-L1, which contribute to decreased local T-cell function. Some tumor cells themselves can present this inhibitory ligand, and expression of PD-L1, as with renal cancer, is associated with a poorer clinical outcome. Blockade of this PD-L1/PD-1 interaction using decoy receptors or antibodies is effective in improving immune therapies in animal models and when translated into human cancer immunotherapy trials has yielded impressive antitumor responses (see later).

Approximately 80% of human cancers do not respond to currently available modern immunotherapies. Since there is a positive correlation between tumor mutational burden and immune checkpoint agents, cancers with few mutations may not present an adequate density of neoAg to the immune system. Impairment of antigen processing or antigen-presenting machinery (e.g., mutation in β ₂ microglobulin) may also reduce neoAg display. Immunoediting and antigen loss, the progressive loss of more immunogenic cancer cell clones under the selective pressure of an evolving adaptive immune response, may yield more poorly immunogenic subclones that escape immune control.

Other immunity-avoidance mechanisms may be operative in the tumor microenvironment for cancers with an otherwise adequate threshold of neoAg density. The presence of immune suppressor cell populations within the tumor microenvironment Treg, myeloid-derived suppressor cells and M2-polarized tumor associates macrophages may dominantly suppress otherwise functional neoAg-reactive T cells.

Treg comprise a small subpopulation of CD4+ T cells (5% to 10%) that constitutively express the α chain of the IL-2 receptor CD25; most of these cells also express transcription factor Foxp3 (a forkhead-winged helix family member) and GITR (glucocorticoid-induced TNF receptor), as well as CTLA-4. These cells, Treg cells, produce immunosuppressive cytokines such as IL-10 and transforming growth factor-β (TGF-β) and can also inhibit through cell contact–dependent mechanisms. Mice or humans with a genetic mutation in Foxp3 lack Treg cells and develop a fulminant and lethal autoimmune disorder. Animal studies have clearly shown that Treg cells are responsible for suppressing the self-reactive T-cell repertoire, and the clinical manifestations from genetic loss of Foxp3 suggest that this may also be true in humans. Human Treg cells are enriched in tumor specimens and in draining lymph nodes of many solid tumors, and there is emerging evidence supporting a dominant role in suppressing self-reactive antitumor immune responses. Moderating Treg cell function could potentially favor antitumor immune responses. The use of lymphodepleting strategies before adoptive cell therapy (described later), which clearly enhances the antitumor biology of adoptively transferred T cells, may in part act through depletion of host resident Treg cells.

Myeloid-derived suppressor cells, which include granulocyte and immature myelomonocytic precursors, are expanded in settings of inflammation and cancer and elaborate immunosuppressive factors that include TGF-β, arginase 1, and inducible nitric oxide syntheses. Some strategies under current investigation seek to pharmacologically reduce their infiltration into and function within the tumor microenvironment.

Tumors themselves, and at times tumor stroma, can produce immunosuppressive substances; a prominent factor is TGF-β. TGF-β directly inhibits CTL activation, cytokine production, helper T-cell responses, and activation of DC and can promote the differentiation of Treg cells. Inhibition of TGF-β can have a salutary effect on antitumor immunity. T cells rendered insensitive to TGF-β signaling using a dominant-negative receptor have enhanced function in vivo. Neutralizing antibodies, small molecule inhibitors, and engineered T cells are currently under study in clinical trials. Vascular endothelial growth factor (VEGF) is important in angiogenesis but can also inhibit the function of DC. Thus, anti-VEGF therapy could also function through an immune mechanism. An isoform of the enzyme cyclooxygenase 2 is overexpressed in many tumors and catalyzes the synthesis of prostaglandin E2. Prostaglandin E2 has a generally adverse impact on the immune system, particularly on DC and T-cell function.

The enzymes indoleamine 2, 3-dioxgenase and arginase metabolize the essential amino acids L -trytophan and arginine, respectively. Their depletion impairs T-cell and DC function. Specific small molecule inhibitors of these enzymes are being studied preclinically and clinically.