Robert L. Ferris, MD, PhD
Faculty and Disclosures
CME Released: 03/26/2015
The immune system plays a key role in the development, establishment, and progression of cancer. A greater understanding of the dysregulation and evasion of the immune system in the evolution and progression of melanomas, lung, kidney, and head and neck cancers provide the basis for improved therapies and outcomes for patients. Cancer cells evade the host immune system through manipulation of their own immunogenicity, production of immunosuppressive mediators, and promotion of immunomodulatory cell types. Through the tumor`s influence on the microenvironment, the immune system can be exploited to promote metastasis, angiogenesis, and growth. This brief overview will address the concept of cancer immunosurveillance by the immune system, and immune escape by immune infiltrating cells in the tumor microenvironment. Current immunotherapeutic strategies for various cancers and emerging results from ongoing clinical trials are discussed.
Introduction
Cancer immunotherapy is based on the premise that tumors can be recognized as foreign matter rather than as the self, and can be effectively attacked by an activated immune system. We have reached a greater understanding of the immune system, its dysregulation, evasion, its ability to harbor tumors and support their growth and metastasis through various avenues of research. There has been a recent renaissance in the idea that nascent premalignant cells are destroyed by the immune system before tumor formation can occur (immune surveillance). Derangements in the immune system or alterations in the transformed cells may allow immune escape, which then enables the cancer to grow.
Tumor progression depends upon the acquisition of traits that allow cancer cells to evade immune surveillance and suppress an effective immune response. That cancer is an immunosuppressive disease is seen by the lower absolute lymphocyte count (ALC) compared with healthy subjects,[1] impaired natural killer (NK) cell activity,[2,3] and poor antigen-presenting function.[4,5] Impairment of tumor-infiltrating T lymphocytes has also been reported in various cancers.[6,7] In addition, suppressive regulatory T cells (Treg) have been linked to cancer tumor progression; Treg cells secrete suppressive cytokines such as transforming growth factor-β (TGF-β) and interleukin (IL)-10, express cytotoxic T-lymphocyte antigen 4 (CTLA-4), and correlate with tumor progression.[8] Immunomodulatory therapies that interfere with immune suppressive signals in cancer patients have shown therapeutic promise. The recent clinical efficacy of the US Food and Drug Administration (FDA)-approved monoclonal antibody (mAb) targeting immune checkpoint receptors, including anti-CTLA-4 and anti-programmed cell death protein 1 (PD-1), provide further promise for patient benefits as positive clinical data emerge.
Cancer Immunosurveillance and Immunoediting
The most telling evidence that "cancer immunosurveillance" is real as hypothesized by Burnet and Thomas[70] is from patients undergoing organ transplantation. These patients, who are immunosuppressed to avoid rejection, demonstrate increased risk of many tumors with no known viral etiology, such as lung, head and neck,[9] pancreatic, endocrine, colon cancer, and melanoma.[10] The discoveries that NK cells provide an approach for innate immune protection from tumor,[11] and that interferon (IFN)-γ and its proapoptotic effect on tumor growth gave additional support to the potential for immune clearance of cancer cells.[12,13] Cancer immunoediting suggests a dynamic evolutionary progress whereby immune surveillance of cancers provides selective pressure on tumor cells that can evade the immune system (negative selection).[14] Thus, successful tumor formation occurs only after the cancer has discovered a means by which it can evade the immune system.
Immune Evasion of Virus-Associated Cancer
A clinically relevant model for understanding immune escape is virus infection and immune evasion in virus-associated cancers. Viral interference using IFN and other signaling pathways is a critical component in avoiding adaptive and innate immune response. Interferons link the innate immune response to the adaptive immune response by activation of immature dendritic cells (DCs), CD8+ T cells, and virus-specific antibody production.[15,16] Interferon alpha (IFN-α) and IFN-β have immunostimulatory properties, are produced in response to virally infected cells, and execute their antiviral effects through inhibition of messenger RNA (mRNA), NK cell stimulation, and inhibition of viral protein expression.[15] Interferon-γ activates leukocyte migration, antigen presentation, and inflammation, and is primarily produced by effector cells. Therefore, antiviral immune response critically depends on inflammatory signaling, further supporting the importance of IFN and other inflammatory signaling pathways in permitting progression of virally associated cancers. Danger signals such as toll-like receptors (TLRs) present on inflammatory cells and are able to detect so-called pathogen-associated molecular patterns (PAMPs)[17] to stimulate these IFNs. Furthermore, viruses interfere with antigen presentation to reduce adaptive immune response, and suppress signal transducer and activator of transcription (STAT)1 signaling inhibition by IFN pathways, causing downregulation of human leukocyte antigen (HLA) class I antigen processing machinery (APM).[17,18]
Immune Escape and Immunosuppression in Cancers
In the normal process, cancer cells will produce oncogenic antigens, proteins that are "non-self." These antigens are immunogenic and initiate an immune response -- presentation on major histocompatibility complex (MHC) by antigen-presenting cells (APCs), recognition by T-cell receptors (TCRs), and induction of cytotoxic CD8+ T cells (Figure 2). In order for cancer cells to avoid detection by the immune system, they reduce their inherent immunogenicity (Table 1) and they actively suppress the antitumor immune response (Figure 1). A key component for the immune system's recognition of different or altered cell antigens is the MHC -- part of the human leucocyte antigen (HLA) complex. This MHC presents processed tumor antigenic peptides to T lymphocytes.[5] Tumor cells can reduce T-cell-mediated recognition by altering MHC class I expression. Using next-generation cancer sequencing, The Cancer Genome Atlas (TCGA) group[19] have reported mutations in specific MHC alleles, β2 microglobulin (β2m), and APM components. Changes in the chromosome[20] and defects in protein expression[4] of the HLA/APM-encoding genes themselves can cause selective loss of HLA and APM component expression in a substantial fraction of cancers, resulting in poor antigen presentation, and are correlated with poor prognosis.[21,22] Conversely, a complete loss of MHC expression may result in T-cell evasion, but now represents a strong trigger for NK cell activation. The absence of MHC removes a key inhibitory signal for NK cells, which, in contrast to the function of cytotoxic T cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The NK cells provide rapid responses to viral-infected cells and respond to tumor formation, acting at around 3 days after infection. These cells are unique, however, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction.[23] Therefore, tumor cells must employ multiple mechanisms to evade immune surveillance, at the same time avoiding a complete loss of MHC expression.
Table 1. Mechanisms of Immune Escape in Cancer
*Development of T-cell tolerance to persistent HPV infection, or overexpressed/mutated oncogenic antigens
*Increased PD-L1 expression on tumors and increased PD-1 expression in cytotoxic T lymphocytes
*Downregulation of interferon regulatory factors and activated STAT1
*Inhibition of inflammatory cytokines and transcription factors
*Downregulation or mutation of HLA class I and APM components
Figure 1. Tumor cell immune evasion and exploitation by cellular and soluble mediators in the tumor microenvironment.
Tumor cells secrete several small molecules and cytokines that depress NK, DC, and T-cell function (red downward arrows) and induce immunosuppressive myeloid-derived suppressor cells (MDSC) and regulatory T cells. MHC downregulation and defects in the antigen presentation machinery impairs T-cell recognition. Fas ligand is expressed, which kills T cells. Chemokine receptors aid in metastasis of the cancer cell to lymph nodes.
APM = antigen processing machinery; HLA = human leukocyte antigen; HPV = human papillomavirus; PD-1 = programmed cell death protein 1; PD-L1 = programmed death ligand 1; STAT = signal transducer and activator of transcription.
Although MHC expression is not absent in tumor cells, nevertheless they do not process and present these mutated proteins on the MHC class I molecule.[5] Thus, cancer cells that express MHC I and tumor antigen can still evade T-cell recognition through decreased expression or mutation of APM components, but still maintain nonspecific MHC I expression in order to avoid recognition by NK cells. In addition to altered expression of MHC, tumor cells express Fas ligand, which can interact with Fas and transduce a powerful apoptotic signal to activated T cells, allowing for immune evasion[24] by eliminating tumor-infiltrating T lymphocytes (TILs). In summary, the immunosuppressive effects of tumor cells include downregulation of MHC and APM components and beneficial STAT1 activation, leading to suppressive STAT3 signaling, cytokines, and ligands on cancer cells.
The Role of Immune Checkpoint Receptors
An important group of molecules that has emerged is the group of immune checkpoint receptors (ICR). As part of the immune system's strategy to control overreaction to inflammatory responses and to limit autoimmunity, several receptors have been identified as "brakes" on the immune system. These receptors are expressed on T cells and/or show inhibitory regulation upon stimulation. They include CTLA-4, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin protein-3 (TIM-3), and PD-1. Each of these receptors has corresponding ligands on the APC. Binding of these receptors to their respective ligands PD-L1 (B7-H1, CD274) induces a loss of function (cytotoxicity) of cytotoxic T cells (CTLs).[25] A member of the B7 receptor family, CTLA-4 is expressed on CD4+, CD8+ and Treg cells,[26] which competes with CD28 for stimulatory ligands CD80 and CD86. Another receptor, LAG-3, was shown to enhance Treg function.[27] Another marker or mediator for immunosuppression, TIM-3, is still being investigated,[28] but studies have correlated TIM-3 expression levels with poor clinical outcome.[29] Understanding these mechanisms has facilitated the establishment of immunotherapies, as outlined below.
Establishment of a Cancer-Promoting Tumor Microenvironment
The fact that some cancers arise at sites of chronic inflammation was first noted by Virchow more than a century ago. Infiltration of inflammatory mediators and a complex milieu of cytokines including TGF-β, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-23, and tumor necrosis factor-α (TNFα), as well as chemokines that are "chemotactic cytokines" may be exploited by tumor cells. More recent developments link many of those cytokines to the formation of suppressive immune cells like myeloid-derived suppressive cells (MDSC), Treg, tumor associated macrophages (TAMs) and their effectors, which are exploited and promoted by the tumor microenvironment. Additionally, tumors express some of the ligands to the immune inhibitory receptors; for example, PD-L1 is upregulated in multiple tumor cell lines and cancer cells.[30] This way, these ligands can bind to their receptors (PD-L1/PD-1) and promote inhibitory function and reduce T cell cytotoxicity.
Cytokines
Cytokines produced by cancer cells that suppress immune function[31] include TGF-β, which suppresses NK and T-cell activation and is a key cytokine in the differentiation of Treg.[32] Interleukin-6 signals via STAT3 to inhibit DC maturation and NK cell, T cell, neutrophil, and macrophage activation.[33] A transcription factor, STAT3, is also involved in several other immunosuppressive pathways, such as IL-10 signaling,[34] suppression of dendritic cells,[35] downregulation of IL-12,[36] and generation of Treg.[37] Prostaglandin E2 (PGE2) is a prosurvival, proangiogenic molecule that is produced by many cancers.[38-40] These processes of MHC downregulation, cytokine release, and contributing factors from Treg result in cytotoxic T-cell anergy. Lymphocytes are anergic when they fail to respond to a non-self-specific antigen. Anergy is 1 of 3 processes that induce tolerance, modifying the immune system to prevent cancer destruction. Vascular endothelial growth factor (VEGF), which is primarily thought of as a promoter of angiogenesis, is overexpressed in 90% of cancers[41] and functions to increase the ratio of immature to mature DC in the tumor microenvironment, which is thought to also contribute to T-cell dysfunction and inactivation.[42] Toll-like receptors (TLRs) stimulate the production of proinflammatory cytokines such as TNF-α and IFN-γ with a T-cell stimulating effect, resulting in a type 1 helper (TH1) response.
Cellular Immune Components of the Tumor Microenvironment: Myeloid-Derived Suppressor Cells, Regulatory T Cells, and
Tumor-Associated Macrophages
Myeloid-derived suppressor cells (MDSC) are diverse cells of myeloid origin with T-cell suppressive functions.[43] Initial studies in cancer found that MDSC inhibit activated T cells. The basal levels of MDSC increase with age and may contribute to increased tumor frequency and growth rate increase with age.[44] Treatments such as antibody depletion, retinoic acid, gemcitabine, and STAT3 blockade diminish MDSC, restore immune surveillance, increase T-cell activation, and improve efficacy of immunotherapy.
A subset of suppressor T cells (ie, Treg) was relatively recently identified, and exist to prevent autoimmunity. This subpopulation of CD4+ T cells also express CD25, CTLA-4, and CD39.[45,46] These Treg promote cancer progression by causing anergy, apoptosis, and cell cycle arrest of activated T cells via production of IL-10, TGF-β, and direct cell-to-cell contact.[47] They also inhibit the action of dendritic cells, NK cells, and B cells.[48] In cancer patients, Treg are increased in the peripheral blood and among T cells infiltrating the tumor and are more potent, resulting in an immunosuppressed state.[26,49,50] Also, Treg numbers are inversely proportional to DC and CD8+ T cell numbers in cancer.[51,52]
In the tumor microenvironment, TAM may be strongly antitumor, and possess a so-called M1 phenotype characterized by the production of IFNγ and other type 1 cytokines. Alternatively activated macrophages (M2) force a type 2 helper (TH2) response, with production of interleukins such as IL-4 and IL-13 that permit tumor growth. Worse clinical outcome correlates with TAM infiltrating tumors, which are closely associated with the M2 phenotype. A macrophage that could become type 1 would have a proinflammatory effect whereas a type 2 would have an anti-inflammatory effect.[71] This dual role has implications on tumor immunity or in immune escape of tumors. The type 2 macrophage participates in immune escape. These TAMs have been demonstrated to produce epidermal growth factor (EGF), IL-6, IL-10, and have been implicated in angiogenesis, local tumor progression, and metastasis.[53] Through these immune/inflammatory cells and mediators, cancer induces an immune suppressed state via multiple potent mechanisms, which is a barrier to effective cancer eradication.[54]
There are other important factors, in addition to host-dependent factors, that contribute to the development and progression of cancers. Localized upregulation of PD-L1 -- leading to T-cell anergy upon ligation of PD-1 on T lymphocytes -- is important for tumor microenvironment development and escape.[55,56] Physiologically, the presence of checkpoint receptors such as PD-1 or CTLA-4 limits an overly robust immune response, in order to protect from autoimmune reactivity.[55,57] Elevated PD-1 expression levels were observed on CD8+ virus-specific tumor lymphocytes,[58] correlating with clinical outcome. Unexpectedly, patients with high PD-1 expressing T-cell infiltration showed a significant 5-year overall survival (93.9%) compared with those patients with low PD-1 expressing T cell infiltration (63.6%)(P= 0.025; HR, 0.13).[58] This potentially conflicting observation may reflect a greater overall antitumor immune response, since pro-inflammatory conditions can stimulate PD-L1 expression. Interestingly, PD-L1 expression of tumor tissue was not correlated to clinical outcome.[59] As a result, the quality and quantity of TILs determines the antitumor response.
Immune Checkpoints and Inhibitors
T-cell activation occurs through a combination of T-cell receptor engagement with the MHC and costimulatory molecules.
Figure 2. Stimulatory and inhibitory reactions in the tumor microenvironment.
The duration and extent of immune responses, for example to infections, is regulated by "immune checkpoints" or inhibitory pathways that prevent excessive inflammatory responses as well as development of autoimmunity. Immune checkpoints have also been shown to play an important role in the tumor microenvironment and can be manipulated as a mechanism of tumor immune evasion.[59] The immune checkpoint pathways are mediated by ligand and receptor interactions; examples include CTLA-4 and its ligands CD80 and CD84, and PD-1 and its ligands PD-L1 and PD-L2. Therapy with blocking antibody to CTLA-4 results in rejection of murine cancers (Figure 3).[60] A mAb against CTLA-4, ipilimumab, was the first drug in this class to demonstrate clinical benefit and was approved by the FDA for patients with metastatic melanoma.[61] Tremelimumab is also available for CLTA-4 targeting. More recently, antibodies against PD-1 or PD-L1 have demonstrated clinical efficacy, alone[62-64] or in combination with ipilimumab.[65]
Figure 2. Stimulatory and inhibitory reactions in the tumor microenvironment.
CD28/B7 interactions promote TCR recognition of the MHC-antigen presentation and result in T-cell cytotoxicity (+). CTLA4/B7, PD1-PD-L1 interactions between the T-cell and the DC act as "brakes" on the immune system as indicated by (-). Interactions between the tumor cell and T cell (PD-1/PD-L1) decrease T-cell activity and reduce tumor recognition and elimination.
Figure 3: Promoting antitumor activity with checkpoint inhibitors
Negative signals (-) dampening the immune response against tumor can be eliminated by the use of checkpoint inhibitors anti-PD1 (nivolumab, pembrolizumab), anti-PD-L1 (MPDL3280A) and anti-CTLA4 (ipilimumab and tremelimumab) as shown above.
PD-L1 pathway targeting in cancer
The 55 kD type I transmembrane protein PD-1 (CD279) is a member of the CD28 family of T-cell costimulatory receptors that also includes CD28, CTLA-4, ICOS, and BTLA. It contains an intracellular membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal immunoreceptor tyrosine-based switch motif (ITSM). Two ligands specific for PD-1 have been identified: PD-L1 (B7-H1/CD274) and PD-L2 (B7-DC/CD273). Both PD-L1 and PD-L2 have been shown to downregulate T-cell activation upon binding to PD-1 in murine and human systems. Specifically, PD-1 delivers a negative signal, suppressing type 1-based antitumor immunity,[66] and is primarily expressed on activated T cells, B cells, and myeloid cells. Potentially, PD-1 blockade can activate anti-self T-cell responses, but these responses are variable and dependent upon various host genetic factors. Thus, PD-1 blockade results in a reproducible enhancement of proliferation and IFN release.
Upon binding to PD-L1 ligand, PD-1 activation inhibits T cell function and proliferation.[66] Tumor immune evasion can occur by high tumor expression of PD-L1 and/or tumor immune infiltration by PD-1+ T lymphocytes. Preliminary analyses indicate that PD-L1 is expressed in 40% to 60% of many cancers, and that tumor infiltration by PD-1+ T cells is a beneficial prognostic marker (indicative of an ongoing immune response), and thus disrupting PD-L1-mediated suppression of these activated cells may be clinically valuable. These data strongly support a role for PD-1 inhibition in the therapy of cancer. The mAbs targeting PD-1 and CTLA-4 (and other co-inhibitory receptors) (Figure 3), as well as their being combined with agonistic receptor ligation (such as CD137, OX-40 and CD7 stimulation) are being investigated in clinical trials, and in 2014 two anti-PD-1 mAbs were FDA approved for melanoma, and in 2015 for non-small-cell lung cancer (NSCLC). Combinations of multiple checkpoint or tumor-targeted antibodies (Abs) are ongoing or in development.
A different group of receptors with a modulating effect on immune cells are the killer-cell immunoglobulin-like receptors (KIRs). They interact with MHC I molecules and regulate immune response. Most of these receptors have a suppressive effect on cytotoxicity, particularly "turning off" NK cells when HLA is present on tumor cells. Thus, anti-KIR Abs might remove a major inhibitory signal on NK cells. Ongoing trials are investigating an anti-KIR mAb in combination with the anti-CTLA-4 mAb ipilimumab (NCT01750580) or anti PD-1 mAb nivolumab (NCT01714739).
Checkpoint receptor-targeted strategies in combination with chemotherapy or TA-targeted mAbs
Here I will provide 1 interesting example of the role of antibody therapy with checkpoint receptor strategies. Cetuximab therapy alters expression of checkpoint receptors on circulating and intratumoral TILs. Specifically, the frequency of Treg suppressor cells that express CTLA-4 and PD-1 are enriched in the tumor microenvironment.[67] Furthermore, cetuximab therapy increases the frequency of CD4+CD25hiCD39+FOXP3+ Treg, indicating that this treatment expands Treg in patients with cancer. In addition, CTLA-4+/CD39+ cells were significantly increased among the majority of CD4+FOXP3+ Treg from patients prior to and after cetuximab treatment, indicating that CTLA-4 targeting may provide enhanced benefit in cetuximab-treated patients. Recent data in NSCLC indicate that the epidermal growth factor receptor (EGFR) pathway may contribute to regulation of PD-L1 expression.[68]
These emerging data support the incorporation of checkpoint inhibitory mAb into conventional cancer therapy, either to deplete Treg or to disrupt the PD-1/PD-L1 suppressive signal transmitted to CD8+ effector T lymphocytes. These suppressed NK cells and T cells express the negative regulatory PD-1 receptor at higher levels, and generating greater inhibitory signals in TILs, providing a strong rationale for combining cetuximab with anti-PD-1 mAb therapy in a curative setting in which traditional cytotoxic chemotherapy may impart deleterious effects on the generation and proliferation of beneficial antitumor lymphocyte responses.
Costimulatory agonistic strategies
In addition to blocking negative regulatory receptors on lymphocytes, another strategy has emerged to enhance and trigger positive, costimulatory signals using agonistic Abs and small molecules. So far, the investigation of tumor necrosis factor receptor (TNFR)-targeting mAb in clinical trials for cancer is in phase 1 trials. Because of the important costimulatory pathways for immune cell activation, substances like CP-870,893, an IgG2 CD40 agonist, OX40 mAb, an IgG2 OX40 agonist or urelumab, and an IgG4 CD137 agonist have been investigated with cetuximab or with nivolumab in clinical trials,[69] which are currently enrolling cancer patients.
Conclusion
Cancer immunology is a rapidly evolving field and only recently have we begun to understand the complex interaction between cancer and the host immune system. Tumor cells demonstrate several methods to exploit the immune system to help promote angiogenesis, derive pro-survival and proliferative signals, and induce metastasis and tumor progression. At the same time, cancers are able to cloak themselves from the immune system by self-modification and by immunosuppression of the host. Recent results from clinical trials show evidence for effective anticancer immunotherapies. Because of the manifold tumor evasion strategies and hence different response rates for treatments, it is crucial to develop combination therapies for cancer treatment. These insights and better understanding of the workings of the immune system have allowed the recent explosion of several promising immunotherapeutic agents that are currently in clinical use or currently in development.
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