The PD-1 Interactome.

T cell activation is a fine-tuned process that involves T cell receptor and costimulation signals. To prevent undue activation of T cells, inhibitory molecules including PD-1 (programmed death 1) are induced and function as brakes for T cell signaling. In a steady state, the interaction of PD-1 with its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) maintains peripheral immune tolerance. However, the expression of PD-L1 on tumor cells and interaction with PD-1 on T cells dampen anti-tumor immunity. Therapeutic inhibitors of the PD-1 pathway have revolutionized tumor immunotherapy. Unfortunately, the majority of patients do not develop sustained anti-tumor responses. However, the knowledge about unique PD-1 interactions and their role in mediating PD-1 inhibitory signals is currently limited. Advances in the mechanistic understanding of the molecular and signaling integration of the PD-1 pathway could unleash the great potential in tumor immunotherapy by allowing the development of combinatorial approaches that target not only PD-1 and its ligands but also its unique downstream signal mediators. In this review, the current advances in understanding the mechanisms of extracellular and intracellular PD-1 interactions and their significance in potential future therapeutic approaches are discussed.

Publisher Correction: Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.

Acquired Resistance of EGFR-Mutated Lung Cancer to Tyrosine Kinase Inhibitor Treatment Promotes PARP Inhibitor Sensitivity.

Lung cancers with oncogenic mutations in the epidermal growth factor receptor (EGFR) invariably acquire resistance to tyrosine kinase inhibitor (TKI) treatment. Vulnerabilities of EGFR TKI-resistant cancer cells that could be therapeutically exploited are incompletely understood. Here, we describe a poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor-sensitive phenotype that is conferred by TKI treatment in vitro and in vivo and appears independent of any particular TKI resistance mechanism. We find that PARP-1 protects cells against cytotoxic reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Compared to TKI-naive cells, TKI-resistant cells exhibit signs of increased RAC1 activity. PARP-1 catalytic function is required for PARylation of RAC1 at evolutionarily conserved sites in TKI-resistant cells, which restricts NOX-mediated ROS production. Our data identify a role of PARP-1 in controlling ROS levels upon EGFR TKI treatment, with potentially broad implications for therapeutic targeting of the mechanisms that govern the survival of oncogene-driven cancer cells.

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer.

The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation.

The immune system maintains a critically organized network to defend against foreign particles, while evading self-reactivity simultaneously. T lymphocytes function as effectors and play an important regulatory role to orchestrate the immune signals. Although central tolerance mechanism results in the removal of the most of the autoreactive T cells during thymic selection, a fraction of self-reactive lymphocytes escapes to the periphery and pose a threat to cause autoimmunity. The immune system evolved various mechanisms to constrain such autoreactive T cells and maintain peripheral tolerance, including T cell anergy, deletion, and suppression by regulatory T cells (TRegs). These effects are regulated by a complex network of stimulatory and inhibitory receptors expressed on T cells and their ligands, which deliver cell-to-cell signals that dictate the outcome of T cell encountering with cognate antigens. Among the inhibitory immune mediators, the pathway consisting of the programed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) plays an important role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells. However, the PD-1:PD-L1/L2 pathway also mediates potent inhibitory signals to hinder the proliferation and function of T effector cells and have inimical effects on antiviral and antitumor immunity. Therapeutic targeting of this pathway has resulted in successful enhancement of T cell immunity against viral pathogens and tumors. Here, we will provide a brief overview on the properties of the components of the PD-1 pathway, the signaling events regulated by PD-1 engagement, and their consequences on the function of T effector cells.

Epigenetic regulation of cancer biology and anti-tumor immunity by EZH2.

Polycomb group proteins regulate chromatin structure and have an important regulatory role on gene expression in various cell types. Two polycomb group complexes (Polycomb repressive complex 1 (PRC1) and 2 (PRC2)) have been identified in mammalian cells. Both PRC1 and PRC2 compact chromatin, and also catalyze histone modifications. PRC1 mediates monoubiquitination of histone H2A, whereas PRC2 catalyzes methylation of histone H3 on lysine 27. These alterations of histones can lead to altered gene expression patterns by regulating chromatin structure. Numerous studies have highlighted the role of the PRC2 catalytic component enhancer of zeste homolog 2 (EZH2) in neoplastic development and progression, and EZH2 mutations have been identified in various malignancies. Through modulating the expression of critical genes, EZH2 is actively involved in fundamental cellular processes such as cell cycle progression, cell proliferation, differentiation and apoptosis. In addition to cancer cells, EZH2 also has a decisive role in the differentiation and function of T effector and T regulatory cells. In this review we summarize the recent progress regarding the role of EZH2 in human malignancies, highlight the molecular mechanisms by which EZH2 aberrations promote the pathogenesis of cancer, and discuss the anti-tumor effects of EZH2 targeting via activating direct anti-cancer mechanisms and anti-tumor immunity.

Corrigendum: Regulation of T Cell Differentiation and Function by EZH2.

[This corrects the article on p. 172 in vol. 7, PMID: 27199994.].

Clinical significance of T cell metabolic reprogramming in cancer.

Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy.

NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression.

Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert its anti-tumor activity; however, the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity in the tumor microenvironment is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα induces tumor cell death in an autocrine manner. A sublethal dose of Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. Furthermore, we determined in a syngeneic sarcoma mouse model that radiation up-regulates IRF3, IFNß, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which are associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Moreover, tumor-infiltrating T cells are in their active form since both the perforin and FasL pathways are activated in irradiated tumor tissues. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Therefore, radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation-mediated tumor suppression.

The role of metabolic reprogramming in T cell fate and function.

T lymphocytes undergo extensive changes in their metabolic properties during their transition through various differentiation states, from naïve to effector to memory or regulatory roles. The cause and effect relationship between metabolism and differentiation is a field of intense investigation. Many recent studies demonstrate the dependency of T cell functional outcomes on metabolic pathways and the possibility of metabolic intervention to modify these functions. In this review, we describe the basic metabolic features of T cells and new findings on how these correlate with various differentiation fates and functions. We also highlight the latest information regarding the main factors that affect T cell metabolic reprogramming.

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation.

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid ß-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development.

IFN regulatory factor 8 represses GM-CSF expression in T cells to affect myeloid cell lineage differentiation.

During hematopoiesis, hematopoietic stem cells constantly differentiate into granulocytes and macrophages via a distinct differentiation program that is tightly controlled by myeloid lineage-specific transcription factors. Mice with a null mutation of IFN regulatory factor 8 (IRF8) accumulate CD11b(+)Gr1(+) myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs), indicating an essential role of IRF8 in myeloid cell lineage differentiation. However, IRF8 is expressed in various types of immune cells, and whether IRF8 functions intrinsically or extrinsically in regulation of myeloid cell lineage differentiation is not fully understood. In this study, we report an intriguing finding that, although IRF8-deficient mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b(+)Gr1(+) MDSCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit no abnormal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and MDSC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased MDSC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased MDSC accumulation in the recipient chimeric mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. Our data determine that, in addition to its intrinsic function as an apoptosis regulator in myeloid cells, IRF8 also acts extrinsically to repress GM-CSF expression in T cells to control myeloid cell lineage differentiation, revealing a novel mechanism that the adaptive immune component of the immune system regulates the innate immune cell myelopoiesis in vivo.

GCN2-dependent metabolic stress is essential for endotoxemic cytokine induction and pathology.

Activated inflammatory macrophages can express indoleamine 2,3-dioxygenase (IDO) and thus actively deplete their own tryptophan supply; however, it is not clear how amino acid depletion influences macrophage behavior in inflammatory environments. In this report, we demonstrate that the stress response kinase GCN2 promotes macrophage inflammation and mortality in a mouse model of septicemia. In vitro, enzymatic amino acid consumption enhanced sensitivity of macrophages to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) with significantly increased interleukin 6 (IL-6) production. Tryptophan withdrawal induced the stress response proteins ATF4 and CHOP/GADD153; however, LPS stimulation rapidly enhanced expression of both proteins. Moreover, LPS-driven cytokine production under amino acid-deficient conditions was dependent on GCN2, as GCN2 knockout (GCN2KO) macrophages had a significant reduction of cytokine gene expression after LPS stimulation. To test the in vivo relevance of these findings, monocytic-lineage-specific GCN2KO mice were challenged with a lethal dose of LPS intraperitoneally (i.p.). The GCN2KO mice showed reduced inflammatory responses, with decreased IL-6 and IL-12 expression correlating with significant reduction in animal mortality. Thus, the data show that amino acid depletion stress signals (via GCN2) synergize with proinflammatory signals to potently increase innate immune responsiveness.

Epigenetics and colorectal cancer pathogenesis.

Colorectal cancer (CRC) develops through a multistage process that results from the progressive accumulation of genetic mutations, and frequently as a result of mutations in the Wnt signaling pathway. However, it has become evident over the past two decades that epigenetic alterations of the chromatin, particularly the chromatin components in the promoter regions of tumor suppressors and oncogenes, play key roles in CRC pathogenesis. Epigenetic regulation is organized at multiple levels, involving primarily DNA methylation and selective histone modifications in cancer cells. Assessment of the CRC epigenome has revealed that virtually all CRCs have aberrantly methylated genes and that the average CRC methylome has thousands of abnormally methylated genes. Although relatively less is known about the patterns of specific histone modifications in CRC, selective histone modifications and resultant chromatin conformation have been shown to act, in concert with DNA methylation, to regulate gene expression to mediate CRC pathogenesis. Moreover, it is now clear that not only DNA methylation but also histone modifications are reversible processes. The increased understanding of epigenetic regulation of gene expression in the context of CRC pathogenesis has led to development of epigenetic biomarkers for CRC diagnosis and epigenetic drugs for CRC therapy.

Deregulation of apoptotic factors Bcl-xL and Bax confers apoptotic resistance to myeloid-derived suppressor cells and contributes to their persistence in cancer.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.

NF-κB directly regulates Fas transcription to modulate Fas-mediated apoptosis and tumor suppression.

Fas is a member of the death receptor family. Stimulation of Fas leads to induction of apoptotic signals, such as caspase 8 activation, as well as "non-apoptotic" cellular responses, notably NF-κB activation. Convincing experimental data have identified NF-κB as a critical promoter of cancer development, creating a solid rationale for the development of antitumor therapy that suppresses NF-κB activity. On the other hand, compelling data have also shown that NF-κB activity enhances tumor cell sensitivity to apoptosis and senescence. Furthermore, although stimulation of Fas activates NF-κB, the function of NF-κB in the Fas-mediated apoptosis pathway remains largely undefined. In this study, we observed that deficiency of either Fas or FasL resulted in significantly increased incidence of 3-methylcholanthrene-induced spontaneous sarcoma development in mice. Furthermore, Fas-deficient mice also exhibited significantly greater incidence of azoxymethane and dextran sodium sulfate-induced colon carcinoma. In addition, human colorectal cancer patients with high Fas protein in their tumor cells had a longer time before recurrence occurred. Engagement of Fas with FasL triggered NF-κB activation. Interestingly, canonical NF-κB was found to directly bind to the FAS promoter. Blocking canonical NF-κB activation diminished Fas expression, whereas blocking alternate NF-κB increased Fas expression in human carcinoma cells. Moreover, although canonical NF-κB protected mouse embryo fibroblast (MEF) cells from TNFα-induced apoptosis, knocking out p65 diminished Fas expression in MEF cells, resulting in inhibition of FasL-induced caspase 8 activation and apoptosis. In contrast, knocking out p52 increased Fas expression in MEF cells. Our observations suggest that canonical NF-κB is a Fas transcription activator and alternate NF-κB is a Fas transcription repressor, and Fas functions as a suppressor of spontaneous sarcoma and colon carcinoma.

Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo.

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.