Figure S5: Immunohistochemical staining of CD8+ T cell infiltration level in colon carcinoma tissues from 10 patients. The brown color indicates specific CD8+ T cells.

Table S2. Scores of CD4+ and CD8+ peritumoral lymphocytes, and tumor GPR109A protein levels in 10 human CRC specimens.

Figure S1. Silencing STAT1 diminishes IFN-gamma-induced GPR109A expression in human colon carcinoma cells.

<p>Table S3. Differential expression of inflammatory genes in human colon carcinoma</p>

Programmed Death-1 In article 2100758, Boussiotis, Patsoukis, Wang, and Bardhan show that Venus-based biomolecular fluorescence complementation detects Programmed Death-1 (PD-1) dimerization by confocal microscopy. HEK-293 cells are transfected with PD-1-VenusYFP-N-terminus and PD-1-VenusYFP-C-terminus together with SHP-2 and either kinase active (top) or kinase inactive (bottom) Fyn. YFP signal (yellow) from VenusYFP structural complementation, indicating PD-1 dimerization at the plasma membrane, is generated only in the presence of kinase active Fyn (red: Rhodamine Phalloidin; blue: DAPI nuclear staining).

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.

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.

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.

PD-1 is an inhibitory receptor expressed on activated T-cells. SHP-2 tyrosine phosphatase interacts with PD-1 and is critical for PD-1-mediated inhibition. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 may interact with either ITIM or ITSM of PD-1. We sought to identify the precise motif of PD-1 that is mandatory for SHP-2 interaction and PD-1 inhibitory function. Using GST-SHP-2 fusion protein we determined that PD-1 interacted with SHP-2 after PD-1 ligation with simultaneous TCR/CD3-mediated activation. This interaction required phosphorylation of the ITSM and was abrogated when the ITSM tyrosine Y248 was mutated to phenylalanine. In contrast, when the ITIM tyrosine Y223 was mutated, interaction of PD-1 with SHP-2 remained unaffected. Based on these findings, we hypothesized that phosphorylation of Y248 might be indicative of PD-1-mediated inhibitory signaling. We generated an antibody specific for Y248 and examined expression and function of PD-1pY248+ T cells in peripheral blood from healthy humans. PD-1pY248+ cells were detected within the CD4 but mostly CD8T cell populations, mainly in central memory and effector memory subsets and in much lower extent in terminally differentiated effectors. Although PD-1high did not correlate with altered ability of CD8 T cells to produce effector cytokines, PD-1pY248+ expression correlated with impaired production of IFN-g and TNFa in response to TCR/CD3+CD28-mediated stimulation. Thus, PD-1pY248 might serve as a biomarker indicative of PD-1 mediated inhibitory signaling in patients with chronic infections and cancer.

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.

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.

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 (T-Regs). 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.

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.

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, IFNβ, 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.

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

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.

Abstract PD-1 inhibits T cell responses but the biochemical mechanisms are poorly understood. PD-1 cytoplasmic tail contains an ITIM and an ITSM motif. SHP-2 tyrosine phosphatase interacts with the ITSM and has a critical role in PD-1-mediated inhibition. Although phosphatases are considered negative regulators of activation, SHP-2 deficient T cells display impaired activation of MAPK in response to TCR/CD3 stimulation. Conversely, SHP-2 mutations leading to gain of function induce activation of Ras-MAPK pathway in AML. Thus, SHP-2 may activate some signaling pathways. We investigated how PD-1: SHP-2 interaction inhibits T cell activation. A key event required for activation of the TCR/CD3 signaling cascade is the downregulation of Csk-mediated inactivating phosphorylation of Lck on Tyr-505. Growth factor stimulation of epithelial cells recruits SHP-2 to the plasma membrane and abrogates Csk-mediated inactivating phosphorylation of Src kinase. We determined that TCR/CD3 stimulation downregulated Lck phosphorylation on Tyr-505 but this was prevented by PD-1 ligation. Because Lck is localized in membrane lipid rafts, we examined whether SHP-2 might be recruited to the rafts. TCR/CD3 stimulation resulted in translocation of SHP-2 to the lipid rafts. In contrast, PD-1 ligation recruited SHP-2 to the PD-1 ITSM abrogating translocation of SHP-2 to the rafts and resulted in enhanced Lck phosphorylation on Tyr-505. Our results unravel a previously unidentified mechanism via which PD-1 inhibits T cell activation by sequestering SHP-2 and preventing its translocation to the site on the plasma membrane where SHP-2 plays a key role in activating the TCR signaling cascade by reversing Csk-mediated inactivating phosphorylation of Lck.

PD-1 is a checkpoint receptor, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. PD-1 blockade promotes anti-tumor responses by eliminating PD-1 mediated inhibitory signaling. PD-1-mediated inhibition relies on the interaction of its ITSM motif with SHP-2 but the mechanism is unknown. We examined how PD-1:SHP-2 interaction inhibits T-cell responses. Pull-down assays revealed that PD-1 interacted with GST-SHP-2 fusion protein after PD-1 ligation with simultaneous TCR/CD3-mediated activation and this interaction was mediated via the SH2 domains of SHP-2. SHP-2 contains two SH2 domains (N-SH2 and C-SH2). To determine which SH2 domain is important for PD-1 interaction, we mutagenized the functional sites of N-SH2 and C-SH2. We transfected COS cells with cDNA of SHP-2 wild type or each SH2 mutant together with PD-1 and TCR proximal kinase Fyn, which is required for phosphorylation of PD-1 ITSM. Immunoprecipitation experiments showed that mutagenesis of either SH2 domain abrogated PD-1:SHP-2 interaction, indicating that both SH2 domains are required for interaction with PD-1. We confirmed this finding by Biacore studies. These results indicate that SHP-2 brings together two ITSM-phosphorylated PD-1 molecules by interaction with N-SH2 and C-SH2 domains. Culture of human T cells in the presence of a soluble dimeric PD-L1 inhibited T cell proliferation and IFN-γ production whereas monomeric PD-L1 had the opposite effect. Our results reveal a new mechanism of PD-1:SHP-2 interaction with implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to enhance T cell activation by disrupting PD-1 homodimerization.