CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Human skin-resident CD8+ T cells require RUNX2 and RUNX3 for induction of cytotoxicity and expression of the integrin CD49a.

The integrin CD49a marks highly cytotoxic epidermal-tissue-resident memory (TRM) cells, but their differentiation from circulating populations remains poorly defined. We demonstrate enrichment of RUNT family transcription-factor-binding motifs in human epidermal CD8+CD103+CD49a+ TRM cells, paralleled by high RUNX2 and RUNX3 protein expression. Sequencing of paired skin and blood samples revealed clonal overlap between epidermal CD8+CD103+CD49a+ TRM cells and circulating memory CD8+CD45RA-CD62L+ T cells. In vitro stimulation of circulating CD8+CD45RA-CD62L+ T cells with IL-15 and TGF-ß induced CD49a expression and cytotoxic transcriptional profiles in a RUNX2- and RUNX3-dependent manner. We therefore identified a reservoir of circulating cells with cytotoxic TRM potential. In melanoma patients, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and improved patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM cells, providing immunosurveillance of infected and malignant cells.

Bcl11b sustains multipotency and restricts effector programs of intestinal-resident memory CD8+ T cells.

The networks of transcription factors (TFs) that control intestinal-resident memory CD8+ T (TRM) cells, including multipotency and effector programs, are poorly understood. In this work, we investigated the role of the TF Bcl11b in TRM cells during infection with Listeria monocytogenes using mice with post-activation, conditional deletion of Bcl11b in CD8+ T cells. Conditional deletion of Bcl11b resulted in increased numbers of intestinal TRM cells and their precursors as well as decreased splenic effector and circulating memory cells and precursors. Loss of circulating memory cells was in part due to increased intestinal homing of Bcl11b-/- circulating precursors, with no major alterations in their programs. Bcl11b-/- TRM cells had altered transcriptional programs, with diminished expression of multipotent/multifunctional (MP/MF) program genes, including Tcf7, and up-regulation of the effector program genes, including Prdm1. Bcl11b also limits the expression of Ahr, another TF with a role in intestinal CD8+ TRM cell differentiation. Deregulation of TRM programs translated into a poor recall response despite TRM cell accumulation in the intestine. Reduced expression of MP/MF program genes in Bcl11b-/- TRM cells was linked to decreased chromatin accessibility and a reduction in activating histone marks at these loci. In contrast, the effector program genes displayed increased activating epigenetic status. These findings demonstrate that Bcl11b is a frontrunner in the tissue residency program of intestinal memory cells upstream of Tcf1 and Blimp1, promoting multipotency and restricting the effector program.

Rapid Enhancer Remodeling and Transcription Factor Repurposing Enable High Magnitude Gene Induction upon Acute Activation of NK Cells.

Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.

CD155: A Multi-Functional Molecule in Tumor Progression.

CD155 is an adhesion molecule belonging to the Nectin/Nectin-like family often overexpressed on tumor cells and involved in many different processes such as cell adhesion, migration and proliferation. In contrast to these pro-tumorigenic functions, CD155 is also a ligand for the activating receptor DNAM-1 expressed on cytotoxic lymphocytes including Natural Killer (NK) cells and involved in anti-tumor immune response. However, during tumor progression inhibitory receptors for CD155 are up-regulated on the surface of effector cells, contributing to an impairment of their cytotoxic capacity. In this review we will focus on the roles of CD155 as a ligand for the activating receptor DNAM-1 regulating immune surveillance against cancer and as pro-oncogenic molecule favoring tumor proliferation, invasion and immune evasion. A deeper understanding of the multiple roles played by CD155 in cancer development contributes to improving anti-tumor strategies aimed to potentiate immune response against cancer.

The Ubiquitin-proteasome pathway regulates Nectin2/CD112 expression and impairs NK cell recognition and killing.

Nectin2 is a member of immunoglobulin-like cell adhesion molecules and plays a prominent role in the establishment of adherens and tight junctions. It is also upregulated on the surface of tumor and virus-infected cells where it functions as a ligand for the activating receptor CD226, thus contributing to cytotoxic lymphocyte-mediated recognition and killing of damaged cells. Little is currently known about the regulation of Nectin2 expression and, in particular, whether posttranscriptional and posttranslational mechanisms are involved. Here, we analyzed Nectin2 expression on a panel of human tumor cell lines and primary cultures and we found that Nectin2 is mainly expressed in cytoplasmic pools. Moreover, we demonstrated that ubiquitination of Nectin2 promotes its degradation and is responsible for protein intracellular retention. Indeed, inhibition of the ubiquitin pathway results in increased Nectin2 surface expression and enhances tumor cell susceptibility to NK cell cytotoxicity. Our results demonstrate a previously unknown mechanism of Nectin2 regulation revealing that the ubiquitin pathway represents a potential target of intervention in order to increase susceptibility to NK cell-mediated lysis.

A skewed pool of resident T cells triggers psoriasis-associated tissue responses in never-lesional skin from patients with psoriasis.

BACKGROUND: Resident T cells are implicated in the maintenance and recurrence of psoriatic lesions. Whether skin that has not yet experienced psoriasis in patients with established disease harbors pathogenic T cells is less investigated. OBJECTIVE: We sought to analyze the composition of resident T cells and T cell-driven tissue responses in skin never affected by psoriasis from patients with mild disease. METHODS: Never-lesional skin from patients with psoriasis (NLP) was collected from those with mild disease. T-cell profiles were assessed by using confocal imaging and flow cytometry. Tissue responses to T-cell stimulation were measured by using multiplex and NanoString technology. RESULTS: T-cell activation ex vivo triggered psoriasiform and type I interferon tissue responses in NLP psoriasis. Accordingly, keratinocytes from NLP responded to IFN-γ stimulation with myxovirus 1 (MX1) expression and IFN-α release. Additionally, CCR6-expressing resident T cells poised to produce IFN-γ and IL-17 were enriched in epidermis from NLP, whereas dermal tissue responses and T-cell compositions were similar to those in healthy skin. Finally, keratinocytes from NLP exposed to IL-17 and skin explants exposed to common fungal antigens responded with upregulation of the CCR6 ligand CCL20. CONCLUSION: Epidermal resident T cells capable of triggering psoriasiform tissue responses accumulate in epidermis from NLP. Our global analysis of NLP reveals that microbial interplay with genetically predisposed keratinocytes might shape the local pool of resident T cells.

Natural killer cells in inflammation and autoimmunity.

First described 40 years ago, natural killer (NK) cells represent the founding members of the innate lymphoid cell (ILC) family. They were initially defined by their ability to kill cancer cells of hematopoietic origin. More recently, NK cells are recognized not only for their ability to kill infected or malignant cells, but also for mediating cytotoxicity against a range of normal immune cells. They thereby play an important physiological role in controlling immune responses and maintaining homeostasis. Besides cytotoxic activity, NK cells activation is accompanied by secretion of pro-inflammatory cytokines. Hence, NK cells have the potential to act both in driving inflammation and in restricting adaptive immune responses that may otherwise lead to excessive inflammation or even autoimmunity. Here, we highlight how NK cell activity is linked to inflammasome activation and review new molecular insights to the roles of NK cells in inflammation and autoimmunity. Furthermore, in light of new insights to NK cell differentiation and memory, we deliberate on how distinct NK cell subsets may impact immunoregulatory functions. Hypothetically, memory-like or adaptive NK cells could drive NK cell-mediated autoreactive diseases. Together, new findings underscore the complex yet important physiological roles of NK cells in both promoting inflammation and exerting immunoregulation and maintenance of immune homeostasis. Insights raise intriguing questions as to how NK cells themselves maintain self-tolerance.

NCR+ ILC3 maintain larger STAT4 reservoir via T-BET to regulate type 1 features upon IL-23 stimulation in mice.

Innate lymphoid cells (ILCs) producing IL-22 and/or IL-17, designated as ILC3, comprise a heterogeneous subset of cells involved in regulation of gut barrier homeostasis and inflammation. Exogenous environmental cues in conjunction with regulated expression of endogenous factors are key determinants of plasticity of ILC3 toward the type 1 fate. Herein, by using mouse models and transcriptomic approaches, we defined at the molecular level, initial events driving ILC3 expressing natural cytotoxicity receptors (NCR+ ILC3) to acquire type 1 features. We observed that NCR+ ILC3 exhibited high basal expression of the signal-dependent transcription factor STAT4 due to T-BET, leading to predisposed potential for the type 1 response. We found that the prototypical inducer of type 3 response, IL-23, played a predominant role over IL-12 by accessing STAT4 and preferentially inducing its phosphorylation in ILC3 expressing T-BET. The early effector program driven by IL-23 was characterized by the expression of IL-22, followed by a production of IFN-γ, which relies on STAT4, T-BET and required chromatin remodeling of the Ifng locus. Altogether, our findings shed light on a feed-forward mechanism involving STAT4 and T-BET that modulates the outcome of IL-23 signaling in ILC3.

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells.

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.

Subset- and tissue-defined STAT5 thresholds control homeostasis and function of innate lymphoid cells.

Innate lymphoid cells (ILCs) patrol environmental interfaces to defend against infection and protect barrier integrity. Using a genetic tuning model, we demonstrate that the signal-dependent transcription factor (TF) STAT5 is critical for accumulation of all known ILC subsets in mice and reveal a hierarchy of STAT5 dependency for populating lymphoid and nonlymphoid tissues. We apply transcriptome and genomic distribution analyses to define a STAT5 gene signature in natural killer (NK) cells, the prototypical ILC subset, and provide a systems-based molecular rationale for its key functions downstream of IL-15. We also uncover surprising features of STAT5 behavior, most notably the wholesale redistribution that occurs when NK cells shift from tonic signaling to acute cytokine-driven signaling, and genome-wide coordination with T-bet, another key TF in ILC biology. Collectively, our data position STAT5 as a central node in the TF network that instructs ILC development, homeostasis, and function and provide mechanistic insights on how it works at cellular and molecular levels.

Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.

BACKGROUND: Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells. METHODS: Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138+ MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays. RESULTS: Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA. CONCLUSIONS: These findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.

Regulation of NKG2D Expression and Signaling by Endocytosis.

NKG2D is an activating receptor that can bind to a large number of stress-induced ligands that are expressed in the context of cancer or viral infection. This receptor is expressed on many cytotoxic lymphocytes, and plays a crucial role in antitumor and antiviral immune responses. However, exposure to NKG2D ligand-expressing target cells promotes receptor endocytosis, ultimately leading to lysosomal receptor degradation and impairment of NKG2D-mediated functions. Interestingly, before being degraded, internalized receptors can signal from the endosomal compartment, leading to the appropriate activation of cellular functional programs. This review summarizes recent findings on ligand-induced receptor internalization, with particular emphasis on the role of endocytosis in the control of both NKG2D-mediated intracellular signaling and receptor degradation.

Ubiquitin-dependent endocytosis of NKG2D-DAP10 receptor complexes activates signaling and functions in human NK cells.

Cytotoxic lymphocytes share the presence of the activating receptor NK receptor group 2, member D (NKG2D) and the signaling-competent adaptor DNAX-activating protein 10 (DAP10), which together play an important role in antitumor immune surveillance. Ligand stimulation induces the internalization of NKG2D-DAP10 complexes and their delivery to lysosomes for degradation. In experiments with human NK cells and cell lines, we found that the ligand-induced endocytosis of NKG2D-DAP10 depended on the ubiquitylation of DAP10, which was also required for degradation of the internalized complexes. Moreover, through combined biochemical and microscopic analyses, we showed that ubiquitin-dependent receptor endocytosis was required for the activation of extracellular signal-regulated kinase (ERK) and NK cell functions, such as the secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. These results suggest that NKG2D-DAP10 endocytosis represents a means to decrease cell surface receptor abundance, as well as to control signaling outcome in cytotoxic lymphocytes.

Regulation of fc receptor endocytic trafficking by ubiquitination.

Most immune cells, particularly phagocytes, express various receptors for the Fc portion of the different immunoglobulin isotypes (Fc receptors, FcRs). By binding to the antibody, they provide a link between the adaptive immune system and the powerful effector functions triggered by innate immune cells such as mast cells, neutrophils, macrophages, and NK cells. Upon ligation of the immune complexes, the downstream signaling pathways initiated by the different receptors are quite similar for different FcR classes leading to the secretion of preformed and de novo synthesized pro-inflammatory mediators. FcR engagement also promotes negative signals through the combined action of several molecules that limit the extent and duration of positive signaling. To this regard, ligand-induced ubiquitination of FcRs for IgE (FcεR) and IgG (FcγR) has become recognized as a key modification that generates signals for the internalization and/or delivery of engaged receptor complexes to lysosomes or cytoplasmic proteasomes for degradation, providing negative-feedback regulation of Fc receptor activity. In this review, we discuss recent advances in our understanding of the molecular mechanisms that ensure the clearance of engaged Fcε and Fcγ receptor complexes from the cell surface with an emphasis given to the cooperation between the ubiquitin pathway and endosomal adaptors including the endosomal sorting complex required for transport (ESCRT) in controlling receptor internalization and sorting along the endocytic compartments.

c-Cbl regulates MICA- but not ULBP2-induced NKG2D down-modulation in human NK cells.

The NKG2D activating receptor on human NK cells mediates "altered self" recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane-bound NKG2DLs can lead to down-modulation of receptor expression and impairment of NKG2D-mediated cell functions. Here, we evaluated whether different cell-bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down-modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down-modulation than ULBP2, leading to a severe impairment of NKG2D-dependent NK-cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c-Cbl direct MICA-induced but not ULBP2-induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti-tumor strategies to restore a normal level of NKG2D receptors on human NK cells.