SOCS1 is a critical checkpoint in immune homeostasis, inflammation and tumor immunity.

The Suppressor of Cytokine Signaling (SOCS) family proteins are important negative regulators of cytokine signaling. SOCS1 is the prototypical member of the SOCS family and functions in a classic negative-feedback loop to inhibit signaling in response to interferon, interleukin-12 and interleukin-2 family cytokines. These cytokines have a critical role in orchestrating our immune defence against viral pathogens and cancer. The ability of SOCS1 to limit cytokine signaling positions it as an important immune checkpoint, as evidenced by the detection of detrimental SOCS1 variants in patients with cytokine-driven inflammatory and autoimmune disease. SOCS1 has also emerged as a key checkpoint that restricts anti-tumor immunity, playing both a tumor intrinsic role and impacting the ability of various immune cells to mount an effective anti-tumor response. In this review, we describe the mechanism of SOCS1 action, focusing on the role of SOCS1 in autoimmunity and cancer, and discuss the potential for new SOCS1-directed cancer therapies that could be used to enhance adoptive immunotherapy and immune checkpoint blockade.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

Enhanced nitric oxide production by macrophages treated with a cell-penetrating peptide conjugate.

The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.

Determining Activation Status of Natural Killer Cells Following Stimulation via Cytokines and Surface Receptors.

Stimulation of Natural Killer (NK) cells with cytokines, target cell interaction, or antibody mediated activation of receptors on the NK cell surface enables the dissection of specific signaling intermediates in different activation pathways. NK cell activation status is commonly measured by production of interferon gamma (IFNγ) and expression of the degranulation marker LAMP-1 (CD107a). Cytotoxic potency can also be assessed by the production of perforin, granzymes, and tumor necrosis factor alpha (TNFα). NK cell receptor mediated activation by antibodies requires crosslinking of the receptor-specific antibodies; thus, in vitro activation assays are performed by binding antibodies to cell culture plates. All parameters can be measured by flow cytometry.

Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Natural Killer Cells: Tumor Surveillance and Signaling.

Natural killer (NK) cells play a pivotal role in cancer immunotherapy due to their innate ability to detect and kill tumorigenic cells. The decision to kill is determined by the expression of a myriad of activating and inhibitory receptors on the NK cell surface. Cell-to-cell engagement results in either self-tolerance or a cytotoxic response, governed by a fine balance between the signaling cascades downstream of the activating and inhibitory receptors. To evade a cytotoxic immune response, tumor cells can modulate the surface expression of receptor ligands and additionally, alter the conditions in the tumor microenvironment (TME), tilting the scales toward a suppressed cytotoxic NK response. To fully harness the killing power of NK cells for clinical benefit, we need to understand what defines the threshold for activation and what is required to break tolerance. This review will focus on the intracellular signaling pathways activated or suppressed in NK cells and the roles signaling intermediates play during an NK cytotoxic response.

Large-scale survey and database of high affinity ligands for peptide recognition modules.

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large-scale peptide-phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM-ligand complexes, indicating that a large majority of the phage-derived peptides are likely to target natural peptide-binding sites and could thus act as inhibitors of natural protein-protein interactions. The complete dataset has been assembled in an online database (http://www.prm-db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.

RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Natural killer cells and anti-tumor immunity.

Immune checkpoint inhibitors harness the power of the immune system to fight cancer. The clinical success achieved with antibodies against the inhibitory T cell receptors PD-1 and CTLA4 has focused attention on the possibility of manipulating other immune cells, in particular those involved in innate immunity. Here we review the role of innate lymphoid cells (ILCs) and their contribution to tumor immunity. As the prototypical ILC, the natural killer (NK) cell has an intrinsic ability to detect and kill cancer cells. NK cells are dependent on the cytokine interleukin (IL)-15 for their development and effector activity. We discuss the role of the Suppressor of cytokine (SOCS) proteins in negatively regulating IL-15 and NK cell responses and the potential for targeting these small intracellular regulators as new immune checkpoints.

SOCS-mediated immunomodulation of natural killer cells.

Natural killer (NK) cells are innate immune cells with an intrinsic ability to detect and kill infected and cancerous cells. The success of therapies targeting immune checkpoints on CD8 cells has intensified interest in harnessing the cytolytic effector functions of NK cells for new cancer treatments. NK cell development, survival and effector activity is dependent on exposure to the cytokine interleukin (IL)-15. The suppressor of cytokine (SOCS) proteins (CIS; SOCS1-7) are important negative regulators of cytokine signaling, and both CIS and SOCS2 are reported to have roles in regulating NK cell responses. Their immunomodulatory effects on NK cells suggest that these SOCS proteins are promising targets that can potentially form the basis of novel cancer therapies. Here we discuss the role of NK cells in tumor immunity as well as review the role of the SOCS proteins in regulating IL-15 signaling and NK cell function.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Characterisation of a novel coumarin-based fluorescent probe for monitoring nitric oxide production in macrophages.

Nitric oxide (NO) is an important effector molecule in host defence against bacterial pathogens. The development of fluorescence imaging to monitor NO production in vitro and in vivo will increase our understanding of its biological role. Recently, a novel 'trappable' fluorescent blue 'turn-on' Cu(II)-complexed coumarin-based probe (CB) has been developed to detect NO. In this study, CB was investigated to evaluate its ability to detect NO in macrophages. Using confocal microscopy, NO was successfully detected in macrophages in the presence of stimuli that induce nitric oxide synthase (iNOS), the enzyme responsible for production of NO. The time dependence and subcellular compartmentalisation of CB in macrophages were evaluated. The probe can be trapped within cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections.

Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis.

The cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy.

Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling.

Influenza virus infections have a significant impact on global human health. Individuals with suppressed immunity, or suffering from chronic inflammatory conditions such as COPD, are particularly susceptible to influenza. Here we show that suppressor of cytokine signaling (SOCS) five has a pivotal role in restricting influenza A virus in the airway epithelium, through the regulation of epidermal growth factor receptor (EGFR). Socs5-deficient mice exhibit heightened disease severity, with increased viral titres and weight loss. Socs5 levels were differentially regulated in response to distinct influenza viruses (H1N1, H3N2, H5N1 and H11N9) and were reduced in primary epithelial cells from COPD patients, again correlating with increased susceptibility to influenza. Importantly, restoration of SOCS5 levels restricted influenza virus infection, suggesting that manipulating SOCS5 expression and/or SOCS5 targets might be a novel therapeutic approach to influenza.

CIS is a potent checkpoint in NK cell-mediated tumor immunity.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase.

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II ß-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.