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.

Just-in-time interprofessional training: lessons from the NHS Nightingale London.

The Coronavirus 2019 (COVID-19) pandemic placed significant pressure on healthcare systems across the globe, with many clinicians redeployed in unfamiliar specialties or disciplines. In England, a just-in-time interprofessional training and education programme was rapidly established to upskill nearly 2,500 people who volunteered to work at the NHS Nightingale London Hospital. Of the 488 respondents in our evaluation, representing a 20% response rate, most felt confident and safe to start work in NHS Nightingale London. Key findings were: streaming of learners should be driven by predicted shared roles in the workplace and previous experience; in situ training to experience how teams work together in the real clinical setting was well received; and online learning should be focused on essential learning. A just-in-time interprofessional training programme can be effective in upskilling and redeploying healthcare staff in emergency situations, and can be useful for supporting staff redeployment or upskilling across the NHS more widely.

The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo.

Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.

In Semliki Forest virus encephalitis, suppressor of cytokine signaling 4 (SOCS4) is an essential modulator of immune responses that mediates the balance between immunopathology and virus clearance.

Central nervous system virus infections are a major cause of morbidity and mortality worldwide and a significant global public health concern. As in many tissues, inflammation and immune responses in the brain, despite their protective roles, can also be harmful. Control of brain inflammation is important in many neurological diseases from encephalitis to multiple sclerosis and neurogenerative disease. The suppressors of cytokine signaling (SOCS) proteins are a key mechanism controlling inflammatory and immune responses across all tissues including the brain. Using a mouse model system, we demonstrate that lack of SOCS4 results in changes in the pathogenesis and clinical outcome of a neurotropic virus infection. Relative to wild-type mice, SOCS4-deficient mice showed accelerated clearance of virus from the brain, lower levels of persisting viral RNA in the brain, increased neuroinflammation and more severe neuropathology. We conclude that, in the mouse brain, SOCS4 is a vital regulator of antiviral immunity that mediates the critical balance between immunopathology and virus persistence.

Design and characterization of a heterobifunctional degrader of KEAP1.

The Kelch-like ECH-associated protein 1 (KEAP1) - nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway senses reactive oxygen species and regulates cellular oxidative stress. Inhibiting KEAP1 to activate the NRF2 antioxidant response has been proposed as a promising strategy to treat chronic diseases caused by oxidative stress. Here, we developed a proteolysis targeting chimera (PROTAC) that depletes KEAP1 from cells through the ubiquitin-proteasome pathway. A previously developed KEAP1 inhibitor and thalidomide were incorporated in the heterobifunctional design of the PROTAC as ligands for KEAP1 and CRBN recruitment, respectively. Optimization of the chemical composition and linker length resulted in PROTAC 14 which exhibited potent KEAP1 degradation with low nanomolar DC50 in HEK293T (11 nM) and BEAS-2B (<1 nM) cell lines. Furthermore, PROTAC 14 increased the expression of NRF2 regulated antioxidant proteins and prevented cell death induced by reactive oxygen species. Together, these results established a blueprint for further development of KEAP1-targeted heterobifunctional degraders and will facilitate the study of the biological consequences of KEAP1 removal from cells. This approach represents an alternative therapeutic strategy to existing treatments for diseases caused by oxidative stress.

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.

SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine.

Suppressor of cytokine signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here, we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine-phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

Suppressor of Cytokine Signalling 5 (SOCS5) Modulates Inflammatory Responses during Alphavirus Infection.

CNS viral infections are one of the major causes of morbidity and mortality worldwide and a significant global public health concern. Uncontrolled inflammation and immune responses in the brain, despite their protective roles, can also be harmful. The suppressor of cytokine signalling (SOCS) proteins is one of the key mechanisms controlling inflammatory and immune responses across all tissues including the brain. SOCS5 is highly expressed in the brain but there is little understanding of its role in the CNS. Using a mouse model of encephalitis, we demonstrate that lack of SOCS5 results in changes in the pathogenesis and clinical outcome of a neurotropic virus infection. Relative to wild-type mice, SOCS5-deficient mice had greater weight loss, dysregulated cytokine production and increased neuroinflammatory infiltrates composed predominantly of CD11b+ cells. We conclude that in the brain, SOCS5 is a vital regulator of anti-viral immunity that mediates the critical balance between immunopathology and virus persistence.

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.

Optimization of Phosphotyrosine Peptides that Target the SH2 Domain of SOCS1 and Block Substrate Ubiquitination.

Suppressor of cytokine signaling 1 (SOCS1) has emerged as a potential therapeutic target in inflammatory and viral diseases. SOCS1 operates via its kinase inhibitory region, Src homology 2 (SH2) domain, and SOCS box to negatively regulate the Janus kinase/signal transducers and activators of transcription signaling pathway. In this study, we utilized native phosphotyrosine peptide substrates as a starting point to iteratively explore the requirement of each amino acid position to target the SH2 domain of SOCS1. We show that Met, Thr, Thr, Val, and Asp in the respective -1, +1, +2, +3, and +5 positions within the peptide substrate are favored for binding to the SOCS1-SH2 domain and identifying several phosphotyrosine peptides that have potent SOCS1 binding affinity with IC50 values ranging from 20 to 70 nM and greater than 100-fold selectivity against the closely related SOCS family proteins, CIS, SOCS2, and SOCS3. The optimized phosphotyrosine peptide was shown to stabilize SOCS1 in a thermal shift assay using cell lysates and inhibited SOCS1-mediated ubiquitination of a target substrate in a biochemical assay. Collectively, these data provide the framework to develop cell-permeable peptidomimetics that further investigate the potential of the SOCS1-SH2 domain as a therapeutic target in inflammatory and viral diseases.

Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands.

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.

Development of an ethogram/guide for identifying feline emotions: a new approach to feline interactions and welfare assessment in practice.

BACKGROUND: An accurate assessment of feline behaviour is essential in reducing the risk of handler injury and evaluating/improving feline welfare within veterinary practices. However, inexperience and/or suboptimal education in feline behaviour may cause many veterinary professionals to be ill equipped for this. In addition, busy veterinary professionals may not have time to thoroughly search the literature to remediate this deficiency. Upon searching the literature, terms such as aggression and stress predominate, but these do not completely represent the rich mental lives that cats are now understood to have. Emotions have recently emerged as an alternative approach to animal behaviour/welfare assessment. However, few resources describe how to identify them, and positive emotions are particularly neglected. In addition, no simple, broad, and concise guide to feline emotions currently exists within the research literature. Therefore, this research aimed to develop a straightforward and clear reference guide to feline emotions (ethogram) to aid veterinary professionals in interpreting feline behaviour in practice and for use in veterinary education. RESULTS: Five primary emotions were identified and defined for domestic species (fear, anger/rage, joy/play, contentment and interest). A feline emotions guide (feline emotions ethogram) was created. Three hundred and seventy-two images were captured of feline behaviours indicative of emotional states. Of these, ten of the best quality and most representative images were selected to illustrate the guide (two of each emotional state). The feline emotions guide and its associated images were subsequently validated by two feline behaviour experts. CONCLUSIONS: Following slight modifications, the emotions definitions yielded during the feline ethogram design process may be transferable to other domestic species. The feline emotions ethogram/guide itself may be particularly helpful for distinguishing immediate motivations and customising patient care within short- term veterinary contexts. Hence, its use may improve feline welfare and feline handling/interactions. However, the guide will need to be reliability tested/ tested in the field and may require adaptation as the feline emotions' knowledge base grows. In addition, novices may benefit from exposure to more images of feline emotional state, particularly those involving mixed emotions. Freely available online images and videos may be sourced and used to supplement the accompanying image bank.

Does the UKCAT predict performance in medical and dental school? A systematic review.

OBJECTIVES: For the first time, this systematic review provides a summary of the literature exploring the relationship between performance in the UK Clinical Aptitude Test (UKCAT) and assessments in undergraduate medical and dental training. DESIGN: In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis, relevant studies were identified through systematic literature searches. Electronic searches were carried out on EBSCO, EMBASE, Educational Resources Information Centre, SCOPUS, Web of Knowledge. Studies which included the predictive validity of selection criteria including some element of the UKCAT were considered. RESULTS: 22 papers were identified for inclusion in the study. Four studies describe outcomes from dental programmes with limited results reported. 18 studies reported on relationships between the UKCAT and performance in undergraduate medical training. Of these, 15 studies reported relationships between the UKCAT cognitive tests and undergraduate medical assessments. Weak relationships (r=0.00-0.29) were observed in 14 of these studies; four studies reported some moderate relationships (r=0.30-0.49). The strongest relationships with performance in medical school were observed for the UKCAT total score and the verbal reasoning subtest. Relationships with knowledge-based assessments scores were higher than those for assessments of skills as the outcome. Relationships observed in small (single and double centre studies) were larger than those observed in multicentre studies. CONCLUSION: The results indicate that UKCAT scores predict performance in medical school assessments. The relationship is generally weak, although noticeably stronger for both the UKCAT total score and the verbal reasoning subtest. There is some evidence that UKCAT continues to predict performance throughout medical school. We recommend more optimal approaches to future studies. This assessment of existing evidence should assist medical/dental schools in their evaluation of selection processes.

"It's going to be hard you know " Teachers' perceived role in widening access to medicine.

Medical schools worldwide undertake widening access (WA) initiatives (e.g. pipeline, outreach and academic enrichment programmes) to support pupils from high schools which do not traditionally send high numbers of applicants to medicine. UK literature indicates that pupils in these schools feel that their teachers are ill-equipped, cautious or even discouraging towards their aspiration and/or application to medicine. This study aimed to explore teachers' perspectives and practices to include their voice in discussions and consider how medical schools might best engage with them to facilitate WA. Interviews were conducted with high school teachers in three UK regions, working in schools targeted by WA initiatives. Data were analysed thematically using template analysis, using a largely data-driven approach. Findings showed that although medicine was largely seen as a prestigious and worthwhile career, teachers held reservations about advocating this above other choices. Teachers saw it as their role to encourage pupils to educate themselves about medicine, but to ultimately allow pupils to make their own decisions. Their attitudes were influenced by material constraints in their schools, and the perception of daunting, long and emotionally difficult admissions requirements, with low chances of success. Medical schools may wish to work with teachers to understand their hesitations and help them develop the mindset required to advocate a challenging and unfamiliar career, emphasising that this encouragement can further the shared goal of empowering and preparing pupils to feel capable of choosing medicine. Reciprocally, medical schools should ensure pupils have fair opportunities for access, should they choose to apply.

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.