A combination of stromal PD-L1 and tumoral nuclear ß-catenin expression as an indicator of colorectal carcinoma progression and resistance to chemoradiotherapy in locally advanced rectal carcinoma.

Programmed cell death-1 (PD-1) and its ligand (PD-L1) are significant mediators of immune suppression in the tumor microenvironment. We focused on the immunological impact of PD-1/PD-L1 signaling during tumor progression in colorectal carcinoma (CRC) and its association with resistance to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal carcinoma (LAd-RC). Histopathological and immunohistochemical analyses of 100 CRC cases (including 34 RC) without NCRT and 109 NCRT-treated LAd-RC cases were performed. Membranous tumoral PD-L1 expression was identified in 9 of 100 (9%) CRC cases, including 1 of 34 (2.9%) RC cases, but PD-L1 immunopositivity was not associated with any clinicopathological factors, with the exception of deficient mismatch repair (dMMR) status. In contrast, stromal PD-L1+ immune cells, which frequently exhibited coexpression of PD-1 and CD8 markers, were significantly correlated with tumor vessel invasion, nuclear ß-catenin+ tumor budding cancer stem cell (CSC)-like features, and unfavorable prognosis. In the LAd-RC cases, stromal CD8+ (but not PD-L1+) immune cell infiltration in pretreatment-biopsied samples was significantly and positively associated with therapeutic efficacy. After NCRT, tumoral PD-L1 expression was observed in only 2 of 83 (2.4%) tumors, independent of dMMR status, whereas high stromal PD-L1+ and tumoral nuclear ß-catenin positivity were significantly linked to a poor response to NCRT and high tumor budding features. In addition, high stromal PD-L1 immunoreactivity was significantly associated with poorer overall survival. In conclusion, a combination of stromal PD-L1+ immune cells and nuclear ß-catenin+ tumor budding may contribute to tumor progression in CRC and resistance to NCRT in LAd-RC, through formation of niche-like lesions that exhibit immune resistance and CSC properties.

Nuclear cGAS: guard or prisoner?

cGAS, an innate immune sensor of cellular stress, recognizes double-stranded DNA mislocalized in the cytosol upon infection, mitochondrial stress, DNA damage, or malignancy. Early models suggested that cytosolic localization of cGAS prevents autoreactivity to nuclear and mitochondrial self-DNA, but this paradigm has shifted in light of recent findings of cGAS as a predominantly nuclear protein tightly bound to chromatin. This has raised the question how nuclear cGAS is kept inactive while being surrounded by chromatin, and what function nuclear localization of cGAS may serve in the first place? Cryo-EM structures have revealed that cGAS interacts with nucleosomes, the minimal units of chromatin, mainly via histones H2A/H2B, and that these protein-protein interactions block cGAS from DNA binding and thus prevent autoreactivity. Here, we discuss the biological implications of nuclear cGAS and its interaction with chromatin, including various mechanisms for nuclear cGAS inhibition, release of chromatin-bound cGAS, regulation of different cGAS pools in the cell, and chromatin structure/chromatin protein effects on cGAS activation leading to cGAS-induced autoimmunity.

Cytoplasmic DNA sensing by KU complex in aged CD4+ T cell potentiates T cell activation and aging-related autoimmune inflammation.

Aging is associated with DNA accumulation and increased homeostatic proliferation of circulating T cells. Although these attributes are associated with aging-related autoimmunity, their direct contributions remain unclear. Conventionally, KU complex, the regulatory subunit of DNA-dependent protein kinase (DNA-PK), together with the catalytic subunit of DNA-PK (DNA-PKcs), mediates DNA damage repair in the nucleus. Here, we found KU complex abundantly expressed in the cytoplasm, where it recognized accumulated cytoplasmic DNA in aged human and mouse CD4+ T cells. This process enhanced T cell activation and pathology of experimental autoimmune encephalomyelitis (EAE) in aged mice. Mechanistically, KU-mediated DNA sensing facilitated DNA-PKcs recruitment and phosphorylation of the kinase ZAK. This activated AKT and mTOR pathways, promoting CD4+ T cell proliferation and activation. We developed a specific ZAK inhibitor, which dampened EAE pathology in aged mice. Overall, these findings demonstrate a KU-mediated cytoplasmic DNA-sensing pathway in CD4+ T cells that potentiates aging-related autoimmunity.

Attenuation of canonical NF-κB signaling maintains function and stability of human Treg.

Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy.

Localization of CDR2L and CDR2 in paraneoplastic cerebellar degeneration.

OBJECTIVE: Identify the subcellular location and potential binding partners of two cerebellar degeneration-related proteins, CDR2L and CDR2, associated with anti-Yo-mediated paraneoplastic cerebellar degeneration. METHODS: Cancer cells, rat Purkinje neuron cultures, and human cerebellar sections were exposed to cerebrospinal fluid and serum from patients with paraneoplastic cerebellar degeneration with Yo antibodies and with several antibodies against CDR2L and CDR2. We used mass spectrometry-based proteomics, super-resolution microscopy, proximity ligation assay, and co-immunoprecipitation to verify the antibodies and to identify potential binding partners. RESULTS: We confirmed the CDR2L specificity of Yo antibodies by mass spectrometry-based proteomics and found that CDR2L localized to the cytoplasm and CDR2 to the nucleus. CDR2L co-localized with the 40S ribosomal protein S6, while CDR2 co-localized with the nuclear speckle proteins SON, eukaryotic initiation factor 4A-III, and serine/arginine-rich splicing factor 2. INTERPRETATION: We showed that Yo antibodies specifically bind to CDR2L in Purkinje neurons of PCD patients where they potentially interfere with the function of the ribosomal machinery resulting in disrupted mRNA translation and/or protein synthesis. Our findings demonstrating that CDR2L interacts with ribosomal proteins and CDR2 with nuclear speckle proteins is an important step toward understanding PCD pathogenesis.

A proof of evidence supporting abnormal immunothrombosis in severe COVID-19: naked megakaryocyte nuclei increase in the bone marrow and lungs of critically ill patients.

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

Exportin 1 inhibition as antiviral therapy.

Coronavirus 2019 (COVID-19; caused by Severe Acute Respiratory Syndrome Coronavirus 2; SARS-CoV-2) is a currently global health problem. Previous studies showed that blocking nucleocytoplasmic transport with exportin 1 (XPO1) inhibitors originally developed as anticancer drugs can quarantine key viral accessory proteins and genomic materials in the nucleus of host cell and reduce virus replication and immunopathogenicity. These observations support the concept of the inhibition of nuclear export as an effective strategy against an array of viruses, including influenza A, B, and SARS-CoV. Clinical studies using the XPO1 inhibitor selinexor as a therapy for COVID-19 infection are in progress.

Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance.

Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, forms large aggregate-like assemblies visualized as nuclear foci. Here we demonstrate that Aire utilizes its caspase activation recruitment domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization also makes Aire susceptible to interaction with promyelocytic leukemia protein (PML) bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs the transcriptional activity of Aire, while dispersing PML bodies with a viral antagonist restores this activity. Our study thus reveals a new regulatory role of PML bodies in Aire function, and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated protein quality control.

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Protein Phosphatase PP1 Negatively Regulates IRF3 in Response to GCRV Infection in Grass Carp (Ctenopharyngodon idella).

Protein phosphatase-1 (PP1) has an important role in many cell functions, such as cell differentiation, development, immune response and tumorigenesis. However, the specific role of PP1 in the antiviral response in fish remains to be elucidated. In this study, the PPP1R3G homolog was identified in the grass carp (Ctenopharyngodon idella) and its role in defence against the GCRV infection was investigated. Phylogenetic analysis demonstrated that CiPPP1R3G clustered with homologues from other teleosts. Temporal expression analysis in vivo revealed that the expression level of CiPPP1R3G was significantly up-regulated in response to GCRV infection in grass carps, especially in the intestine and head-kidney. Cellular distribution analysis revealed that CiPPP1R3G was located in the nucleus and cytoplasm. Overexpression of CiPPP1R3G significantly negatively regulated the expression of CiIRF3, thus inhibiting its activation. In summary, we systematically analyzed the PPP1R3G gene in grass carp and illustrated its function as a negative regulator in the anti-GCRV immune responses.

Methods for measuring HMGB1 release during immunogenic cell death.

The exodus of the alarmin high mobility group box 1 (HMGB1) from the nucleus constitutes a crucial cellular danger signal and manifests as a sequential process in which HMGB1 first exits the nucleus into the cytoplasm and then is secreted or passively released through the permeabilized plasma membrane. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate innate immune responses. Here, we describe a discovery pipeline for the identification of pharmacological agents endowed with HMGB1 releasing properties. The "retention using selective hooks" (RUSH) system in which a streptavidin-NLS3 fusion protein serves as a nuclear hook to sequester streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP) allowed for synchronizing HMGB1 increase. Thus, exclusively in the presence of biotin, which liberates HMGB1-SBP-GFP from its nuclear hook, immunogenic cell death (ICD) inducers such as anthracyclines are able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system facilitates the identification of HMGB1 releasing agents in medium- to high-throughput screening assays.

Different measures of HMGB1 location in cancer immunology.

HMGB1 is the most abundant non-histone nuclear protein. It regulates transcriptional access to open areas of chromatin and limits release of DNA with apoptotic death, serving to both inhibit apoptosis and promote DNA repair. When HMGB1 is translocated to the cytosol with many types of cellular stress, it is a powerful inducer of autophagy. It can also be released by activated immune cells and damaged or dying cells into the extracellular space, where it acts as a damage associated molecular pattern (DAMP) molecule, contributing to the pathogenesis and progression of cancer. Here, the most common methodologies to not only measure HMGB1 but also to effectively determine its subcellular localization, which dictates many of HMGB1's different functions, are reviewed.

Macrophage-Specific NF-κB Activation Dynamics Can Segregate Inflammatory Bowel Disease Patients.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.

A versatile flow-based assay for immunocyte-mediated cytotoxicity.

Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.

Activin-A in the regulation of immunity in health and disease.

The TGF-ß superfamily of cytokines plays pivotal roles in the regulation of immune responses protecting against or contributing to diseases, such as, allergy, autoimmunity and cancer. Activin-A, a member of the TGF-ß superfamily, was initially identified as an inducer of follicle-stimulating hormone secretion. Extensive research over the past decades illuminated fundamental roles for activin-A in essential biologic processes, including embryonic development, stem cell maintenance and differentiation, haematopoiesis, cell proliferation and tissue fibrosis. Activin-A signals through two type I and two type II receptors which, upon ligand binding, activate their kinase activity, phosphorylate the SMAD2 and 3 intracellular signaling mediators that form a complex with SMAD4, translocate to the nucleus and activate or silence gene expression. Most immune cell types, including macrophages, dendritic cells (DCs), T and B lymphocytes and natural killer cells have the capacity to produce and respond to activin-A, although not in a similar manner. In innate immune cells, including macrophages, DCs and neutrophils, activin-A exerts a broad range of pro- or anti-inflammatory functions depending on the cell maturation and activation status and the spatiotemporal context. Activin-A also controls the differentiation and effector functions of Th cell subsets, including Th9 cells, TFH cells, Tr1 Treg cells and Foxp3+ Treg cells. Moreover, activin-A affects B cell responses, enhancing mucosal IgA secretion and inhibiting pathogenic autoantibody production. Interestingly, an array of preclinical and clinical studies has highlighted crucial functions of activin-A in the initiation, propagation and resolution of human diseases, including autoimmune diseases, such as, systemic lupus erythematosus, rheumatoid arthritis and pulmonary alveolar proteinosis, in allergic disorders, including allergic asthma and atopic dermatitis, in cancer and in microbial infections. Here, we provide an overview of the biology of activin-A and its signaling pathways, summarize recent studies pertinent to the role of activin-A in the modulation of inflammation and immunity, and discuss the potential of targeting activin-A as a novel therapeutic approach for the control of inflammatory diseases.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses.

DNA viruses typically eject genomic DNA into the nuclei of host cells after entry. It is unclear, however, how nuclear pathogen-derived DNA triggers innate immune responses. We report that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) recognizes pathogenic DNA and amplifies interferon-α/ß (IFN-α/ß) production. Upon DNA virus infection, nuclear-localized hnRNPA2B1 senses viral DNA, homodimerizes, and is then demethylated at arginine-226 by the arginine demethylase JMJD6. This results in hnRNPA2B1 translocation to the cytoplasm where it activates the TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway, leading to IFN-α/ß production. Additionally, hnRNPA2B1 facilitates N 6-methyladenosine (m6A) modification and nucleocytoplasmic trafficking of CGAS, IFI16, and STING messenger RNAs. This, in turn, amplifies the activation of cytoplasmic TBK1-IRF3 mediated by these factors. Thus, hnRNPA2B1 plays important roles in initiating IFN-α/ß production and enhancing stimulator of interferon genes (STING)-dependent cytoplasmic antiviral signaling.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.