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The histone methyltransferase enhancer of zeste homolog 2 (EZH2)-mediated epigenetic regulation of T cell differentiation in acute infection has been extensively investigated. However, the role of EZH2 in T cell exhaustion remains under-explored. Here, usingin vitroexhaustion models, we demonstrated that transient inhibition of EZH2 in T cells before the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetastat, delayed their dysfunctional progression and maintained T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetestat induced T cell epigenetic reprogramming and increased the expression of the self-renewing T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. In a murine melanoma model, T cells pre-treated with tazemetastat exhibited a superior response to anti-PD-1 blockade therapy after adoptive transfer. Collectively, these data unveil the potential of transient epigenetic reprogramming as a potential intervention to be combined with checkpoint blockade for immune therapy.

Background: Checkpoint inhibitors have achieved a wide adoption for treatment of advanced cancer, but the majority of patients fail to respond. Among the underlying causes are redundant mechanisms driving cytotoxic lymphocyte dysfunction. Motivated by the discovery of JAK inhibitors as hit compounds in a screen for the reversal of T cell exhaustion, this study investigates the hypothesis that JAK inhibition could enhance the efficacy of checkpoint inhibitors. Methods: Murine immunocompetent models of cancer were utilized (A20, EL4, LLC1, MC38). The combination therapy of systemic treatment with the JAK inhibitor ruxolitinib with anti-PD1 + anti-CTLA4 was evaluated for efficacy and biomarkers compared to checkpoint inhibitors (ICI) alone. Tumor-infiltrating, blood and lymphoid organ immune cells were phenotyped using single-cell transcriptomics, functional assays and flow cytometry. The combination therapy was clinically tested in an investigator-initiated Phase I/II clinical trial of ruxolitinib with nivolumab in relapsed or refractory Hodgkin lymphoma (NCT03681561). Patients who previously failed to respond to ICI received ruxolitinib for 1 week then nivolumab every 4 weeks concurrent with ruxolitinib twice daily. Peripheral blood was collected at baseline, 1 week after ruxolitinib and 4 weeks after 1st nivolumab dose. Hematologic and transcriptomic analyses were performed on peripheral blood samples. Results: The ruxolitinib + ICI combination was superior to ICI in 3/4 of the tumor models examined in controlling tumor growth. Tumor sizes were reduced by >50% in the MC38 (mean volume 123.1 vs 283.2 mm3, ICI + rux vs ICI, respectively, n=9 per group, ANOVA p=0.0094), LLC1 and A20 models (survival hazard ratio 0.24, ICI + rux vs ICI, n=8 per group, p=0.048). Remarkably, we observed a broad shift of tumor monocytes and granulocytes from a suppressive into an immunostimulatory state characterized by the expression of MHC molecules and the ability to stimulate T cell proliferation. Hodgkin lymphoma patients in the ruxolitinib with nivolumab trial exhibited an interim disease control rate of 76% (13/17). Ruxolitinib treatment in these patients did not impair T cell cytokine production but significantly reduced the neutrophil-to-lymphocyte ratio (NLR, mean difference -0.82, n=14, p=0.0023) and the expression of myeloid derived suppressor cell markers in monocytes (normalized enrichment score -1.74, n=14, q Conclusions: The combination of ruxolitinib with ICI was effective in preclinical models of cancer and in a Phase I/II Hodgkin lymphoma clinical trial. Unexpectedly, JAK inhibitor-mediated reprogramming of myeloid cells and the associated enhanced T cell activity may be important for the observed efficacy. Citation Format: Jaroslav Zak, Isaraphorn Pratumchai, Brett S. Marro, Kristi L. Marquardt, Michael B. Oldstone, Judith A. Varner, Veronika Bachanova, John R. Teijaro. Reprogramming myeloid cells by JAK inhibition to enhance checkpoint blockade immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5743.

Unleashing anti-tumor T cell activity by checkpoint inhibition is effective in many cancer patients but clinical response rates remain limited. Myeloid derived suppressor cells erode antitumor lymphocyte numbers and function, and correlate with resistance to checkpoint inhibitors. By screening small molecule libraries, we identified JAK inhibitors’ ability to rescue T cell function. Despite its documented immune suppressive properties, the prototypical JAK inhibitor ruxolitinib enhanced the efficacy of immune checkpoint blockade in cancer. This effect correlated with loss of suppressive gene expression, and acquisition of immunostimulatory molecular markers and T cell stimulatory activity in myeloid cells. In preclinical models, ruxolitinib significantly improved the function and increased the total numbers of activated tumor-infiltrating NK and CD4 T cells compared to checkpoint blockade alone and the efficacy was conditional on granulocytic cells. In addition to myeloid reprogramming in the tumor, ruxolitinib blunts G-CSF signaling in the bone marrow to prevent expression of suppressive and chemotaxis genes in neutrophils. In a clinical trial of Hodgkin lymphoma patients resistant to checkpoint inhibitors, treatment with ruxolitinib significantly reduced neutrophil-to-lymphocyte ratios and levels of suppressive markers in myeloid cells but increased numbers of cytokine-producing T cells. These results support the therapeutic potential of JAK inhibition in combination with checkpoint inhibitors in cancer and highlight the potential of reshaped myeloid immunity to improve immunotherapy.One sentence summary: Ruxolitinib reshapes myeloid immunity to synergize with checkpoint inhibitors

Bispecific T-cell engager (BiTE)-based cancer therapies that activate the cytotoxic T cells of a patient’s own immune system have gained momentum with the recent FDA approval of Blinatumomab for treating B cell malignancies. However, this approach has had limited success in targeting solid tumors. Here, we report the development of BiTE-sialidase fusion proteins that enhance tumor cell susceptibility to BiTE-mediated cytolysis by T cells via targeted desialylation at the BiTE-induced T cell-tumor cell interface. Targeted desialylation results in better immunological synapse formation, T-cell activation and effector function. As a result, BiTE-sialidase fusion proteins show remarkably increased efficacy in inducing T-cell-dependent tumor cell cytolysis in response to target antigens compared to the parent BiTE molecules alone. This enhanced function is seen bothin vitroand inin vivoxenograft and syngeneic solid tumor mouse models. Our findings highlight BiTE-sialidase fusion proteins as promising candidates for the development of next-generation bispecific T-cell engaging molecules for cancer immunotherapy.

Summary: Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering T cell activity. In humans, several persisting viruses as well as cancers exploit IR signaling by upregulating IR ligands, resulting in suppression of T cell function (i.e., exhaustion). This allows escape from immune surveillance and continuation of disease. Here, we report the design, implementation, and results of a phenotypic high-throughput screen for molecules that modulate CD8+ T cell activity. We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted T cells. Analysis of our top hit, ingenol mebutate, a protein kinase C (PKC) inducing diterpene ester, reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells. Collectively, these results demonstrate a disease-relevant methodology for identifying modulators of T cell function and reveal new targets for immunotherapy. : Discovery of pharmacologic drugs that target exhausted T cells is essential to overcome the limitations of current checkpoint blockade therapies. Marro et al. utilize a high-throughput screening method to identify small-molecule modulators of T cells and describe a role for protein kinase C in resurrecting T cell effector activity. Keywords: T cell exhaustion, chronic infection, high-throughput flow cytometry, checkpoint blockade, CD8 T cell, PKC, ingenol mebutate

Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer.

Type 1 interferon provides potent antiviral defense but unchecked IFN-I signaling can lead to pathology or immune suppression. The mechanisms by which IFN-I contributes to disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of USP18 enzymatic function during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, pulmonary cytokine amplification, lung vascular leakage and death. Lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A knock-in mice and required Isg15. Mechanistically, interrupting the enzymes that conjugate/deconjugate ISG15 led to accumulation of ISG15 which was accompanied by inflammatory neutrophil accumulation and lung pathology. Moreover, neutrophil depletion reversed pathological manifestations, morbidity and mortality in Usp18C61A mice. Our results suggest that dysregulated ISG15 signaling during viral infection can produce lethal immune pathology and could be a therapeutic target during severe viral infections with pulmonary pathological manifestations.

Cytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion of Usp18. However, lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A mice which were dependent on Isg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A mice. In summary, we reveal that the enzymatic function of Usp18 is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.

IFNAR1 signaling in NK cells compromises cellular and humoral immune responses to hamper virus control., Inhibition of type 1 interferon (IFN-I) signaling promotes the control of persistent virus infection, but the underlying mechanisms remain poorly understood. Here, we report that genetic ablation of Ifnar1 specifically in natural killer (NK) cells led to elevated numbers of T follicular helper cells, germinal center B cells, and plasma cells and improved antiviral T cell function, resulting in hastened virus clearance that was comparable to IFNAR1 neutralizing antibody treatment. Antigen-specific B cells and antiviral antibodies were essential for the accelerated control of LCMV Cl13 infection following IFNAR1 blockade. IFNAR1 signaling in NK cells promoted NK cell function and general killing of antigen-specific CD4 and CD8 T cells. Therefore, inhibition of IFN-I signaling in NK cells enhances CD4 and CD8 T cell responses, promotes humoral immune responses, and thereby facilitates the control of persistent virus infection.

Background: Recent studies demonstrated that classical Hodgkin lymphoma (cHL) is characterized by chromosome 9p24.1 amplification with associated overexpression of PD-1 ligands as well as JAK2 tyrosine kinase activation. Immune evasion mediated by the PD-1/PD-1 ligand axis can be inhibited with IgG4 agonist antibody specific for human PD-1 nivolumab which has shown excellent overall response rate (ORR) of over 60% in relapsed cHL. JAK2 induces PD-1 ligand expression and augments tumor cell proliferation. JAK2 signaling can be inhibited with clinical grade small molecules (i.e. ruxolitinib). Given this biology, we designed a Phase I/II study to target both pathways in cHL patients who had failed PD-1 blocking agents. Methods: This is a Phase I/II, multicenter, open-label, dose escalation/dose-expansion study to evaluate the safety and tolerability of ruxolitinib when combined with nivolumab (fixed dose 3mg/kg IV every 2 weeks) in patients with relapsed or refractory (R/R) cHL (NCT03681561). Eligible patients had to fail prior check-point inhibitor (CPI; nivolumab or pembrolizumab). Three dose levels of ruxolitinib were tested: 10 mg orally twice a day (bid), 15 mg bid, and 20 mg bid. Planned duration of therapy was 2 years. The primary objective was to find the maximum tolerated dose (MTD) of ruxolitinib when given with nivolumab and characterize the safety and tolerability of this combination. The phase 2 objective was to evaluate the overall response rate (ORR). Results: We enrolled 19 patients. Median age was 38 years (range 22-76); 68% were males. Patients were a median of 3.4 years from the initial diagnosis (range 0.9-16.7 years), 89% had stage III-IV disease and all had experienced progressive disease following CPI. 17 (89%) had prior autologous HCT and 1 had prior allogeneic HCT. All patients received nivolumab combined with ruxolitinib, and the highest ruxolitinib dose 20mg BID (MTD) was reached without DLTs. The combination was well tolerated; most AEs were grade 1 and 2. Only 3 patients required hospitalization (2 had pneumonia, one for disease progression). Three patients experienced immune mediated adverse events (LFT elevation: 1 Gr 1 and 1 Gr 2; 1 Gr 3 pneumonitis) and all were reversible. Three subjects were not evaluable for response (2 patients who received < 1 month of therapy due to rapid disease progression and Gr 2 immune hepatitis and 1 patient with short follow-up is not yet evaluable for response). Median follow-up was 13 months (range 3-27 months). In 16 patients evaluable for response, best ORR was 75% (12 of 16); including 3 CRs (19%), 2 PRs (13%), 6 patients had stable disease (SD, 44%) with tumor bulk reduction ranging from 10-45%. One patient had indeterminate response (negative biopsy of new PET-avid site). Duration of responses were 12.5 months (ranged from 3.7 to 20.4 months); 5 responders continue study therapy and 2 completed 2 years; 1 went on to autologous HCT in PR, and 3 progressed while on therapy after 6, 6 and 23 months. One patient died after later experimental therapy. Progression-free survival at 1 year was 64% (95%CI 34-84%). Conclusions: Inhibition of JAK2 combined blockade of the PD-1 pathway represent complementary rational therapeutic targets in cHL. Therapy combining ruxolitinib with nivolumab is well tolerated and yield encouragingly high remission rates and durable responses in patients who had all failed previous CPI. Pharmacodynamic and correlative analyses on the mechanism of synergism are underway. Disclosures Bachanova: KaryoPharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; FATE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janakiram: ADC Therapeutics: Research Funding; FATE Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding; Kyowa Kirin Therapeutics: Honoraria. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Farooq: Kite, a Gilead Company: Honoraria. OffLabel Disclosure: ruxolitinib for Hodgkin lymphoma

Contains fulltext : 206572.pdf (Publisher’s version ) (Open Access) We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

CXCR5+ TCF1+ CD8+ T cells sustain responses during persistent viral infection and mediate the proliferative burst following anti-PD1 treatment. Huang et al. show that IL-27 supports rapid division of these cells by competing with type 1 interferon for STAT1, driving IRF1 expression and preventing cell death., Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer., Graphical Abstract

Immune checkpoint blockade (ICB) therapy has emerged as viable first-line treatment for cancers such as melanoma. Despite the striking long-term clinical benefits that were previously unattainable, the effectiveness of ICB therapy remains limited in a majority of patients and cancer types. This is due in part to insufficient activation and/or restoration of anti-tumor T cell responses. Thus, understanding the signaling pathways that potentiate T cell exhaustion is essential for developing pharmacologic drugs that reactivate T cells and overcome the limitations of current ICB therapy. To this end, we developed a biologically relevant phenotypic screening system using the LCMV-CL13 infection model coupled with high-throughput flow cytometry to identify small-molecules that resurrect hypofunctional T cells. We discovered 19 hits following a screen of a manually curated collection of 12,000 repurposed drugs. Among the lead compounds identified were janus kinase (JAK) inhibitors including the anti-myelofibrosis drug ruxolitinib. Phenotypic rescue of exhausted T cells in vitro following ruxolitinib treatment resulted in a unique transcriptional signature that diverged from anti-PD-L1 treatment. Lineage-defining genes known to preserve precursor exhausted CD8+ T cells, including transcription factor 7 (Tcf7), were significantly upregulated following exposure to ruxolitinib. Mechanistically, ruxolitinib attenuated the cumulative pSTAT signaling signature within resurrected cells to enhance their survival. Collectively, these results demonstrate a disease-relevant framework for identifying small-molecule modulators of dysfunctional T cells and suggest that JAKs are viable targets for cancer immunotherapy.

ISG15 is one of the strongest type I interferon (IFN-I) stimulated genes (ISGs) which is conjugated to proteins in an enzymatic process termed ISGylation. Protein ISGylation is subsequently reversed by the enzyme Usp18 which acts in a de-ubiqutinase fashion to remove SIG15 from target proteins. Many proteins are ISGylated following IFN-I signaling however, immunological studies have been limited to the anti-viral properties of ISG15 conjugation while its regulation of innate and adaptive immune responses has been poorly studied. Here using Usp18−/− and enzymatically inactive (Usp18C61A/C61A Knock-in) mouse models, we show that hyper-ISGylation during chronic viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. The pathology associated with chronic infection required T cells but not T cell-intrinsic Usp18 expression. We reveal that lack of Usp18 in LysM+ cells reproduced the pathological manifestations observed in Usp18−/− or Usp18C61A/C61A mice. Using single cell sequencing, we observed that hyper-ISGylation during chronic infection altered neutrophil maturation and promoted an inflammatory neutrophil lung infiltrate. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A/C61A mice. In summary, we reveal Usp18’s enzymatic function is crucial for regulating inflammatory neutrophil differentiation and preventing immune hyperactivation during chronic viral infection. Moreover, our results suggest that hyper-ISGylation may drive the inflammatory pathology observed in Usp18-null patients and indicate that regulation of ISG15 expression or conjugation could lessen disease.

Emerging evidence indicates that B lymphocytes regulate immunity by producing antibodies, presenting antigens and secreting cytokines that provide signals to other cells to modulate both humoral and cellular immunity. Here, we show that B cells produce IL-27 upon activation by TLR4 and CD40 in the presence of IL-21 in vitro. Using Lymphocytic choriomeningitis virus (LCMV) Clone 13 as a model of a persistent viral infection, we identify plasma B cells as major source of IL-27. Further, we show that IL-27-producing B cells are key players in the regulation of immunity. Mice with conditional B cell–specific IL-27p28 knockout were unable to control persistent LCMV infection and failed to respond to anti-IFNAR1 immunotherapy. The impaired immunity found in these mice was associated with reduced virus-specific IgG2a/2c. In addition, these mice displayed reduced virus-specific CD4+ T cells and T follicular helper (Tfh) cells, suggesting that B-cell-derived cytokines regulate immunity by providing signals to T cells. The effects of Il27ra deletion from T cells mirrors the phenotypes observed in mice lacking B-cell-derived IL-27. In addition, IL-27ra deficient (KO) Tfh cells lost their ability to produce IL-21, resulting in aberrant production of virus-specific antibodies to LCMV clone 13 in IL-27ra KO mice. Finally, RNA-sequencing of virus-specific CD4+ T cells identified IL-27R-dependent genes during persistent LCMV infection. Collectively, our data reveal a central role of plasma cell-derived IL-27 in the regulation of T cell functions. Importantly, our study describes cellular interactions between T and B cells and highlights the importance of virus-specific IgG2a/2c during persistent LCMV infection.

High-throughput screening (HTS) techniques have enabled large scale image-based studies, but extracting biological insights from the imaging data in an exploratory setting remains a challenge. Existing packages for this task either require expert annotations, which can bias the outcome of the study, or are completely unsupervised, failing to leverage the information present in the assay design. We present HTX, an interactive tool to aid in the exploration of large microscopy data sets by allowing the visualization of entire image-based assays according to visual similarities between the samples in an intuitive and navigable manner. Underlying HTX are a collection of novel algorithmic techniques for deep texture descriptor learning, 2D data visualization, adversarial suppression of batch effects, and backprop-based image saliency estimation.We demonstrate that HTX can exploit the screen meta-data in order to learn screen-specific image descriptors, which are then used to quantify the visual similarity between samples in the assay. Given these similarities and the different visualization resources of HTX, it is shown that screens of small-molecule libraries on cell data can be easily explored, reproducing the results of previous studies where highly-specific domain knowledge was required.

Significance Type 1 diabetes (T1D) is an increasing medical health problem caused by autoimmune T cells killing insulin-producing β cells in the islets of Langerhans. When the majority of β cells are destroyed, unless exogenous insulin is administered, ketoacidosis and death follow. However, providing exogenous insulin does not prevent the resultant complications of stroke, heart disease, visual impairment, or faulty wound healing, indicating the need to preserve β cells in the prediabetic stage to insure endogenous insulin production. We show that blockage of IFN-α signaling prior to clinical T1D disease by antibody or a sphingosine-1 receptor agonist prevents autoaggressive T cells from entering the islets and killing β cells. The result is aborting T1D by acting at the prediabetic stage.

Overpasses are essential to establish permeability of highways for the wildlife. Width and length are the key technical parameters influencing the overpass effectiveness. It is crucial to secure the proper technology and correct water management on its surface. Wildlife behaviour and their requirements for the above stated technical parameters of overpasses have also been investigated. In all cases, the overpass performance increases with enlarging the width and drop with an increasing length. The goal is therefore to achieve a balanced compromise allowing sufficient functionality while also sustaining reasonable costs. Data envelopment analysis (DEA) provides a method to select a “good” solution in the sense of sufficient overpass performance with moderate total expenses. Overpass performance calculations were made on the original methodology pioneered by the author. Overall expenses were provided based on actual construction costs in the Czech Republic, including expenses for preparations, design, construction, maintenance, and demolition. The results show that DEA has proven to be a very appropriate method for identifying suitable solutions that can be effective in designing measures to secure the highway’s permeability for the wildlife.

B cells regulate immune responses by both generating antibodies and through cell surface interactions and cytokine production that provide signals to various cells to modulate humoral and cell-mediated immunity. During persistent viral infection, IL-27R is required for viral control which correlates with impaired anti-viral CD4+ T cell responses and lower LCMV-specific antibody production. Here, using mice with conditional B cell–specific IL-27 p28 deletion, we identify IL-27-producing B cells as key players in promoting control of persistent viral infection. Mice in which B cells lacked IL-27 p28 expression lost the ability to control persistent LCMV infection. The impaired immunity was associated with the reduction of GC B cells, plasma cells and Tfh cells. In addition, these mice also displayed reduced virus-specific CD8 T cells and lost the ability to expand CXCR5+ follicular CD8 T cells following anti-IFNAR1 treatment, suggesting that B-cell-derived IL-27 p28 regulates immunity by providing signals to T cells. To assess the role of IL-27 signaling in T cells we generated IL-27R conditional KO mice by mating IL-27Ra-floxed mice with CD4 and Granzyme B-Cre strains. Mice in which IL-27Ra was conditionally ablated in all T cells lost the ability to control persistent LCMV infection. In addition, these mice produced significantly lower titers of virus-specific IgG2a throughout the course of infection despite similar levels of virus-specific IgG1. Finally, we identify plasma cells expressing the transcription factor Blimp1 as a major source of IL-27 p28 during persistent LCMV infection. Overall, our study highlights the importance of IL-27-producing B cells in regulating T cell responses and control of persistent viral infection.

Eliminating hematopoietic stem cells (HSCs) with DNA damage is necessary to maintain the homeostasis of HSCs, but the mechanisms underlying this apoptotic elimination are unclear. Now in Cell Stem Cell, Yamashita et al. (2015) show that Aspp1 coordinates with p53 to protect HSC pool integrity, guarding against hematological malignancies.

Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.Cell Death and Differentiation advance online publication, 22 July 2016; doi:10.1038/cdd.2016.76.

Background: The Czech Pneumological and Phthisiological Society (CPPS) has prepared new national guidelines. Diagnosis: The essential requirement for the diagnosis of COPD is the presence of a post-bronchodilator expiratory airflow limitation, defined as FEV1/VC < LLN. GOLD classification allows us to classify each patient into one of the four well known categories.The most common clinical presentation are bronchitic and/or emphysemateous phenotypes. Next variants of COPD syndrome consist of: frequent exacerbators, COPD and asthma overlap syndrome (ACOS), bronchiectatic COPD and COPD with pulmonary cachexia. A clinically apparent phenotype can be found especially in B and D categories. Therapy: Treatment of COPD patients is generally determined by the disease categories, and the presence of phenotype(s) of the disease, while considering the presence of complications and comorbidities. CPPS treatment recommendations can be divided into four steps. Conclusion: The CPPS aimed to place these guidelines into the context of the healthcare system in the Czech Republic. Emphasis is placed on personalized care, targeting the symptoms and phenotypes of each patient, taking into consideration comorbidities.

SummaryRegulation of nuclear import is fundamental to eukaryotic biology. The majority of nuclear import pathways are mediated by importin-cargo interactions. Yet not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Here, we identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway and is frequently used by AR-containing transcriptional regulators, especially those regulating NF-κB/p53.

Epithelial to mesenchymal transition (EMT), and the reverse mesenchymal to epithelial transition (MET), are known examples of epithelial plasticity that are important in kidney development and cancer metastasis. Here we identify ASPP2, a haploinsufficient tumour suppressor, p53 activator and PAR3 binding partner, as a molecular switch of MET and EMT. ASPP2 contributes to MET in mouse kidney in vivo. Mechanistically, ASPP2 induces MET through its PAR3-binding amino-terminus, independently of p53 binding. ASPP2 prevents β-catenin from transactivating ZEB1, directly by forming an ASPP2-β-catenin-E-cadherin ternary complex and indirectly by inhibiting β-catenin's N-terminal phosphorylation to stabilize the β-catenin-E-cadherin complex. ASPP2 limits the pro-invasive property of oncogenic RAS and inhibits tumour metastasis in vivo. Reduced ASPP2 expression results in EMT, and is associated with poor survival in hepatocellular carcinoma and breast cancer patients. Hence, ASPP2 is a key regulator of epithelial plasticity that connects cell polarity to the suppression of WNT signalling, EMT and tumour metastasis.

Background. COPD is a global concern. Currently, several sets of guidelines, statements and strategies to managing COPD exist around the world. Methods. The Czech Pneumological and Phthisiological Society (CPPS) has commissioned an Expert group to draft recommended guidelines for the management of stable COPD. Subsequent revisions were further discussed at the National Consensus Conference (NCC). Reviewers’ comments contributed to the establishment of the document’s final version. Diagnosis. The hallmark of the novel approach to COPD is the integrated evaluation of the patient’s lung functions, symptoms, exacerbations and identifications of clinical phenotype(s). The CPPS defines 6 clinically relevant phenotypes: frequent exacerbator, COPD-asthma overlap, COPD-bronchiectasis overlap, emphysematic phenotype, bronchitic phenotype and pulmonary cachexia phenotype. Treatment. Treatment recommendations can be divided into four steps. 1 st step = Risk exposure elimination: reduction of smoking and environmental tobacco smoke (ETS), decrease of home and occupational exposure risks. 2 nd step = Standard treatment: inhaled bronchodilators, regular physical activity, pulmonary rehabilitation, education, inhalation training, comorbidity treatment, vaccination. 3 rd step = Phenotype-specific therapy: PDE4i, ICS+LABA, LVRS, BVR, AAT augmentation, physiotherapy, mucolytic, ABT. 4 th step = Care for respiratory insufficiency and terminal COPD: LTOT, lung transplantation, high intensity-NIV and palliative care. Conclusion. Optimal treatment of COPD patients requires an individualised, multidisciplinary approach to the patient’s symptoms, clinical phenotypes, needs and wishes. The new Czech COPD guideline reflects and covers these requirements.

Cancer and developmental disorders (DD) share dysregulated cellular processes such as proliferation and differentiation. There are well-known genes implicated in both in cancer and DD. In this study, we aim to quantify this genetic connection using publicly available data. We found that among DD patients, germline damaging de novo variants are more enriched in cancer driver genes than non-drivers. We estimate that cancer driver genes comprise about a third of DD risk genes. Additionally, de novo likely-gene-disrupting (LGD) variants are more enriched in tumor suppressors, and about 40% of implicated de novo damaging missense variants are located in cancer somatic mutation hotspots, indicating that many genes have a similar mode of action in cancer and DD. Our results suggest that we can view tumors as natural laboratories for assessing the deleterious effects of mutations that are applicable to germline variants and identification of causal genes and variants in DD.

ydroxylation is an emerging modification catalysed by a family of ‘2-oxoglutarate (2OG)-dependent oxygenases’ that includes the Collagen and HIF hydroxylases, TET 5hmC demethylases, and JmjC histone demethylases. These enzymes are widely implicated in cancer due to roles in hypoxia signalling, extracellular matrix formation, and epigenetics, and because of their inhibition by oncometabolites. Although other sub-families of 2OG-oxygenases exist they are less well characterised and their role in cancer remains largely unexplored.

Despite their usefulness as fluorophores and synthetic precursors, efficient and reliable routes to coumarin-8-carbaldehydes are lacking. We describe here a high-yielding continuous flow synthesis that requires no manual intermediate purification or work-up, giving access to multigram quantities of the aldehyde product.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Snake bites caused by viperid snakes of Atheris genus are extremely rare, envenoming of a bite of related viper Proatheris superciliaris was described only once in the literature. The present case study depicts the envenoming of a 57 years old Czech man, a private herpetologist, who was bitten to his finger. He developed painful local reaction, nausea, hematuria, hypertension, chest and lumbar pain. Coagulopathy and thrombocytopenia subsequently developed as well as acute renal failure, hepatic and lung lesion. Intensive care therapy was purely symptomatic and supportive as no antisera exists. Treatment included haemodialysis, substitution of fresh frozen plasma and platelets. Patient completely recovered during 1 month.

The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.