bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒05‒03
thirty papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research

  1. Cell Signal. 2020 Apr 22. pii: S0898-6568(20)30131-5. [Epub ahead of print] 109654
    Chen M, Huang M, Chen W, Jiang X, Su W, Wang X, Tang N, She F, Chen Y.
      Gallbladder carcinoma has a high degree of malignancy. No effective treatment exists for patients with advanced tumors. The second mitochondria-derived activator of caspases (Smac) is the antagonist of the inhibitors of apoptosis protein. Smac mimetics are a class of effective tumor-targeted drugs undergoing clinical trials. However, studies on the effect of Smac mimetics on gallbladder cancer are unavailable. In this study, Smac mimetics can promote tumor necrosis factor-α (TNF-α) to inhibit the proliferation of gallbladder cancer cells and activate the apoptotic pathway, thereby promoting the ubiquitination of Lys48 on Receptor interacting protein kinase-1 (RIPK1) and leading to proteasomal degradation that causes damage to RIPK1 protein integrity. The formation of complex I (RIPK1, tumor necrosis factor 1-associated death domain protein, and TNF receptor-associated factor 2) is inhibited. Then, nonubiquitinated RIPK1 binds with the Fas-associated death domain and caspase-8 to form complex II and promotes the death receptor pathway of apoptosis. Animal experiments further verify that TNF-α combined with Smac mimetics can inhibit the growth of transplanted tumors and induce the apoptosis of transplanted tumor cells. This research provides a new direction for the targeted therapy of gallbladder cancer.
    Keywords:  Apoptosis; Gallbladder carcinoma; RIPK1; Smac mimetic; TNF-α
  2. J Leukoc Biol. 2020 Apr 30.
    Field NS, Moser EK, Oliver PM.
      The E3 ubiquitin ligase Itch has long been appreciated to be a critical suppressor of inflammation, first identified as a regulator of Th2 differentiation and lung inflammation. Recent studies have revealed novel roles for this protein in mouse and human disease, and it is now clear that Itch also limits the function of other lymphocytes, innate immune cells, and nonhematopoietic cells to regulate immunity. In addition to Th2 cells, Itch also regulates Th17 and regulatory T cells. Itch regulates humoral immunity through direct roles in T follicular helper cells and T follicular regulatory cells, and B cells. Furthermore, Itch limits innate immune responses, such as macrophage cytokine production. Through these cell-intrinsic functions, Itch regulates the interplay between innate and adaptive immune cells, resulting in profound autoinflammation in Itch-deficient mice. Whereas Itch deficiency was previously thought to be an extremely rare occurrence humans, whole exome sequencing of patients with unexplained autoimmune disease has revealed at least two additional cases of Itch deficiency in the last year alone, each caused by distinct mutations within the Itch gene. The recent identification of these patients suggests that Itch mutations may be more common than previously thought, and demonstrates the need to understand how this protein regulates inflammation and autoimmune disease.
    Keywords:  T cell differentiation; autoantibody production; autoinflammation; cytokine production; ubiquitin
  3. Cell Death Differ. 2020 Apr 27.
    Fullstone G, Bauer TL, Guttà C, Salvucci M, Prehn JHM, Rehm M.
      The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.
  4. Cells. 2020 Apr 27. pii: E1083. [Epub ahead of print]9(5):
    Gasic I, Groendyke BJ, Nowak RP, Yuan JC, Kalabathula J, Fischer ES, Gray NS, Mitchison TJ.
      Dysregulation of microtubules and tubulin homeostasis has been linked to developmental disorders, neurodegenerative diseases, and cancer. In general, both microtubule-stabilizing and destabilizing agents have been powerful tools for studies of microtubule cytoskeleton and as clinical agents in oncology. However, many cancers develop resistance to these agents, limiting their utility. We sought to address this by developing a different kind of agent: tubulin-targeted small molecule degraders. Degraders (also known as proteolysis-targeting chimeras (PROTACs)) are compounds that recruit endogenous E3 ligases to a target of interest, resulting in the target's degradation. We developed and examined several series of α- and β-tubulin degraders, based on microtubule-destabilizing agents. Our results indicate, that although previously reported covalent tubulin binders led to tubulin degradation, in our hands, cereblon-recruiting PROTACs were not efficient. In summary, while we consider tubulin degraders to be valuable tools for studying the biology of tubulin homeostasis, it remains to be seen whether the PROTAC strategy can be applied to this target of high clinical relevance.
    Keywords:  PROTAC; microtubule; tubulin
  5. Cell Death Differ. 2020 Apr 27.
    Lalaoui N, Merino D, Giner G, Vaillant F, Chau D, Liu L, Kratina T, Pal B, Whittle JR, Etemadi N, Berthelet J, Gräsel J, Hall C, Ritchie ME, Ernst M, Smyth GK, Vaux DL, Visvader JE, Lindeman GJ, Silke J.
      Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.
  6. Int J Mol Sci. 2020 Apr 28. pii: E3116. [Epub ahead of print]21(9):
    Ha J, Kim M, Seo D, Park JS, Lee J, Lee J, Park SH.
      p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.
    Keywords:  apoptosis; cell survival; nuclear factor-κB; p62; tumor necrosis factor α; ubiquitin-specific protease 20
  7. PLoS Pathog. 2020 Apr 27. 16(4): e1008458
    Cammarata-Mouchtouris A, Nguyen XH, Acker A, Bonnay F, Goto A, Orian A, Fauvarque MO, Boutros M, Reichhart JM, Matt N.
      The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by IL-1β in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.
  8. J Med Chem. 2020 Apr 30.
    Hanan EJ, Liang J, Wang X, Blake R, Blaquiere N, Staben ST.
      The discovery and development of targeted protein degraders has become an important area of research in the field of medicinal chemistry. Inducing degradation of a target protein presents several advantages relative to simple inhibition including a potential for extended duration of action and more profound pharmacology. Whilst engineered heterodimeric molecules have recently been a major focus within industry and academia, this perspective highlights examples of targeted protein degradation observed for smaller, monomeric molecules. Methods and tools for evaluating protein degradation as well as a discussion of physical properties of monomeric vs. engineered heterodimeric degraders are presented.
  9. Int J Mol Sci. 2020 Apr 25. pii: E3036. [Epub ahead of print]21(9):
    Sewduth RN, Baietti MF, Sablina AA.
      Ubiquitination is a versatile and dynamic post-translational modification in which single ubiquitin molecules or polyubiquitin chains are attached to target proteins, giving rise to mono- or poly-ubiquitination, respectively. The majority of research in the ubiquitin field focused on degradative polyubiquitination, whereas more recent studies uncovered the role of single ubiquitin modification in important physiological processes. Monoubiquitination can modulate the stability, subcellular localization, binding properties, and activity of the target proteins. Understanding the function of monoubiquitination in normal physiology and pathology has important therapeutic implications, as alterations in the monoubiquitin pathway are found in a broad range of genetic diseases. This review highlights a link between monoubiquitin signaling and the pathogenesis of genetic disorders.
    Keywords:  deubiquitinases; genetic diseases; monoubiquitin signaling; protein complex formation; ubiquitin ligase; ubiquitin system; vesicular trafficking
  10. ACS Chem Biol. 2020 Apr 30.
    Maneiro MA, Forte N, Shchepinova MM, Kounde CS, Chudasama V, Baker JR, Tate EW.
      Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity and do not discriminate between cells of different types. Here, we describe a strategy for selective protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab-PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internalization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, while sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to the release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.
  11. Molecules. 2020 Apr 23. pii: E1948. [Epub ahead of print]25(8):
    Manda S, Lee NK, Oh DC, Lee J.
      A focused PROTAC library was developed to degrade both IGF-1R and Src proteins, which are associated with various cancers. PROTACs with IGF-1R and Src degradation potentials were synthesized by tethering different inhibitor warhead units and the E3 ligase (CRBN) recruiting-pomalidomide with various linkers. The designed PROTACs 12a-b inhibited the proliferation and migration of MCF7 and A549 cancer cells with low micromolar potency (1-5 μM) in various cellular assays.
    Keywords:  IGF-1R; PROTACs; Src; anticancer activity; protein degradation
  12. Annu Rev Immunol. 2020 Apr 26. 38 171-202
    Chen YL, Hardman CS, Yadava K, Ogg G.
      Innate lymphocyte populations are emerging as key effectors in tissue homeostasis, microbial defense, and inflammatory skin disease. The cells are evolutionarily ancient and carry conserved principles of function, which can be achieved through shared or unique specific mechanisms. Recent technological and treatment advances have provided insight into heterogeneity within and between individuals and species. Similar pathways can extend through to adaptive lymphocytes, which softens the margins with innate lymphocyte populations and allows investigation of nonredundant pathways of immunity and inflammation that might be amenable to therapeutic intervention. Here, we review advances in understanding of innate lymphocyte biology with a focus on skin disease and the roles of commensal and pathogen responses and tissue homeostasis.
    Keywords:  innate; lymphocytes; skin
  13. Front Oncol. 2020 ;10 439
    Liang L, Cheng C, Hu G, Wang X, Liu J, Yan Z, Zeng W, Xia Y.
      Recent studies showed that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) induces the proliferation of squamous cell carcinoma (SCC) cells. However, the precise mechanism underlying such effect of TWEAK remains unclear. This study was designed to elucidate the role of cellular inhibitor of apoptosis 1 (cIAP1) in TWEAK-induced proliferation of SCC cells. Human SCC cells (SCC-13, A431, and SCC-9) were cultured in vitro, receiving the stimulation of TWEAK or TNF-related apoptosis-inducing ligand (TRAIL). We found that TWEAK induced cytoplasmic cIAP1 importation and RIP1 ubiquitination in cells, followed by the activation of canonical nuclear factor kappa B signals. MV1, a cIAP1 inhibitor, abrogated TWEAK-induced proliferation of these cells. Moreover, the interaction between TWEAK and its receptor, fibroblast growth factor-inducible 14 (Fn14), enhanced the expression of TRAIL receptor types 3 and 4 (TRAIL-R3/4). Furthermore, the transfection of TRAIL-R3/4 siRNA abrogated the promotion effect of TWEAK on SCC-13 cell proliferation and cIAP1 expression. Therefore, TWEAK/Fn14 interaction promotes the proliferation of SCC cells through activating cIAP1 signals. Targeting the downstream cIAP1 signals might attenuate the effect of TWEAK on SCC cells.
    Keywords:  Fn14; TWEAK; cIAP1; proliferation; squamous cell carcinoma
  14. Cleve Clin J Med. 2020 May;87(5): 288-299
    Pasadyn SR, Knabel D, Fernandez AP, Warren CB.
      Biologic therapies have become widely used but often cause cutaneous adverse effects. The authors discuss the cutaneous adverse effects of tumor necrosis factor (TNF) alpha inhibitors, epidermal growth factor receptor (EGFR) inhibitors, small-molecule tyrosine kinase inhibitors (TKIs), and cell surface-targeted monoclonal antibodies, including how to manage these reactions and when to refer to a dermatologist.
  15. Cancers (Basel). 2020 Apr 23. pii: E1045. [Epub ahead of print]12(4):
    Xu S, Lam KP.
      Multiple myeloma (MM) has emerged as the next most likely oncological or hematological disease indication amenable for cellular immunotherapy. Much of the attention has been focused on B cell maturation antigen (BCMA) as a unique cell surface protein on myeloma cells that is available for monoclonal antibodies, antibody drug conjugates (ADCs), T-cell redirecting bispecific molecules, and chimeric antigen receptor (CAR) T cell targeting. BCMA is a member of the tumor necrosis factor receptor (TNFR) superfamily that binds two ligands B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) and mediates the growth and survival of plasma and MM cells. Interestingly, transmembrane activator and CAML interactor (TACI), another TNFR superfamily member, also binds the same ligands and plays largely overlapping roles as BCMA in normal plasma and malignant MM cells. In this article, we review the biology of TACI, focusing on its role in normal B and plasma cells and malignant MM cells, and also discuss various ways to incorporate TACI as a potential target for immunotherapies against MM.
    Keywords:  TACI; immunotherapy; multiple myeloma; plasma cell
  16. Annu Rev Immunol. 2020 Apr 26. 38 249-287
    Ghilardi N, Pappu R, Arron JR, Chan AC.
      Since the birth of biotechnology, hundreds of biotherapeutics have been developed and approved by the US Food and Drug Administration (FDA) for human use. These novel medicines not only bring significant benefit to patients but also represent precision tools to interrogate human disease biology. Accordingly, much has been learned from the successes and failures of hundreds of high-quality clinical trials. In this review, we discuss general and broadly applicable themes that have emerged from this collective experience. We base our discussion on insights gained from exploring some of the most important target classes, including interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), IL-6, IL-12/23, IL-17, IL-4/13, IL-5, immunoglobulin E (IgE), integrins and B cells. We also describe current challenges and speculate about how emerging technological capabilities may enable the discovery and development of the next generation of biotherapeutics.
    Keywords:  autoimmunity; biotherapeutics; clinical trial; inflammation
  17. Biomolecules. 2020 Apr 23. pii: E650. [Epub ahead of print]10(4):
    Petushkova AI, Zamyatnin AA.
      Proteolytic enzymes play a crucial role in metabolic processes, providing the cell with amino acids through the hydrolysis of multiple endogenous and exogenous proteins. In addition to this function, proteases are involved in numerous protein cascades to maintain cellular and extracellular homeostasis. The redox regulation of proteolysis provides a flexible dose-dependent mechanism for proteolytic activity control. The excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) in living organisms indicate pathological conditions, so redox-sensitive proteases can swiftly induce pro-survival responses or regulated cell death (RCD). At the same time, severe protein oxidation can lead to the dysregulation of proteolysis, which induces either protein aggregation or superfluous protein hydrolysis. Therefore, oxidative stress contributes to the onset of age-related dysfunction. In the present review, we consider the post-translational modifications (PTMs) of proteolytic enzymes and their impact on homeostasis.
    Keywords:  apoptosis; autophagy; caspases; homeostasis; proteases; reactive oxygen species; regulated cell death
  18. Life Sci Alliance. 2020 Jun;pii: e202000735. [Epub ahead of print]3(6):
    Heilig R, Dilucca M, Boucher D, Chen KW, Hancz D, Demarco B, Shkarina K, Broz P.
      Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). Gsdmd deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1-driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd-deficient cells is caused by a synergistic effect of rapid caspase-1-driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1-dependent inflammatory disease.
  19. Paediatr Drugs. 2020 Apr 30.
    Nguyen QD, Starling CT, Hebert AA.
      Tumor necrosis factor alpha (TNF) inhibitors have had a significant impact in medicine since the approval of the first drug of its class by the US FDA in 1998. New clinical data and indications have emerged for TNF inhibitors in recent years. Currently, four TNF inhibitors have been approved by the US FDA for dermatology, two of which include US FDA-approved pediatric use. In particular, growing evidence supports the use of etanercept and adalimumab as attractive therapies for pediatric psoriasis. Data for use of etanercept in treating toxic epidermal necrolysis and either etanercept or infliximab for Kawasaki disease is expanding. In addition, there have been clinical reports on the use of TNF inhibitors to treat a variety of other pediatric dermatologic conditions. To help clinicians keep pace with the new data provided by many pediatric dermatology studies involving TNF inhibitors, this review provides an overview of the use of TNF inhibitors in the treatment of pediatric plaque psoriasis, hidradenitis suppurativa, atopic dermatitis, pyoderma gangrenosum, toxic epidermal necrolysis, and Kawasaki disease. For TNF inhibitors with little data in the pediatric population, data on adult use is discussed. Furthermore, the review summarizes available clinical data on efficacy, safety, and tolerability of agents currently available.
  20. Int J Mol Sci. 2020 Apr 27. pii: E3070. [Epub ahead of print]21(9):
    Labbozzetta M, Notarbartolo M, Poma P.
      Multidrug resistance (MDR), of the innate and acquired types, is one of major problems in treating tumor diseases with a good chance of success. In this review, we examine the key role of nuclear factor-kappa B (NF-κB) to induce MDR in three tumor models characterized precisely by innate or acquired MDR, in particular triple negative breast cancer (TNBC), hepatocellular carcinoma (HCC), and acute myeloid leukemia (AML). We also present different pharmacological approaches that our group have employed to reduce the expression/activation of this transcriptional factor and thus to restore chemo-sensitivity. Finally, we examine the latest scientific evidence found by other groups, the most significant clinical trials regarding NF-κB, and new perspectives on the possibility to consider this transcriptional factor a valid drug target in neoplastic diseases.
    Keywords:  MDR; NF-κB; cancer; drug target
  21. Cell Mol Biol Lett. 2020 ;25 28
    Bagheri S, Safaie Qamsari E, Yousefi M, Riazi-Rad F, Sharifzadeh Z.
      Background: Adoptive T-cell therapy (ACT) using autologous tumor-reactive T lymphocytes has considerable potential for cancer immunotherapy. In ACT, T cells are isolated from cancer patients and then stimulated and expanded in vitro by cytokines and costimulatory molecules. 4-1BB is an important costimulatory protein belonging to the TNF receptor superfamily. It is involved in T-cell survival, proliferation and activation. Agonistic anti-4-1BB monoclonal antibodies have been introduced as appropriate tools for ACT.Methods: Here, various single-chain fragment variable (scFv) antibodies were used to activate T cells isolated from peripheral blood via immune magnetic isolation. The T cells were stimulated with IL-2 and anti-CD-3 mAb and then treated with agonistic anti-4-1BB scFvs. The results showed the remarkable effects of anti-41BB scFvs on the functional properties of T cells, including their activation, proliferation and cytokine production. The flow cytometry analysis revealed a considerable increase in the expression of the T-cell activation marker CD69. Moreover, T-cell proliferation was evidenced in treated cells by CFSE labeling compared to the control groups.
    Result: Anti-4-1BB scFvs significantly increased IFN-γ and IL-2 mRNA and protein expression in T cells, but exhibited no stimulatory effect on IL-4 expression. These findings show that anti-4-1BB scFvs could evoke a Type I immune response.
    Conclusions: Our results demonstrate that targeting the 4-1BB molecule using agonistic scFvs could be an effective strategy for T-cell stimulation as part of an ACT approach to cancer treatment.
    Keywords:  4-1BB; Immunomodulation; Single-chain fragment antibody; T cell responses; T-cell therapy
  22. Mol Cell. 2020 Apr 22. pii: S1097-2765(20)30228-8. [Epub ahead of print]
    Zasłona Z, O'Neill LAJ.
      Metabolites have functions in the immune system independent of their conventional roles as sources or intermediates in biosynthesis and bioenergetics. We are still in the pioneering phase of gathering information about the functions of specific metabolites in immunoregulation. In this review, we cover succinate, itaconate, α-ketoglutarate, and lactate as examples. Each of these metabolites has a different story of how their immunoregulatory functions were discovered and how their roles in the complex process of inflammation were revealed. Parallels and interactions are emerging between metabolites and cytokines, well-known immunoregulators. We depict molecular mechanisms by which metabolites prime cellular and often physiological changes focusing on intra- and extra-cellular activities and signaling pathways. Possible therapeutic opportunities for immune and inflammatory diseases are emerging.
  23. Am J Pathol. 2020 Apr 24. pii: S0002-9440(20)30198-X. [Epub ahead of print]
    Liao C, Zhang Q.
      Maintaining oxygen homeostasis is a most basic cellular process for adapting physiological oxygen variations and its abnormality typically leads to various disorders in the human body. The key molecules of the oxygen-sensing system include the transcriptional regulator hypoxia-inducible factor (HIF) which controls a wide range of oxygen responsive target genes (e.g. EPO, VEGF), the certain members of the oxygen/2-oxoglutarate dependent dioxygenases including the HIF proline hydroxylase (PHD, or EglN), and an E3 ubiquitin ligase component for HIF destruction called von Hippel-Lindau (VHL). In this review, we summarize the physiological role and highlight the pathological function for each protein of the oxygen-sensing system. A better understanding of the molecular mechanism will help uncover novel therapeutic targets and develop more effective treatment approaches for related human diseases, including cancer.
  24. Eur J Pharmacol. 2020 Apr 24. pii: S0014-2999(20)30221-1. [Epub ahead of print] 173129
    Gregorczyk I, Maślanka T.
      The main aims of this study were: (1) to investigate whether a blockade of the interaction between the receptor activator of nuclear factor-κB (NF-ĸB) ligand (RANKL) and its receptor RANK may have potential as a novel therapeutic strategy for allergic asthma; (2) to compare the efficacies of the blockade of RANKL/RANK interaction as well as the blockade of NF-κB inhibitor kinase (IKK) and of NF-κB translocation to the nucleus, also in comparison with glucocorticosteroid treatment, in terms of the development of a mouse model of allergic airway inflammation (AAI) and accompanying immune response. The blockade of each of the targets fully prevented the development of AAI. All the tested therapeutic strategies seemed to have a certain advantage over glucocorticosteroids with regard to counteracting the development of AAI. Prevention of the activation and clonal expansion of CD4+ effector T (Teff) cells in the mediastinal lymph nodes (MLNs) constitutes a fundamental event underlying the anti-asthmatic effect induced by the blockade of IKK, NF-κB translocation or of RANKL/RANK interaction. The results indicate that attenuation of the CD11b+CD103-CD11chigh dendritic cell response in the MLNs is an initial but not the main mechanism responsible for this effect. In turn, the direct anti-proliferative action on CD4+ Teff cells seems to constitute the chief mechanism responsible for the anti-asthmatic effect of all the tested therapeutic strategies. A clinical implication is that local inhibition of RANKL/RANK interaction achieved via inhalatory administration of a RANKL antagonist can be considered as a novel therapeutic strategy in treatment of allergic asthma.
    Keywords:  Anti-asthmatic drugs; CD4(+) T cells; Dendritic cells; NF-κB; RANKL/RANK; Treg cells
  25. Immunol Cell Biol. 2020 Apr 30.
    Tarlinton D.
      Plasma cell longevity is dependent on their accessing and residing in so-called survival niches that are predominantly located in the bone marrow. It is proposed that by some process a small fraction of the plasma cells generated in response to new antigen challenges can enter into the long-lived repertoire by displacing existing plasma cells. Several lines of research show that this process is not stochastic as not all resident, long-lived plasma cells appear equally likely to be displaced. The basis of these differences might reside in the niches, the plasma cells or a combination of both that intersect to create a distribution of susceptibility to replacement and lifespans. In this review, I consider factors that might vary in plasma cells and thus influence their access to niches and the ability of newly generated plasma cells to survive over the long-term.
    Keywords:  Antibody; apoptosis; niches; turnover; vaccines
  26. Nature. 2019 May 01.
    White M.
    Keywords:  Careers; Publishing; Research data
  27. Nat Cell Biol. 2020 Apr 27.
    Dawson CA, Pal B, Vaillant F, Gandolfo LC, Liu Z, Bleriot C, Ginhoux F, Smyth GK, Lindeman GJ, Mueller SN, Rios AC, Visvader JE.
      Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
  28. Cell Rep. 2020 Apr 28. pii: S2211-1247(20)30523-4. [Epub ahead of print]31(4): 107574
    Zhou Y, Shao N, Bessa de Castro R, Zhang P, Ma Y, Liu X, Huang F, Wang RF, Qin L.
      Comprehensive evaluation of single T cell functions such as cytokine secretion and cytolysis of target cells is greatly needed in adoptive cell therapy (ACT) but has never been fully fulfilled by current approaches. Herein, we develop a hierarchical loading microwell chip (HL-Chip) that aligns multiple cells and functionalized beads in a high-throughput microwell array with single-cell/bead precision based on size differences. We demonstrate the potential of the HL-Chip in evaluating single T cell functions by three applications: high-throughput longitudinal secretory profiling of single T cells, large-scale evaluation of cytolytic activity of single T cells, and integrated T cell-tumor cell interactions. The HL-Chip is a simple and robust technology that constructs arrays of defined cell/object combinations for multiple measurements and material retrieval.
    Keywords:  T cell function; adoptive cell therapy; cell-bead pairing; cell-cell interaction; cytokine detection; cytotoxicity; microfluidics; single cell
  29. J Hematol Oncol. 2020 May 01. 13(1): 40
    Yu X, Li W, Liu H, Deng Q, Wang X, Hu H, Xu-Monette ZY, Xiong W, Lu Z, Young KH, Wang W, Li Y.
      BACKGROUND: Aberrant activation of DNA damage response (DDR) is a major cause of chemoresistance in colorectal cancer (CRC). CHK1 is upregulated in CRC and contributes to therapeutic resistance. We investigated the upstream signaling pathways governing CHK1 activation in CRC.METHODS: We identified CHK1-binding proteins by mass spectrometry analysis. We analyzed the biologic consequences of knockout or overexpression of TRAF4 using immunoblotting, immunoprecipitation, and immunofluorescence. CHK1 and TRAF4 ubiquitination was studied in vitro and in vivo. We tested the functions of TRAF4 in CHK1 phosphorylation and CRC chemoresistance by measuring cell viability and proliferation, anchorage-dependent and -independent cell growth, and mouse xenograft tumorigenesis. We analyzed human CRC specimens by immunohistochemistry.
    RESULTS: TRAF4 catalyzed the ubiquitination of CHK1 in multiple CRC cell lines. Following DNA damage, ubiquitination of CHK1 at K132 by TRAF4 is required for CHK1 phosphorylation and activation mediated by ATR. Notably, TRAF4 was highly expressed in chemotherapy-resistant CRC specimens and positively correlated with phosphorylated CHK1. Furthermore, depletion of TRAF4 impaired CHK1 activity and sensitized CRC cells to fluorouracil and other chemotherapeutic agents in vitro and in vivo.
    CONCLUSIONS: These data reveal two novel steps required for CHK1 activation in which TRAF4 serves as a critical intermediary and suggest that inhibition of the ATR-TRAF4-CHK1 signaling may overcome CRC chemoresistance.
    Keywords:  Checkpoint kinase 1; Chemoresistance; Colorectal cancer; Tumor necrosis factor receptor-associated factor 4; Ubiquitination