DDX3X loss is an adverse prognostic marker in diffuse large B-cell lymphoma and is associated with chemoresistance in aggressive non-Hodgkin lymphoma subtypes.

Funder: addenbrooke's charitable trust, cambridge university hospital

Functional impact of high extracellular potassium ion on human T-cells

Despite the clinical success of current immunotherapies, there remains a pressing need to fully exploit the power of such treatments and improve their efficacy. Tumor microenvironment (TME) crucially dictates the T-cell anti-tumor immune responses. Yet, individual factors in the TME that drive immune suppression remains to be fully elucidated. Dying/necrotic tumor cells release a substantial amount of intracellular potassium ion ([K+]i), increasing extracellular potassium ion ([K+]e) to 5-10 fold. Here, we investigated the effects of high-[K+]e on the mechanistic and functional aspects of T-cells. We demonstrated via imaging, real-time impedance-based measurements and molecular assays that high-[K+]e impedes T-cell motility (15% inhibition) and possesses a chemotactic influence over T-cells. Moreover, High-[K+]e upregulates the expression of Kv1.3 K+ channel as well as PD-1 in T-cells. Using Jurkat T-cell line as a model, we observed that high-[K+]e reduces T-cell cytokines (IL-2 and IFN-) production and modulates both AMPK and ACC expression and phosphorylation, which are prerequisite factors in cellular metabolism. Taken together, high-[K+]einduced impairment of T-cell functions reported in the current study has implications in T-cell anti-tumor immune responses and immunotherapies.Bachelor of Science in Biological Science

Maladaptive T-Cell Metabolic Fitness in Autoimmune Diseases

Immune surveillance and adaptive immune responses, involving continuously circulating and tissue-resident T-lymphocytes, provide host defense against infectious agents and possible malignant transformation while avoiding autoimmune tissue damage. Activation, migration, and deployment of T-cells to affected tissue sites are crucial for mounting an adaptive immune response. An effective adaptive immune defense depends on the ability of T-cells to dynamically reprogram their metabolic requirements in response to environmental cues. Inability of the T-cells to adapt to specific metabolic demands may skew cells to become either hyporesponsive (creating immunocompromised conditions) or hyperactive (causing autoimmune tissue destruction). Here, we review maladaptive T-cell metabolic fitness that can cause autoimmune diseases and discuss how T-cell metabolic programs can potentially be modulated to achieve therapeutic benefits.Medicine, Faculty ofNon UBCMedicine, Department ofReviewedFacultyResearcherPostdoctora

GSK3β interacts with CRMP2 and Notch1 and controls T-cell motility

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3β in T-cell motility. Inhibition of GSK3β by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3β, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3β activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3β-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3β in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3β inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3β controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.Ministry of Education (MOE)National Research Foundation (NRF)Published versionThis work was supported by the grants from the Singapore Ministry of Education (MOE) Academic Research Fund (AcRF) Tier 1 (2014-T1-001-141 and 2020-T1-001-062) and the National Research Foundation Singapore under its Open Fund Large Collaborative Grant (OFLCG18May-0028) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC)

The steroidal lactone withaferin A impedes T-cell motility by\ua0inhibiting the kinase ZAP70 and subsequent kinome signaling

Not AvailableThe steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found thatWFA dose-dependently (within the concentration range of 0.3–1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhe sion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome anal ysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, sug gesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and im pedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologiesNot Availabl

The steroidal lactone withaferin A impedes T-cell motility by inhibiting the kinase ZAP70 and subsequent kinome signaling

Not AvailableThe steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found thatWFA dose-dependently (within the concentration range of 0.3–1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhe sion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome anal ysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, sug gesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and im pedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologiesNot Availabl

Electrospun aligned PCLl/gelatin scaffolds mimicking the skin ECM for effective antimicrobial wound dressings

Bacterial infections and multidrug-resistant bacteria are major health burdens in wound care. Biocompatible antimicrobial agents, e.g., ε-polylysine (ε-PL), provide a broad spectrum of antibacterial properties and support dermal cell growth. Here, ε-PL was incorporated into polycaprolactone (PCL)/gelatin electrospun scaffolds collected at varying rotation speeds. Then, the samples were crosslinked using dopamine hydrochloride to provide highly proliferative dressings with broad antimicrobial activity. The morphological study showed that the electrospun wound dressings were smooth, continuous, and bead-free, with a mean diameter ranging from 267 ± 7 to 331 ± 8 nm for all random and aligned nanofibers. The fiber alignment of the electrospun PCL/gelatin scaffolds improved their tensile strength and modulus. Moreover, nanofiber mats are highly hydrophilic, which is crucial for an efficient wound dressing. The samples also demonstrated high antimicrobial properties against common wound bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (SA), Escherichia coli (EC), Acinetobacter baumannii (AB), and Pseudomonas aeruginosa (PA). Mammalian cell proliferation and morphology assays involving primary human dermal fibroblasts (hDFs) and immortalized keratinocytes (HaCaT) showed excellent biocompatibility of the electrospun mats and remarkably aligned mats. Furthermore, aligned mats showed more cell migration than randomly oriented mats, which is desirable for more efficient wound healing. Therefore, it can be concluded that aligned PCL/gelatin mats containing ε-PL are promising for potential use in wound dressings. Graphical Abstract: [Figure not available: see fulltext.].Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversityE.R.G. acknowledges the financial support from the Singapore International Graduate Award (SINGA). R.L. thanks funding support from the Duke-NUS Khoo Bridge Funding Award (Duke-NUS-KBrFA/2021/0044). N.K.V. acknowledges funding support from the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 1 Grant (RG26/20) and the Agency for Science, Technology and Research (A*STAR) under its Wound Care Innovation for the Tropics (WCIT) Industry Alignment Fund Pre-Positioning (IAF-PP) Grant (H17/01/a0/0K9). B.H.S.W. and Z.S.P. were provided Ph.D. fellowships by HealthTech NTU and Lee Kong Chian School of Medicine, Nanyang Technological University Singapore

Obstacles for T-lymphocytes in the tumour microenvironment: therapeutic challenges, advances and opportunities beyond immune checkpoint

The tumour microenvironment (TME) imposes a major obstacle to infiltrating T-lymphocytes and suppresses their function. Several immune checkpoint proteins that interfere with ligand/receptor interactions and impede T-cell anti-tumour responses have been identified. Immunotherapies that block immune checkpoints have revolutionized the treatment paradigm for many patients with advanced-stage tumours. However, metabolic constraints and soluble factors that exist within the TME exacerbate the functional exhaustion of tumour-infiltrating T-cells. Here we review these multifactorial constraints and mechanisms - elevated immunosuppressive metabolites and enzymes, nutrient insufficiency, hypoxia, increased acidity, immense amounts of extracellular ATP and adenosine, dysregulated bioenergetic and purinergic signalling, and ionic imbalance - that operate in the TME and collectively suppress T-cell function. We discuss how scientific advances could help overcome the complex TME obstacles for tumour-infiltrating T-lymphocytes, aiming to stimulate further research for developing new therapeutic strategies by harnessing the full potential of the immune system in combating cancer.Ministry of Education (MOE)Ministry of Health (MOH)Nanyang Technological UniversityNational Medical Research Council (NMRC)National Research Foundation (NRF)Published versionThis research was supported, in part, by the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 2 (MOE2017-T2-2-004) and Tier 1 (2020-T1-001-062) grants, and the National Research Foundation Singapore under its Open Fund - Large Collaborative Grant (OFLCG18May-0028) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC). N.F.G. acknowledges funding support from the NMRC Transition Award (NMRC/TA/0051/2016) and the NMRC Centre Grant (TETRAD 2). B.H.S.W. and Z.S.P. were provided with PhD fellowship by HealthTech NTU and Lee Kong Chian School of Medicine, Nanyang Technological University Singapore

DDX3X loss is an adverse prognostic marker in diffuse large B-cell lymphoma and is associated with chemoresistance in aggressive non-Hodgkin lymphoma subtypes

10.1186/s12943-021-01437-0MOLECULAR CANCER20