A Laboratory Model to Study T-Cell Motility

Verma, Navin Kumar; Palapetta, Shyam Mohan; Ong, Seow Theng; Fazil, Mobashar Hussain Urf Turabe; Chalasani, Madhavi Latha Somaraju; Prasannan, Praseetha; Kizhakeyil, Atish; Kelleher, Dermot

doi:10.1007/978-1-4939-9036-8_3

Cited by 3 publications

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References 11 publications

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“…T-cells were stimulated to migrate via LFA-1/ICAM-1 adhesive interactions using our well characterized model system, as described (26,55). Briefly, 96-or 6well flat bottom Nunc™ tissue culture plates (Thermo Fisher Scientific, Singapore) were coated with 5 μg/ml Fc-specific goat anti-human IgG in sterile phosphate buffered saline (PBS) overnight at 4°C.…”

Section: Induction Of Lfa-1/icam-1-stimulated T-cell Migrationmentioning

confidence: 99%

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

Fazil

Chirumamilla

Pérez-Novo

et al. 2021

Journal of Biological Chemistry

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The 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 that WFA 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 adhesion 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 analysis 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, suggesting 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 impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.

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Section: Induction Of Lfa-1/icam-1-stimulated T-cell Migrationmentioning

confidence: 99%

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

Fazil

Chirumamilla

Pérez-Novo

et al. 2021

Journal of Biological Chemistry

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“…We used our well-characterized T-cell migration in vitro model, where primary or cultured T-cells were seeded on rICAM-1coated plates and cells were allowed to migrate as described previously (12). Briefly, 5 µg/ml anti-human IgG (Fc specific) in sterile phosphate buffered saline (PBS, pH 7.2) was coated on 6or 96-well tissue culture plates or 18 mm coverslips overnight at 4°C.…”

Section: Lfa-1/icam-1-induced T-cell Migrationmentioning

confidence: 99%

GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility

et al. 2021

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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.

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“…12 Laemmli sample buffer (5X SDS-PAGE loading buffer) Dissolve in deionised water to a final volume of 1 L. Dilute 1:10 in deionised water before use.…”

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Targeting pro-tumorigenic signaling pathways in hematolymphoid malignancies

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A Laboratory Model to Study T-Cell Motility

Cited by 3 publications

References 11 publications

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

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

GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility

Targeting pro-tumorigenic signaling pathways in hematolymphoid malignancies

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