Experimental Applications of TNF-Reporter Mice with Far-Red Fluorescent Label

Shebzukhov, Yury; Kuchmiy, Anna; Kruglov, Andrey; Zipp, Frauke; Siffrin, Volker; Nedospasov, Sergei A.

doi:10.1007/978-1-4939-0669-7_13

Cited by 5 publications

(2 citation statements)

References 8 publications

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“…The gain in Lifeact-EGFP fluorescence correlates with late phase activation and effector cytokine production. Basal GFP expression enables tracking of T cells before they are activated, providing an advantage over reporters that fluoresce only upon cytokine production [including TNF-α (Shebzukhov et al, 2014) and IL-2 (Naramura et al, 1998)] and hence require additional vital dye labelling; or reporters that monitor the translocation of nuclear factor of activated T cells (NFATs) (Lodygin et al, 2013) that require an additional constitutive nuclear label. It will be interesting to compare the Lifeact-EGFP sensitivity and specificity with the Nur77 EGFP reporter (Au-Yeung et al, 2014), in which GFP is expressed from the immediate early gene Nr4a1 (Nur77) locus and can rapidly and specifically be induced upon antigen receptor triggering.…”

Section: Discussionmentioning

confidence: 99%

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

Niño

Tay

Tearle

et al. 2020

Journal of Cell Science

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It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8 + T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activationinduced gains in fluorescence were also observed in naïve and CD4 + T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

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Section: Discussionmentioning

confidence: 99%

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

Niño

Tay

Tearle

et al. 2020

Journal of Cell Science

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“…Total cells were isolated from spleen, mesenterial, popliteal and auxiliary lymph nodes and CD4 + CD62L + CD44 − CD25 − naïve T cells were purified by magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec, Bergisch Gladbach, Germany) 41 . Th1 and Th2 cells were polarized as described 14 .…”

Section: Methodsmentioning

confidence: 99%

Low level of Lck kinase in Th2 cells limits expression of CD4 co-receptor and S73 phosphorylation of transcription factor c-Jun

Shebzukhov

Stanislawiak

Bezhaeva

et al. 2017

Sci Rep

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The Src-family tyrosine kinase Lck is an enzyme associated with the CD4 and CD8 co-receptors and promoting signaling through the T cell receptor (TCR) complex. The levels of Lck expression and activity change during the development and differentiation of T cells. Here we show that Lck expression is higher in Th1 cells as compared to Th2 cells. Ectopic overexpression of Lck in Th2 cells results in increased expression of CD4 co-receptor and enhanced S73 phosphorylation of transcription factor c-Jun. Our findings indicate that TCR-mediated signaling in Th2 cells may be directly attenuated by Lck protein expression level.

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Microbiota induces expression of tumor necrosis factor in postnatal mouse skin

Yuzhakova

Shirmanova

Bocharov

et al. 2016

Biochemistry Moscow

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Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates many important processes in the body. TNF production in a physiological state supports the structure of lymphoid organs and determines the development of lymphoid cells in hematopoiesis. However, chronic TNF overexpression leads to the development of various autoimmune disorders. Sites of TNF production in the naïve state remain unclear due to the lack of in vivo models. In the present study, we used TNF-2A-Kat reporter mice to monitor the expression of TNF in different tissues. Comparative analysis of tissue fluorescence in TNF-2A-Kat reporter mice and wild type mice revealed constitutive expression of TNF in the skin of naïve adult mice. In the skin of TNF-2A-Kat reporter mouse embryos, no statistically significant differences in the expression of TNF compared to wild type animals were observed. Furthermore, we established that local depletion of microflora with topical antibiotics leads to a reduction in the fluorescence signal. Thus, we assume that the skin microflora is responsible for the expression of TNF in the skin of mice.

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Experimental Applications of TNF-Reporter Mice with Far-Red Fluorescent Label

Cited by 5 publications

References 8 publications

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

Low level of Lck kinase in Th2 cells limits expression of CD4 co-receptor and S73 phosphorylation of transcription factor c-Jun

Microbiota induces expression of tumor necrosis factor in postnatal mouse skin

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