scholarly journals tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging

2019 ◽  
Author(s):  
Daniel J. Nieves ◽  
Geva Hilzenrat ◽  
Jason Tran ◽  
Zhengmin Yang ◽  
Hugh H. MacRae ◽  
...  
Keyword(s):  
T Cell Receptor ◽  
Single Molecule ◽  
Quantitative Imaging ◽  
Cell Receptor ◽  
Great Promise ◽  
Immune Synapse ◽  
Receptor Signalling ◽  
Localisation Microscopy ◽  
Protein Tags ◽  
Dna Docking

AbstractRecently, DNA-PAINT single molecule localisation microscopy (SMLM) has shown great promise for quantitative imaging. However, labelling strategies so far have relied on approaches that are multivalent or affinity-based. Here, we demonstrate tagPAINT - the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA docking strands for single molecule localisation microscopy via DNA-PAINT. We utilised tagPAINT for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

scholarly journals tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging

2019 ◽  
Vol 6 (12) ◽  
pp. 191268
Author(s):  
Daniel J. Nieves ◽  
Geva Hilzenrat ◽  
Jason Tran ◽  
Zhengmin Yang ◽  
Hugh H. MacRae ◽  
...  
Keyword(s):  
Single Molecule ◽  
Quantitative Imaging ◽  
Cell Receptor ◽  
Great Promise ◽  
Immune Synapse ◽  
Localization Microscopy ◽  
Receptor Signalling ◽  
Protein Tags ◽  
Dna Docking ◽  
Single Molecule Localization Microscopy

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

scholarly journals The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

2011 ◽  
Vol 30 (7) ◽  
pp. 1238-1250 ◽  
Author(s):  
Francesc Baixauli ◽  
Noa B Martín-Cófreces ◽  
Giulia Morlino ◽  
Yolanda R Carrasco ◽  
Carmen Calabia-Linares ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Mitochondrial Fission ◽  
Cell Receptor ◽  
Related Protein ◽  
Immune Synapse ◽  
Receptor Signalling

scholarly journals Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1

2021 ◽  
Vol 22 (11) ◽  
pp. 5816
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Gianluca Baldanzi
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Adaptor Protein ◽  
Diacylglycerol Kinase ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Disease Type ◽  
T Cell Response ◽  
Receptor Signalling

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+CDS+thymocytes

Nature ◽  
1995 ◽  
Vol 374 (6521) ◽  
pp. 474-476 ◽  
Author(s):  
Klaus-Dieter Fischer ◽  
Antanina Zmuidzinas ◽  
Sandra Gardner ◽  
Mariano Barbacid ◽  
Alan Bernstein ◽  
...  
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Signalling ◽  
Selection Of

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling

Brain ◽  
2010 ◽  
Vol 133 (2) ◽  
pp. 375-388 ◽  
Author(s):  
Wei Hu ◽  
Stefan Nessler ◽  
Bernhard Hemmer ◽  
Todd N. Eagar ◽  
Lawrence P. Kane ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
T Cell ◽  
Autoimmune Disease ◽  
Prion Protein ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Signalling

Antiproliferative action of interferon-α requires components of T-cell-receptor signalling

Nature ◽  
10.1038/37648 ◽  
1997 ◽  
Vol 390 (6660) ◽  
pp. 629-632 ◽  
Author(s):  
Emanuel F. Petricoin ◽  
Satoshi Ito ◽  
Brandi L. Williams ◽  
Susette Audet ◽  
Louis F. Stancato ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Interferon Α ◽  
Receptor Signalling ◽  
Antiproliferative Action

scholarly journals T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is IP3R type 1 essential for T-cell-receptor signalling?

1998 ◽  
Vol 333 (3) ◽  
pp. 615-619 ◽  
Author(s):  
Junji HIROTA ◽  
Masashi BABA ◽  
Mineo MATSUMOTO ◽  
Teiichi FURUICHI ◽  
Kiyoshi TAKATSU ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Cell Phenotype ◽  
Receptor Signalling ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

Stimulation of T-cells via the T-cell receptor (TCR) complex is accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i). Recently, it was reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of inositol 1,4,5-trisphosphate receptor (IP3R) type 1 (IP3R1), failed to demonstrate increased [Ca2+]i or interleukin-2 production after TCR stimulation and was also resistant to apoptotic stimuli. This cell line lacked IP3R1 expression, but expressed the type-2 and -3 receptors, IP3R2 and IP3R3 respectively [Jayaraman, Ondriasova, Ondrias, Harnick and Marks (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6007–6011, and Jayaraman and Marks (1997) Mol. Cell. Biol. 17, 3005–3012]. The authors concluded that IP3R1 is essential for TCR signalling and suggested that Ca2+ release via IP3R1 is a critical mediator of apoptosis. To establish whether a loss of IP3R1 function in T-cells occurred in vivo and in vitro, we investigated Ca2+ signalling after TCR stimulation and the properties of T-cells using IP3R1-deficient (IP3R1-/-) mice. As IP3R1-/- mice die at weaning, we transplanted bone marrow cells of IP3R1-/- mice into irradiated wild-type mice. Western blot analysis showed that the recipient IP3R1-containing (IP3R1+/+) lymphocytes were replaced by the donor IP3R1-/- lymphocytes after transplantation and that expression of IP3R2 and IP3R3 was unaltered. In contrast with the previous reports, T-cells lacking IP3R1 were able to mobilize Ca2+ from intracellular Ca2+ stores after stimulation via the TCR. We observed no significant differences between IP3R1+/+ and IP3R1-/- T-cells in terms of the number of thymocytes and splenocytes, the proportion of the T-cell phenotype, proliferative response to anti-CD3 monoclonal antibody (mAb) stimulation and cell viability. Therefore IP3R1 is not essential for T-cell development and function.

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