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Article ; Online: Tim-3 Is Not Required for Establishment of CD8+ T Cell Memory to Lymphocytic Choriomeningitis Virus.

Manandhar, Priyanka / Szymczak-Workman, Andrea L / Kane, Lawrence P

Journal of immunology (Baltimore, Md. : 1950)

2023 Volume 212, Issue 3, Page(s) 466–474

Abstract: Tim-3 is a transmembrane protein that is best known for being highly expressed on terminally exhausted CD8+ T cells associated with chronic infection and tumors, although its expression is not limited to those settings. Tim-3 is also expressed by CD8+ T ... ...

Abstract	Tim-3 is a transmembrane protein that is best known for being highly expressed on terminally exhausted CD8+ T cells associated with chronic infection and tumors, although its expression is not limited to those settings. Tim-3 is also expressed by CD8+ T cells during acute infection and by multiple other immune cell types, including CD4+ Th1 and regulatory T cells, dendritic cells, and mast cells. In this study, we investigated the role of Tim-3 signaling on CD8+ T cell memory using a Tim-3 conditional knockout mouse model and mice lacking the signaling portion of the Tim-3 cytoplasmic domain. Together, our results indicate that Tim-3 has at most a modest effect on the formation and function of CD8+ memory T cells.
MeSH term(s)	Animals ; Mice ; CD8-Positive T-Lymphocytes ; Hepatitis A Virus Cellular Receptor 2/genetics ; Hepatitis A Virus Cellular Receptor 2/metabolism ; Lymphocytic Choriomeningitis ; Lymphocytic choriomeningitis virus ; Memory T Cells ; Signal Transduction
Chemical Substances	Hepatitis A Virus Cellular Receptor 2 ; Havcr2 protein, mouse
Language	English
Publishing date	2023-12-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	3056-9
ISSN	1550-6606 ; 0022-1767 ; 1048-3233 ; 1047-7381
ISSN (online)	1550-6606
ISSN	0022-1767 ; 1048-3233 ; 1047-7381
DOI	10.4049/jimmunol.2300401
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Article ; Online: Tim-3 co-stimulation promotes short-lived effector T cells, restricts memory precursors, and is dispensable for T cell exhaustion.

Avery, Lyndsay / Filderman, Jessica / Szymczak-Workman, Andrea L / Kane, Lawrence P

Proceedings of the National Academy of Sciences of the United States of America

2018 Volume 115, Issue 10, Page(s) 2455–2460

Abstract: Tim-3 is highly expressed on a subset of T cells during T cell exhaustion in settings of chronic viral infection and tumors. Using lymphocytic choriomeningitis virus (LCMV) Clone 13, a model for chronic infection, we found that Tim-3 was neither ... ...

Abstract	Tim-3 is highly expressed on a subset of T cells during T cell exhaustion in settings of chronic viral infection and tumors. Using lymphocytic choriomeningitis virus (LCMV) Clone 13, a model for chronic infection, we found that Tim-3 was neither necessary nor sufficient for the development of T cell exhaustion. Nonetheless, expression of Tim-3 was sufficient to drive resistance to PD-L1 blockade therapy during chronic infection. Strikingly, expression of Tim-3 promoted the development of short-lived effector T cells, at the expense of memory precursor development, after acute LCMV infection. These effects were accompanied by increased Akt/mTOR signaling in T cells expressing endogenous or ectopic Tim-3. Conversely, Akt/mTOR signaling was reduced in effector T cells from Tim-3-deficient mice. Thus, Tim-3 is essential for optimal effector T cell responses, and may also contribute to exhaustion by restricting the development of long-lived memory T cells. Taken together, our results suggest that Tim-3 is actually more similar to costimulatory receptors that are up-regulated after T cell activation than to a dominant inhibitory protein like PD-1. These findings have significant implications for the development of anti-Tim-3 antibodies as therapeutic agents.
MeSH term(s)	Animals ; Chronic Disease ; Hepatitis A Virus Cellular Receptor 2/genetics ; Hepatitis A Virus Cellular Receptor 2/immunology ; Lymphocyte Activation/immunology ; Lymphocytic Choriomeningitis ; Lymphocytic choriomeningitis virus/immunology ; Mice ; Phenotype ; Receptors, Antigen, T-Cell/immunology ; Signal Transduction/immunology ; T-Lymphocyte Subsets/immunology ; TOR Serine-Threonine Kinases/metabolism
Chemical Substances	Havcr2 protein, mouse ; Hepatitis A Virus Cellular Receptor 2 ; Receptors, Antigen, T-Cell ; TOR Serine-Threonine Kinases (EC 2.7.1.1) ; mTOR protein, mouse (EC 2.7.1.1)
Language	English
Publishing date	2018-02-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1712107115
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Article ; Online: IFNγ-induction of T

Gocher-Demske, Angela M / Cui, Jian / Szymczak-Workman, Andrea L / Vignali, Kate M / Latini, Julianna N / Pieklo, Gwen P / Kimball, Jesse C / Avery, Lyndsay / Cipolla, Ellyse M / Huckestein, Brydie R / Hedden, Lee / Meisel, Marlies / Alcorn, John F / Kane, Lawrence P / Workman, Creg J / Vignali, Dario A A

Nature immunology

2023 Volume 24, Issue 5, Page(s) 841–854

Abstract: Regulatory T ( ... ...

Abstract	Regulatory T (T
MeSH term(s)	T-Lymphocytes, Regulatory ; Interferon-gamma/metabolism ; Cytokines/metabolism ; CD8-Positive T-Lymphocytes ; Antiviral Agents/metabolism ; Th1 Cells
Chemical Substances	Interferon-gamma (82115-62-6) ; Cytokines ; Antiviral Agents
Language	English
Publishing date	2023-03-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2016987-5
ISSN	1529-2916 ; 1529-2908
ISSN (online)	1529-2916
ISSN	1529-2908
DOI	10.1038/s41590-023-01453-w
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Zs.A 5294: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment.

Banerjee, Hridesh / Nieves-Rosado, Hector / Kulkarni, Aditi / Murter, Benjamin / McGrath, Kyle V / Chandran, Uma R / Chang, Alexander / Szymczak-Workman, Andrea L / Vujanovic, Lazar / Delgoffe, Greg M / Ferris, Robert L / Kane, Lawrence P

Cell reports

2021 Volume 36, Issue 11, Page(s) 109699

Abstract: Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, ... ...

Abstract	Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3
Language	English
Publishing date	2021-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2021.109699
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Article ; Online: The costimulatory activity of Tim-3 requires Akt and MAPK signaling and its recruitment to the immune synapse.

Kataoka, Shunsuke / Manandhar, Priyanka / Lee, Judong / Workman, Creg J / Banerjee, Hridesh / Szymczak-Workman, Andrea L / Kvorjak, Michael / Lohmueller, Jason / Kane, Lawrence P

Science signaling

2021 Volume 14, Issue 687

Abstract: Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others ... ...

Abstract	Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3-mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)-dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.
MeSH term(s)	Hepatitis A Virus Cellular Receptor 2/genetics ; Humans ; Immunological Synapses ; Lymphocyte Activation ; MAP Kinase Signaling System ; Persistent Infection ; Proto-Oncogene Proteins c-akt/genetics
Chemical Substances	HAVCR2 protein, human ; Hepatitis A Virus Cellular Receptor 2 ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1)
Language	English
Publishing date	2021-06-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2417226-1
ISSN	1937-9145 ; 1945-0877
ISSN (online)	1937-9145
ISSN	1945-0877
DOI	10.1126/scisignal.aba0717
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Article ; Online: A Cre-driven allele-conditioning line to interrogate CD4

Andrews, Lawrence P / Vignali, Kate M / Szymczak-Workman, Andrea L / Burton, Amanda R / Brunazzi, Erin A / Ngiow, Shin Foong / Harusato, Akihito / Sharpe, Arlene H / Wherry, E John / Taniuchi, Ichiro / Workman, Creg J / Vignali, Dario A A

Immunity

2021 Volume 54, Issue 10, Page(s) 2209–2217.e6

Abstract: ... ...

Abstract	CD4
MeSH term(s)	Alleles ; Animals ; CD4-Positive T-Lymphocytes/cytology ; CD8-Positive T-Lymphocytes/cytology ; Cell Differentiation/immunology ; Cell Line ; Cell Lineage/immunology ; Gene Editing/methods ; Integrases/genetics ; Mice
Chemical Substances	Cre recombinase (EC 2.7.7.-) ; Integrases (EC 2.7.7.-)
Language	English
Publishing date	2021-09-21
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1217235-2
ISSN	1097-4180 ; 1074-7613
ISSN (online)	1097-4180
ISSN	1074-7613
DOI	10.1016/j.immuni.2021.08.029
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Zs.A 4227: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 69: Show issues

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Article ; Online: Generation of 2A-linked multicistronic cassettes by recombinant PCR.

Szymczak-Workman, Andrea L / Vignali, Kate M / Vignali, Dario A A

Cold Spring Harbor protocols

2012 Volume 2012, Issue 2, Page(s) 251–254

Abstract: The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. ... ...

Abstract	The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.
MeSH term(s)	Gene Expression ; Genes ; Genetic Engineering/methods ; Genetic Vectors ; Open Reading Frames ; Peptides/genetics ; Peptides/metabolism ; Polymerase Chain Reaction/methods ; Protein Biosynthesis ; Protein Processing, Post-Translational ; Proteolysis ; Ribosomes/metabolism
Chemical Substances	Peptides
Language	English
Publishing date	2012-02-01
Publishing country	United States
Document type	Journal Article
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.prot067884
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Article ; Online: Design and construction of 2A peptide-linked multicistronic vectors.

Szymczak-Workman, Andrea L / Vignali, Kate M / Vignali, Dario A A

Cold Spring Harbor protocols

2012 Volume 2012, Issue 2, Page(s) 199–204

Abstract: The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple ... ...

Abstract	The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.
MeSH term(s)	Gene Expression ; Genes ; Genetic Vectors ; Open Reading Frames ; Peptides/genetics ; Peptides/metabolism ; Protein Biosynthesis ; Protein Processing, Post-Translational ; Proteolysis ; Ribosomes/metabolism
Chemical Substances	Peptides
Language	English
Publishing date	2012-02-01
Publishing country	United States
Document type	Journal Article
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.ip067876
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Article ; Online: Verification of 2A peptide cleavage.

Szymczak-Workman, Andrea L / Vignali, Kate M / Vignali, Dario A A

Cold Spring Harbor protocols

2012 Volume 2012, Issue 2, Page(s) 255–257

Abstract	The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.
MeSH term(s)	Blotting, Western ; Cell Line ; Gene Expression ; Genes ; Genetic Engineering/methods ; Genetic Vectors ; Humans ; Open Reading Frames ; Peptides/genetics ; Peptides/metabolism ; Protein Biosynthesis ; Protein Processing, Post-Translational ; Proteolysis ; Ribosomes/metabolism ; Transfection
Chemical Substances	Peptides
Language	English
Publishing date	2012-02-01
Publishing country	United States
Document type	Journal Article
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.prot067892
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Article ; Online: Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity.

Liu, Chang / Somasundaram, Ashwin / Manne, Sasikanth / Gocher, Angela M / Szymczak-Workman, Andrea L / Vignali, Kate M / Scott, Ellen N / Normolle, Daniel P / John Wherry, E / Lipson, Evan J / Ferris, Robert L / Bruno, Tullia C / Workman, Creg J / Vignali, Dario A A

Nature immunology

2020 Volume 21, Issue 9, Page(s) 1010–1021

Abstract: ... Robust ... ...

Abstract	Robust CD8
MeSH term(s)	Animals ; CD8-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Humans ; Immune Checkpoint Proteins/genetics ; Immune Checkpoint Proteins/metabolism ; Immune Tolerance ; Immunity ; Immunologic Memory ; Melanoma, Experimental/immunology ; Mice ; Mice, Knockout ; Neuropilin-1/genetics ; Neuropilin-1/metabolism ; Precursor Cells, T-Lymphoid/immunology ; Programmed Cell Death 1 Receptor/metabolism ; Signal Transduction
Chemical Substances	Immune Checkpoint Proteins ; Pdcd1 protein, mouse ; Programmed Cell Death 1 Receptor ; Neuropilin-1 (144713-63-3)
Language	English
Publishing date	2020-07-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2016987-5
ISSN	1529-2916 ; 1529-2908
ISSN (online)	1529-2916
ISSN	1529-2908
DOI	10.1038/s41590-020-0733-2
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