CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling

1994 ◽  
Vol 14 (12) ◽  
pp. 8078-8084
Author(s):  
B B Niklinska ◽  
D Hou ◽  
C June ◽  
A M Weissman ◽  
J D Ashwell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phosphatase Activity ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Tcr Signaling ◽  
Antigen Receptor Signaling ◽  
Membrane Anchoring

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.

scholarly journals CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

1994 ◽  
Vol 14 (12) ◽  
pp. 8078-8084 ◽  
Author(s):  
B B Niklinska ◽  
D Hou ◽  
C June ◽  
A M Weissman ◽  
J D Ashwell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phosphatase Activity ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Single Amino Acid ◽  
Tcr Signaling ◽  
Antigen Receptor Signaling ◽  
Membrane Anchoring

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.

scholarly journals Themis-associated phosphatase activity controls signaling in T cell development

2018 ◽  
Vol 115 (48) ◽  
pp. E11331-E11340 ◽  
Author(s):  
Monika Mehta ◽  
Joanna Brzostek ◽  
Elijah W. Chen ◽  
Desmond W. H. Tung ◽  
Shuting Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Phosphatase Activity ◽  
Ex Vivo ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
Thymic Development ◽  
Double Deletion

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.

scholarly journals Dynamic regulation of CD45 by tetraspanin CD53

2019 ◽  
Author(s):  
V. E. Dunlock ◽  
A. B. Arp ◽  
E. Jansen ◽  
S. Charrin ◽  
S. J. van Deventer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Tyrosine Phosphatase ◽  
Adaptive Immune Response ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Dynamic Regulation ◽  
And Function

AbstractT cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells.We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP.Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

scholarly journals The tyrosine phosphatase CD148 is excluded from the immunologic synapse and down-regulates prolonged T cell signaling

2003 ◽  
Vol 162 (4) ◽  
pp. 673-682 ◽  
Author(s):  
Joseph Lin ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Phosphatase Domain ◽  
T Cell Clone ◽  
Immunologic Synapse

CD148 is a receptor-like protein tyrosine phosphatase up-regulated on T cells after T cell receptor (TCR) stimulation. To examine the physiologic role of CD148 in TCR signaling, we used an inducible CD148-expressing Jurkat T cell clone. Expression of CD148 inhibits NFAT (nuclear factor of activated T cells) activation induced by soluble anti-TCR antibody, but not by antigen-presenting cells (APCs) loaded with staphylococcal enterotoxin superantigen (SAg) or immobilized anti-TCR antibody. Immunofluorescence microscopy revealed that the extracellular domain of CD148 mediates its exclusion from the immunologic synapse, sequestering it from potential substrates. Targeting of the CD148 phosphatase domain to the immunologic synapse potently inhibited NFAT activation by all means of triggering through the TCR. These data lead us to propose a model where CD148 function is regulated in part by exclusion from substrates in the immunologic synapse. Upon T cell–APC disengagement, CD148 can then access and dephosphorylate substrates to down-regulate prolongation of signaling.

scholarly journals Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 867
Author(s):  
Ling Wu ◽  
Joanna Brzostek ◽  
Shvetha Sankaran ◽  
Qianru Wei ◽  
Jiawei Yap ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Model ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
Barr Virus ◽  
Car T ◽  
Epstein Barr ◽  
Lower Magnitude

Chimeric antigen receptor T cells (CAR-T) utilize T cell receptor (TCR) signaling cascades and the recognition functions of antibodies. This allows T cells, normally restricted by the major histocompatibility complex (MHC), to be redirected to target cells by their surface antigens, such as tumor associated antigens (TAAs). CAR-T technology has achieved significant successes in treatment of certain cancers, primarily liquid cancers. Nonetheless, many challenges hinder development of this therapy, such as cytokine release syndrome (CRS) and the efficacy of CAR-T treatments for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, which has been less studied. To dissect CAR signaling, we designed a CAR that targets an epitope from latent membrane protein 2 A (LMP2 A) of the Epstein–Barr virus (EBV) presented on HLA*A02:01. Because of this, CAR and TCR signaling can be compared directly, allowing us to study the involvement of other signaling molecules, such as coreceptors. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer, but at a lower magnitude, than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. The distinct CAR signaling processes could provide explanations for clinical behavior of CAR-T therapy and suggest ways to improve the technology.

scholarly journals Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X

2014 ◽  
Vol 211 (10) ◽  
pp. 1947-1955 ◽  
Author(s):  
Edwina Naik ◽  
Joshua D. Webster ◽  
Jason DeVoss ◽  
Jinfeng Liu ◽  
Rowena Suriben ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tolerance Induction ◽  
Ubiquitin Ligases ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Deubiquitinating Enzymes ◽  
Tcr Signaling ◽  
Self Tolerance ◽  
A Cell

The T cell hyperproliferation and autoimmune phenotypes that manifest in mice lacking E3 ubiquitin ligases such as Cbl, ITCH, or GRAIL highlight the importance of ubiquitination for the maintenance of peripheral T cell tolerance. Less is known, however, about the deubiquitinating enzymes that regulate T cell proliferation and effector function. Here, we define a cell intrinsic role for the deubiquitinase Usp9X during proximal TCR signaling. Usp9X-deficient T cells were hypoproliferative, yet mice with T cell–specific Usp9x deletion had elevated numbers of antigen-experienced T cells and expanded PD-1 and OX40-expressing populations consistent with immune hyperactivity. Aged Usp9x KO mice developed lupus-like autoimmunity and lymphoproliferative disease, indicating that ubiquitin ligases and deubiquitinases maintain the delicate balance between effective immunity and self-tolerance.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

scholarly journals Loss of tonic T-cell receptor signals alters the generation but not the persistence of CD8+ memory T cells

Blood ◽  
2010 ◽  
Vol 116 (25) ◽  
pp. 5560-5570 ◽  
Author(s):  
Karla R. Wiehagen ◽  
Evann Corbo ◽  
Michelle Schmidt ◽  
Haina Shin ◽  
E. John Wherry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphocytic Choriomeningitis ◽  
Sh2 Domain ◽  
Normal Numbers ◽  
Tcr Signaling ◽  
Tcr Signals

Abstract The requirements for tonic T-cell receptor (TCR) signaling in CD8+ memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain–containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76–dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.

scholarly journals Fyn-Binding Protein (Fyb)/Slp-76–Associated Protein (Slap), Ena/Vasodilator-Stimulated Phosphoprotein (Vasp) Proteins and the Arp2/3 Complex Link T Cell Receptor (Tcr) Signaling to the Actin Cytoskeleton

2000 ◽  
Vol 149 (1) ◽  
pp. 181-194 ◽  
Author(s):  
Matthias Krause ◽  
Antonio S. Sechi ◽  
Marlies Konradt ◽  
David Monner ◽  
Frank B. Gertler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Actin Cytoskeleton ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Signaling ◽  
Tcr Signaling ◽  
Activated T Cells ◽  
Antigen Presenting ◽  
Syndrome Protein

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76–associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3–coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

scholarly journals DNA probes that store mechanical information reveal transient piconewton forces applied by T cells

2019 ◽  
Vol 116 (34) ◽  
pp. 16949-16954 ◽  
Author(s):  
Rong Ma ◽  
Anna V. Kellner ◽  
Victor Pui-Yan Ma ◽  
Hanquan Su ◽  
Brendan R. Deal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Real Time ◽  
Death Receptor ◽  
Cell Receptor ◽  
Dna Probes ◽  
Information Storage ◽  
Single Amino Acid ◽  
Peptide Ligand ◽  
Mechanical Information

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand–receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

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