scholarly journals In Vivo Significance of ITK-SLP-76 Interaction in Cytokine Production

2010 ◽  
Vol 30 (14) ◽  
pp. 3596-3609 ◽  
Author(s):  
Juris A. Grasis ◽  
David M. Guimond ◽  
Nicholas R. Cam ◽  
Krystal Herman ◽  
Paola Magotti ◽  
...  
Keyword(s):  
T Cell ◽  
Biological Significance ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Sh3 Domain ◽  
Live Cells ◽  
Pharmacological Intervention ◽  
Cell Contact

ABSTRACT In vitro data have suggested that activation of the inducible T-cell kinase (ITK) requires an interaction with the adaptor protein SLP-76. One means for this interaction involves binding of the ITK SH3 domain to the polyproline-rich (PR) region of SLP-76. However, the biological significance of this association in live cells and the consequences of its disruption have not been demonstrated. Here, we utilized a polyarginine-rich, cell-permeable peptide that represents the portion of the SLP-76 PR region that interacts with the ITK SH3 domain as a competitive inhibitor to disrupt the association between ITK and SLP-76 in live cells. We demonstrate that treatment of cells with this peptide, by either in vitro incubation or intraperitoneal injection of the peptide in mice, inhibits the T-cell receptor (TCR)-induced association between ITK and SLP-76, recruitment and transphosphorylation of ITK, actin polarization at the T-cell contact site, and expression of Th2 cytokines. The inhibition is specific, as indicated by lack of effects by the polyarginine vehicle alone or a scrambled sequence of the cargo peptide. In view of the role of ITK as a regulator of Th2 cytokine expression, the data underscore the significance of ITK as a target for pharmacological intervention.

scholarly journals Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro

1997 ◽  
Vol 138 (2) ◽  
pp. 271-281 ◽  
Author(s):  
Jes Dietrich ◽  
Jesper Kastrup ◽  
Bodil L. Nielsen ◽  
Niels Ødum ◽  
Carsten Geisler
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tolerance Induction ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cell Receptor ◽  
Coated Vesicle

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.

scholarly journals FVIII-Specific TCR Engineered Human Regulatory T Cells Suppress the Production of FVIII-Specific Responses Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2586-2586
Author(s):  
Yong Chan Kim ◽  
Aihong Zhang ◽  
Jeong-Heon Yoon ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Hemophilia A ◽  
Cell Receptor ◽  
Cell Contact ◽  
Therapeutic Trial

Abstract Expanded antigen-specific engineered regulatory T cells (Tregs) have been proposed for potential clinical application for the treatment of undesirable immune responses, such as inhibitor responses in hemophilia A patients and autoimmune diseases. By providing an antigen-specific T-cell receptor (TCR) to polyclonal natural Tregs, we suggested that antigen-specific engineered Tregs would migrate specifically to particular target tissues and induce antigen-specific immune tolerance in the local milieu. Previously, we developed FVIII C2-specific Tregs using a long-term stabilization protocol in vitro and demonstrated that these stabilized engineered Tregs successfully modulated FVIII-specific T-cell and B-cell immune responses in vitro. Furthermore, these engineered Tregs could suppress T-effectors specific for additional epitopes in local milieu in both a cell contact and contactless manner. From these data, we hypothesized that IL-2 and related signaling pathways are major regulatory mechanisms of the suppression. To further investigate how IL-2R signaling is engaged to control T effectors and Tregs, we followed the phospho-STAT5 status of these cells kinetically. Our results showed clearly that IL-2 from activated T effectors is a key requirement for Treg activation, inducing subsequent blockage of STAT5 signal in T effectors by activated Tregs. As further evidence of the efficacy of these specific Tregs, we then determined whether FVIII C2-specific Tregs could suppress the induction of FVIII inhibitor antibody in vivo. Thus, we transferred FVIII C2-specific human Tregs into HLA DR1 hemophilic mice and challenged them with FVIII in vivo. Our results showed that induction of FVIII-specific antibodies was inhibited for over 8 weeks. Taken together, our results suggest a potential therapeutic trial of FVIII-specific engineered Tregs in hemophilia A. Disclosures Kim: Henry Jackson Foundation: Patents & Royalties: Provisional submitted.

scholarly journals CIN85 drives B cell responses by linking BCR signals to the canonical NF-κB pathway

2011 ◽  
Vol 208 (7) ◽  
pp. 1447-1457 ◽  
Author(s):  
Kohei Kometani ◽  
Takayuki Yamada ◽  
Yoshiteru Sasaki ◽  
Tadashi Yokosuka ◽  
Takashi Saito ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Adaptor Protein ◽  
Cellular Responses ◽  
Cell Receptor ◽  
Antibody Responses ◽  
Cell Responses ◽  
Subsequent Degradation

CIN85, an adaptor protein which binds the C-terminal domain of tyrosine phosphorylated Cbl and Cbl-b, has been thought to be involved in the internalization and subsequent degradation of receptors. However, its physiological function remains unclear. To determine its role in B cells, we used Mb1-cre to generate mice with a B cell–specific deletion of CIN85. These mice had impaired T cell–independent type II antibody responses in vivo and diminished IKK-β activation and cellular responses to B cell receptor (BCR) cross-linking in vitro. Introduction of a constitutively active IKK-β construct corrected the defective antibody responses as well as cellular responses in the mutant mice. Together, our results suggest that CIN85 links the BCR to IKK-β activation, thereby contributing to T cell–independent immune responses.

scholarly journals Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

2001 ◽  
Vol 193 (11) ◽  
pp. 1295-1302 ◽  
Author(s):  
Megan K. Levings ◽  
Romina Sangregorio ◽  
Maria-Grazia Roncarolo
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Major Advance ◽  
Loss Of Function

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

scholarly journals Activation of Hematopoietic Progenitor Kinase 1 Involves Relocation, Autophosphorylation, and Transphosphorylation by Protein Kinase D1

2005 ◽  
Vol 25 (6) ◽  
pp. 2364-2383 ◽  
Author(s):  
Rüdiger Arnold ◽  
Irene M. Patzak ◽  
Brit Neuhaus ◽  
Sadia Vancauwenbergh ◽  
André Veillette ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Hematopoietic Progenitor ◽  
Kinase Activation ◽  
Protein Kinase D1 ◽  
Enzymatic Activation ◽  
Antigen Presenting

ABSTRACT Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-κB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-κB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.

scholarly journals Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

2012 ◽  
Vol 209 (6) ◽  
pp. 1201-1217 ◽  
Author(s):  
Tadashi Yokosuka ◽  
Masako Takamatsu ◽  
Wakana Kobayashi-Imanishi ◽  
Akiko Hashimoto-Tane ◽  
Miyuki Azuma ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Programmed Cell Death ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tyrosine Phosphatase ◽  
Cell Receptor ◽  
Programmed Cell Death 1

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1–mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain–containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1–TCR colocalization within microclusters is required for efficient PD-1–mediated suppression. This inhibitory mechanism also functions in PD-1hi T cells generated in vivo and can be overridden by a neutralizing anti–PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.

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 CCDC88B is a novel regulator of maturation and effector functions of T cells during pathological inflammation

2014 ◽  
Vol 211 (13) ◽  
pp. 2519-2535 ◽  
Author(s):  
James M. Kennedy ◽  
Nassima Fodil ◽  
Sabrina Torre ◽  
Silayuv E. Bongfen ◽  
Jean-Frédéric Olivier ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Coiled Coil ◽  
Cell Receptor ◽  
Pleiotropic Effect ◽  
Experimental Cerebral Malaria ◽  
A Genome ◽  
Important Regulator

We used a genome-wide screen in mutagenized mice to identify genes which inactivation protects against lethal neuroinflammation during experimental cerebral malaria (ECM). We identified an ECM-protective mutation in coiled-coil domain containing protein 88b (Ccdc88b), a poorly annotated gene that is found expressed specifically in spleen, bone marrow, lymph nodes, and thymus. The CCDC88B protein is abundantly expressed in immune cells, including both CD4+ and CD8+ T lymphocytes, and in myeloid cells, and loss of CCDC88B protein expression has pleiotropic effects on T lymphocyte functions, including impaired maturation in vivo, significantly reduced activation, reduced cell division as well as impaired cytokine production (IFN-γ and TNF) in response to T cell receptor engagement, or to nonspecific stimuli in vitro, and during the course of P. berghei infection in vivo. This identifies CCDC88B as a novel and important regulator of T cell function. The human CCDC88B gene maps to the 11q13 locus that is associated with susceptibility to several inflammatory and auto-immune disorders. Our findings strongly suggest that CCDC88B is the morbid gene underlying the pleiotropic effect of the 11q13 locus on inflammation.

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