A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 596-625 ◽  
Author(s):  
Michal Smida ◽  
Anita Posevitz-Fejfar ◽  
Vaclav Horejsi ◽  
Burkhart Schraven ◽  
Jonathan A. Lindquist
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Activity ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Ras Activation ◽  
Anergic T Cells

Abstract In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.

scholarly journals PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

2018 ◽  
Vol 215 (12) ◽  
pp. 3165-3179 ◽  
Author(s):  
Uzodinma U. Uche ◽  
Ann R. Piccirillo ◽  
Shunsuke Kataoka ◽  
Stephanie J. Grebinoski ◽  
Louise M. D’Cruz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Modulation ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Cellular Growth ◽  
Negative Regulator ◽  
Regulatory Subunit ◽  
Kringle Domain

Phosphatidylinositol-3 kinases (PI3Ks) modulate cellular growth, proliferation, and survival; dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or transmembrane inhibitor of PI3K [TrIP]) is a putative transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular domain with homology to the inter-SH2 domain of the PI3K regulatory subunit p85, but the mechanism of TrIP function is poorly understood. We show that both the kringle and p85-like domains are necessary for TrIP inhibition of PI3K and that TrIP is down-modulated from the surface of T cells during T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by mediating oligomerization. Using an inducible knockout mouse model, we show that TrIP-deficient T cells exhibit more robust activation and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is a negative regulator of T cell activation and may represent a novel target for immune modulation.

scholarly journals FOXP3 Isoforms and Human Regulatory T Cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Rebeca Santos ◽  
Baohua Zhou
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Inflammatory Cytokines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Exon 2

Background:  Regulatory T-cells (Tregs) are critical to maintaining immune tolerance, thus prevent autoimmunity and allergic diseases. The master transcription factor FOXP3, expressed in humans as two isoforms through mRNA alternative splicing, controls the development and function of Tregs. However, the functions of the two isoforms remain unclear. We hypothesized that the oligonucleotides, developed by the lab, would efficiently shift FOXP3 to its shorter isoform, lacking exon 2 (FOXP3 ΔE2), which enables us to study how the FOXP3 ΔE2 isoform affects Treg function.      Methods:  Human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and cultured in the presence of 1 µM isoform-shifting oligonucleotides for three weeks. Expression of the FOXP3 isoforms, CD25, CTLA-4, CD40L, was examined through flow cytometry. Another goal was to study how the FOXP3 ΔE2 isoform shift affects the Treg response to inflammatory cytokines.    Results:  The oligonucleotides are highly effective in shifting the FOXP3 to the FOXP3 ΔE2 isoform. The majority of Tregs express only the FOXP3 ΔE2 isoform after two weeks of culturing in 1 µM oligo and had reduced expression of CD25, which plays a role in Treg differentiation, function, and homeostasis. Tregs display reduced expression of CTLA-4, a negative regulator of T-cell activation, but elevated CD40L, which costimulates and regulates the immune response. The FOXP3 ΔE2 isoform shift also sensitizes the Tregs to proinflammatory cytokine stimulation, allowing for increased IL-4, IL-17A, and IFN-γ production.    Conclusion:  Our studies demonstrate that FOXP3 ΔE2 Tregs are less stable because of the lower CD25 expression and less suppressive as determined by lower CTLA-4 but higher CD40L expression. In the inflammatory environment, the FOXP3 ΔE2 Tregs also produce more inflammatory cytokines, which further reduces their regulatory function. This study establishes an understanding of FOXP3ΔE2 Tregs’ role in autoimmunity and provides a potential novel target to treating autoimmune diseases.     Acknowledgements:  This study was funded, in part, with support from the Immunology and Infectious Diseases Training Program Grant funded, in part by T32 AI 060519 from the National Institutes of Health to RS and NIH AI159804 to BZ.  

scholarly journals PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt

2017 ◽  
Author(s):  
Uzodinma Uche ◽  
Andrea L. Szymczak-Workman ◽  
Stephanie Grebinoski ◽  
Ann Piccirillo ◽  
Louise M. D’Cruz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Modulation ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Regulatory Subunit ◽  
Kringle Domain ◽  
Knockout Mouse Model

ABSTRACTPhosphatidylinositol-3 kinases (PI3Ks) modulate numerous cellular functions, including growth, proliferation and survival. Dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or Transmembrane Inhibitor of PI3K – TrIP) is a novel transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular “p85-like” domain with homology to the inter-SH2 domain of the regulatory subunit of PI3K. Although TrIP has been shown to bind to the p110 catalytic subunit of PI3K in fibroblasts, the mechanism by which TrIP functions is poorly understood. We show that both the kringle and “p85-like” domains are necessary for TrIP inhibition of PI3K. We also demonstrate that TrIP protein is down-modulated from the surface of T cells to allow T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by binding an as-yet-unidentified ligand. Using an inducible knockout mouse model that we developed, we show that TrIP-deficient T cells exhibit more robust T cell activation, show a preference for Th1 polarization and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is an important negative regulator of T cell activation and may represent a novel target for immune modulation therapies.

scholarly journals T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

1993 ◽  
Vol 178 (6) ◽  
pp. 2107-2113 ◽  
Author(s):  
A J da Silva ◽  
O Janssen ◽  
C E Rudd
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

Abstract 219: Deficiency of the T Cell Regulator Cbl-B Aggravates Atherosclerosis by Inducing CD8 + T Cell-Mediated Macrophage Death

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Tom T Seijkens ◽  
Holger Winkels ◽  
Marion Gijbels ◽  
Jan A Kuivenhoven ◽  
Ljubica Perisic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aortic Arch ◽  
T Cell Activation ◽  
Cell Activation ◽  
B Cell Lymphoma ◽  
Negative Regulator ◽  
Atherosclerotic Plaques ◽  
Chow Diet ◽  
Plaque Area

Aims: The E3-ligase CBL-B ( Casitas B-cell Lymphoma-B ) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and Results: Here we investigated the effect of CBL-B deficiency in hyperlipidemic Apoe -/- mice in atherosclerosis. At the age of 20 weeks, chow diet-fed Cbl-b -/- Apoe -/- mice showed a significant increase in plaque area in the aortic arch, due to greater macrophage infiltration. Cbl-b -/- Apoe -/- macrophages displayed strong recruitment towards MCP1 and showed an increase in oxidized (ox)LDL uptake. In the aortic root of the same Cbl-b -/- Apoe -/- mice, where more advanced plaques were present than in the aortic arch, plaque area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages, had larger necrotic cores, and harboured more CD8 + T cells. The CD8 + T cells of Cbl-b -/- Apoe -/- mice were less susceptible to apoptosis and less resistant to Treg suppression. The increase in CD8 + T cells in the plaque effected greater macrophage apoptosis, resulting in enhanced necrotic core formation. Moreover, CBL-B gene expression was downregulated in human atherosclerotic plaques, and positively correlated with FoxP3 expression, indicating an atheroprotective effect. Conclusion: CBL-B is an important regulator of innate and adaptive immune reactions in atherosclerosis, by mediating macrophage recruitment and activation, CD8 + T cell activation, and CD8 + T cell-induced macrophage death in atherosclerotic plaques.

scholarly journals Induction of Cytotoxic T Lymphocyte Antigen 4 (Ctla-4) Restricts Clonal Expansion of Helper T Cells

2001 ◽  
Vol 194 (7) ◽  
pp. 893-902 ◽  
Author(s):  
Alden M. Doyle ◽  
Alan C. Mullen ◽  
Alejandro V. Villarino ◽  
Anne S. Hutchins ◽  
Frances A. High ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
T Lymphocyte ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cytotoxic T Lymphocyte ◽  
Clonal Expansion ◽  
Negative Regulator ◽  
Lymphocyte Antigen ◽  
Cell Divisions

Cytotoxic T lymphocyte antigen (CTLA)-4 plays an essential role in immunologic homeostasis. How this negative regulator of T cell activation executes its functions has remained controversial. We now provide evidence that CTLA-4 mediates a cell-intrinsic counterbalance to restrict the clonal expansion of proliferating CD4+ T cells. The regulation of CTLA-4 expression and function ensures that, after ∼3 cell divisions of expansion, most progeny will succumb to either proliferative arrest or death over the ensuing three cell divisions. The quantitative precision of the counterbalance hinges on the graded, time-independent induction of CTLA-4 expression during the first three cell divisions. In contrast to the limits imposed on unpolarized cells, T helper type 1 (Th1) and Th2 effector progeny may be rescued from proliferative arrest by interleukin (IL)-12 and IL-4 signaling, respectively, allowing appropriately stimulated progeny to proceed to the stage of tissue homing. These results suggest that the cell-autonomous regulation of CTLA-4 induction may be a central checkpoint of clonal expansion of CD4+ T cells, allowing temporally and spatially restricted growth of progeny to be dictated by the nature of the threat posed to the host.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

scholarly journals Both Maturation and Survival of Human Dendritic Cells are Impaired in the Presence of Anergic/Suppressor T Cells

2003 ◽  
Vol 10 (1) ◽  
pp. 61-65 ◽  
Author(s):  
L. Frasca ◽  
C. Scottà ◽  
G. Lombardi ◽  
E. Piccolella
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Costimulatory Molecule ◽  
T Cell Suppression ◽  
Cell Suppression ◽  
Anergic T Cells ◽  
Human Dendritic Cells

T cell suppression is a well established phenomenon, but the mechanisms involved are still a matter of debate. Mouse anergic T cells were shown to suppress responder T cell activation by inhibiting the antigen presenting function of DC. In the present work we studied the effects of co-culturing human anergic CD4+T cells with autologous dendritic cells (DC) at different stages of maturation. Either DC maturation or survival, depending on whether immature or mature DC where used as APC, was impaired in the presence of anergic cells. Indeed, MHC and costimulatory molecule up-regulation was inhibited in immature DC, whereas apoptotic phenomena were favored in mature DC and consequently in responder T cells. Defective ligation of CD40 by CD40L (CD154) was responsible for CD95-mediated and spontaneous apoptosis of DC as well as for a failure of their maturation process. These findings indicate that lack of activation of CD40 on DC by CD40L-defective anergic cells might be the primary event involved in T cell suppression and support the role of CD40 signaling in regulating both activation and survival of DC.

CD11b+gr-1+ myeloid Derived Suppressor Cells (MDSC) In Normal Bone Marrow Suppress T Cell Proliferation and Enhance T-Regulatory Function Via a IL10-, IL4- and IDO-Independent Mechanism

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4801-4801 ◽  
Author(s):  
Parvin Forghani ◽  
Wayne Harris ◽  
jian-Ming Li ◽  
M.R. Khorramizadeh ◽  
Edmund Waller
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Regulatory Function ◽  
T Cell Proliferation ◽  
Suppressive Activity ◽  
Ifn Γ

Abstract Abstract 4801 MDSC have been described as an important negative regulators of autologous anti-cancer immune responses. Considering the important role of MDSC in immune regulation in allogenic stem cell and organ transplantation, we undertook an investigation of the mechanism(s) by which MDSC inhibit T–cell activation and proliferation, and tested the hypothesis that local cytokine secretion or IDO activity is required for suppression of T-cell proliferation. Two separate populations CD11bhiGr-1hi and CD11bhi Gr-1int were isolated by high-speed FACS from lineage- BM antigen presenting cells (C57 & BALB/c mice). Both MDSC subsets had potent capacity for in–vitro suppression of CD4+ and CD8+ T cells proliferation in response to anti-CD3/anti-CD28 beads and Con A. A ratio of 0.5/1 MDSC: T-cells were sufficient to inhibit >66% control levels of T-cell proliferation. MDSC isolated from transgenic mice that had been “knocked-out” for IFN-γ and IDO had equivalent suppressive activity as MDSC from wild-type donors. Addition of saturating concentrations of anti IL-10 and IL-4 MAb, or in combination with anti- IFN-γ MAb did not abrogate MDSC-suppressive activity. Ex-vivo culture of MDSC with mitogen-activated T-cells generated two—fold more Fox-p3 T-reg compared with cultures of T cell plus mitogen. Data will be presented regarding the novel role of MDSC involving in the homeostasis regulation of normal T-cell activation and proliferation in non-tumor-bearing mice. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document